SYSTEM AND METHOD FOR HOMOGENOUS GPCR PHOSPHORYLATION AND IDENTIFICATION OF BETA-2 ADRENERGIC RECEPTOR POSITIVE ALLOSTERIC

MODULATORS

RELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional application no. 62/627,678, filed February 7, 2018, and U.S. provisional application no. 62/627,680, filed February 7, 2018, the entire contents of which are incorporated herein by reference.

TECHNIC AL FIELD

[0002] The invention relates to a chimeric G protein-coupled receptor (GPCR) comprising a C-terminus amino acid sequence ligated to a synthetic phosphopeptide, j½ adrenergic receptor positive allosteric modulators, their use in the treatment of diseases or conditions ameliorated by 2 receptor activation, and methods of screening and identifying b ₂ adrenergic receptor positive allosteric modulators.

INCORPORATION -BY -REFEREN CE OF MATERIAL SUBMITTED ELECTRONICALLY

[0003] Incorporated by reference in its entirety herein is a computer-readable

nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 3,600 Bytes ASCII (Text) file named "028193-9284- US01_As_Filed_Sequence_Listing.TXT," created on February 6, 2018.

BACKGROUND OF THE INVENTION

[0004] G protein-coupled receptors (GPCRs), a large family of plasma membrane receptors coupled to guanine nucleotide regulatory proteins, represent one of the most important mechanisms for transducing extracellular signals into specific cellular responses. Their important role m regulating many physiological processes makes them a common therapeutic target of clinically used drugs. The overwhelming majority of these, both agonists and antagonists, bind to the orthosteric binding site on the receptor. This is defined as the site to which the endogenous hgand(s) for the receptor binds, e.g. adrenaline for the adrenergic receptors, histamine for the histamine receptors etc. Most clinically used antagonists are orthosteric binders and exert their effects by competitive inhibition.

[0005] Despite their ability to recognize a vast array of ligands (Lagerstrom MC & Schioth HB (2008) Nat Rev Drug Discov 7(4):339-357), GPCRs have a highly conserved mechanism of action. Ligand binding to the extracellular orthosteric pocket induces conformational changes within the receptor transmembrane (TM) region (Manglik A & Kruse AC (2017) Biochemistry 56(42): 5628-5634), leading to the sequential intracellular coupling of three main transducer proteins: G protein, GPCR kinase (GRK) and b-arrestin (Parr) (Lefkowitz RJ (2013) Angew Chem Int Ed Engl 52(25):6366-6378). More specifically, GPCR-dependent activation of the heterotnmenc G protein leads to the dissociation of the a subunit from the bg subunits, resulting in modulation of second messenger systems, such as cAMP (Neves SR, et al (2002) Science 296(5573): 1636-1639). Subsequent GRK phosphorylation of specific serine/threonine residues within the receptor third intracellular loop (I CL) or carboxyl (C)-terminal tail recruits parr (Benovic JL, et al. (1987) Proc Natl Acad Sci U S A 84(24): 8879-8882). The binding of parr desensitizes GPCR signaling by sterically blocking G protein coupling and promoting receptor internalization through interactions with AP2 and clathrin (Shenoy SK & Lefkowitz RJ (201 1 ) Trends Pharmacol Sci 32(9): 521 -533). Additionally, Parr can directly modulate cell signaling through G protein-independent pathways (Peterson YK & Luttrell LM (2017) Pharmacol Rev 69(3):256-297).

[0006] It is now well established that“biased” GPCR ligands can disproportionately regulate particular branches of receptor signaling, a phenomenon known as biased agonism (Reiter E, et al. (2012) Anna Rev Pharmacol Toxicol 52: 179-197). The selective activation of signaling pathways indicates that, although all three transducers specifically interact with agonist-bound GPCRs, their conformational specificities are not identical. However, the fundamental mechanisms underlying this differential coupling remain obscure, largely because events mediated by different transducers are intricately intertwined. In particular, Parr binds to receptors through a two-step process, initially interacting with GRK-phosphoryiated residues and then coupling to the agonist-activated GPCR TM core (Fig. 1) (Gurevich W & Benovic L (1993) JBiol Chem 268(16): 11628-11638). Biochemical and structural studies have

demonstrated that binding to GPCRs’ phosphorylated tails induces extensive conformational changes in Parr, including the extension of several loops implicated m Parr’s interaction with GPCRs’ TM bundle (Shukla AK, et al. (2013) Nature 497(7447): 137-141).

[0007] Engagement of Parr with GPCRs’ TM cores is believed to mediate particular functions of parr such as receptor desensitization, but efforts to understand the nature and consequences of this interaction have been hampered by its low affinity and its dependence on GRK phosphorylation. Thus, there remains a need for compositions and methods for obtaining uniformly phosphorylated receptors in a cellular context or in vitro, which can be used to identify compounds which modulate GPCR activity'. Such compounds may have therapeutic utility against diseases associated with GPCR dysfunction.

[00Q8] Recently, an increasing number of negative and positive allosteric modulators (NAMs and PAMs) for GPCRs have been described, although to date only two have reached the clinic. Rather than directly stimulating or inhibiting biological effects on their own, these allosteric compounds exert their effects by modulating receptor responsiveness to endogenous agonists. Such allosteric ligands offer a number of potential advantages as drugs including greater specificity amongst closely related receptor subtypes, and maximum or ceiling effects which can reduce adverse actions, amongst others. Such allosteric modulators can also serve as valuable reagents in the research laboratory where, by virtue of their cooperative interactions with orthostenc ligands, they can help to freeze or lock specific receptor conformations so that they can be studied by biophysical techniques.

[0009] The p?.-adrenergic receptor (b?.AK) is a prototypical G protein-coupled receptor (GPCR) that plays important roles in cardiovascular and pulmonary pathophysiologies, and is a key therapeutic target. Conventional drug discovery efforts at P?.ARs have led to the development of ligands that bind exclusively to the receptor’s hormone-binding orthostenc site. On the other hand, targeting the largely unexplored and evolutionarily unique allosteric sites has the potential for developing drugs that are more specific and have fewer side-effects than orthosteric ligands. [0010] Selection of allosteric modulators for GPCRs using the usual cell-based functional assays such as those for cyclic AMP (cAMP) generation or b-arrestin recruitment have a number of disadvantages. For example, it can be laborious and difficult to interpret modulation of a response with these assays, rather than the on or off responses that such assays are better suited to measure. These assays are also subject to a variety of artifacts and have relatively limited compound throughput of ~10 ³ -10 ⁶ .

[0011] In contrast, interaction or affinity-based methods, in which large libraries of self- encoding potential binders are screened against a target protein molecule, circumvent these shortcomings. A particularly powerful approach is the use of DNA encoded small molecule libraries (DELs) containing as many as 1 billion potential ligands. Each molecule in such a library' is covalently linked to a small stretch of nucleotides which serves as a barcode which is used to identify target binders by next generation sequencing. Such approaches work well when applied to soluble protein targets but have been much more difficult to adapt to membrane proteins such as GPCRs. However, using this approach we recently described i solation of the first NAM for the b2- άG6h6¾io receptor (“allosteric b-biocker”) and identified its intracellular binding site on the receptor by x-ray crystallography (Ahn et al. (2017) PNAS 1 14(7): 1708- 1713. dot: 10.1073/pnas.16206451 14; Liu et al. (2017) Nature 548(7668):480-484. dot:

10.1 038/nature23652.).

SUMMARY OF THE INVENTION

[0012] In one aspect, the disclosure provides a complex comprising (i) a chimeric G protein- coupled receptor (GPCR) comprising the amino acid sequence LPETGGG (SEQ ID NO: 1) located within the C -terminus of the GPCR and a synthetic phosphopeptide ligated to SEQ ID NO: 1 ; and (ii) a b-arrestin (Parr) protein bound to the C -terminus of the GPCR.

[0013] In another aspect, the disclosure provides an in vitro method for producing the aforementioned complex, as well as methods for identifying compounds or ligands which hind to and modulate the activity of the complex.

[0014] The present invention also relates to positive allosteric small molecules for the b ₂ AK, identified by affinity-based screening of over 500 million distinct library compounds. Compounds of the invention display positive cooperatively with orthosteric agonists, thus enhancing their binding to the receptor and ability to stabilize its active state. The compounds also exhibit positive cooperativity with G protein and b-arrestin, thus potentiating their stabilization of high-affimty agonist-bound states of the receptor, as well as downstream cAMP production and b-arrestin recruitment to the activated receptor. The positive allosteric activity is specific for the ?.AR compared to its closely related sub-type, the biAK

[0015] In another aspect, provided is a compound according to Formula (I):

[0016] or a pharmaceutically acceptable salt thereof,

[0017] wherein:

[0018] R ¹ is an aryl group optionally substituted with 1-5 substituents independently selected from the group consisting of Ci-ealkyl, Ci-ehaloalkyl, C3-7cycloalkyl, halogen, cyano, -OH, -

OCi-ealkyl, -OCi-ehaloalkyl, M l:. -NHCi-ealkyl, N({ : -r.alky 1 ) ·. -OCn-ecydoalkyl, M IC ^' .

6cycloalkyl, -N(Ci-6a3kyl)(C3-6cycloalkyl), and -N(C3-6cycloalkyl)2, and optionally the aryl is phenyl wherein two substituents join to form a 5- to 7-mernbered non-aromatic fused ring containing 1-2 heteroatom groups selected from NR ^la and O;

0019] l-ialkyl),

[0020] R ^la is H or Ciaalkyl;

[0021] R ^u ’ and R ^!C are each independently hydrogen or Ci-4alkyl, or R ^lb and R ^5c together with the carbon to winch they are attached form a C ₃ -6cycloalkyl ring;

[0022] R ² is hydrogen, Ci-6alkyl, or C3-7cycloalkyl; [0023] R ^" is hydrogen, Ci-ealkyi, C3-7cycloalkyl, or aryl, the aiyl being optionally substituted with 1-5 substituents independently selected from the group consisting of Ci-ealkyl, Ci- ehaloalkyl, Cs-/cycloalkyL halogen, eyano, -OH, -OCi-ealkyl, -QCi-ehaloalkyl, -NH2, -NHCi- ealkyl, -N(Ci-6alkyl) ₂ , -OCs-ecycloalkyl, -NHC3-6cyeloalkyl, -N(Ci-6alkyd)(C ₃ -6cyeloalkyi), and -N (C 3 -6cy cloaiky 1) 2 ;

[0024] alternatively, R ² and R ³ together with the nitrogen to which they are attached form a 4- to 8-membered heterocyclic ring optionally containing one additional heteroatom selected from N, O, and S, and being optionally substituted with 1-4 substituents independently selected from the group consisting of Ci-ealkyl, Ci-ehaloalkyl, halogen, eyano, -OH, oxo, -OCi-ealkyl, - NH _{2, -} NHCi-ealkyl, and -N(C _l -6alkyl) ₂ ;

[0025] R ⁴ is hydrogen, Ci-6alkyl, or C ₃ -?cy cloaiky 1;

[0026] R ⁵ is CHR'”R ^b :

[0027] alternatively, R ⁴ and R ⁵ together with the nitrogen to which they are attached form a 4- to 8-membered heterocyclic ring optionally containing one additional heteroatom selected from N, O, and S, and being optionally substituted with 1-4 substituents independently selected from the group consisting of Ci-ealkyl, Ci-ehaloalkyl, halogen, eyano, -OH, oxo, -OCi-ealkyl, - NII2, -NHCi-ealkyl, and -N(Ci-6alkyi)2;

[0028] R ^5a is aryl or -Ci-salkylene-aryl, wherein each aryl in R ^5a is optionally substituted with 1-5 substituents independently selected from the group consisting of Ci-ealkyl, Ci- sha!oalkyl, C3-7cycloalkyl, halogen, eyano, -OH, -OCi-salkyl, -OCi-shaloalkyl, -NH2, -NHCi- ealkyl, -N(Ci-6alkyl) ₂ , -OC3-6cycloalkyl, -NHC -ecycloalkyl, -N(Ci-6alkyl)(C3-6cy cloaiky 1), and -N(C3-6cycloalkyl) ₂ ;

[0029] R ^5b is X ² or -Ci-ialkylene-X ² ; and

[0030] X ² is -CN, -C(0)OH, -C(0)OCi-4alkyl, -C(0)NH ₂ , -C(0)NHCi-4alkyl, -C(0)N(Ci- 4alkyl)2, -SO2NH2, -S0 ₂ NHCi-4alkyl, or -S02N(Ci- ₄ alkyl) ₂ .

[0031] In another aspect, is provided a pharmaceutical composition comprising a compound according to Formula (I) or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. [0032] In another aspect is provided a method of treating a disease or condition ameliorated by b?. receptor activation comprising administering to a subject in need thereof a therapeutically effective amount of the compound of formula (I), a pharmaceutically acceptable salt or composition thereof.

[0033] In another aspect, the invention provides compounds of formula (I), or a

pharmaceutically acceptable salt thereof for use in treating a disease or condition mediated by a b2 receptor.

[0034] In another aspect, the invention provides the use of a compound of formula (I), or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment a disease or condition mediated by a b2 receptor.

[0035] In a further aspect, the in vention provides kits comprising a compound of formula (I), or a pharmaceutically acceptable salt or composition thereof, and instructions for use.

[0036] Other aspects will become apparent by consideration of the detailed description and accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWING(S)

[0037] Figure 1 shows the two-step binding mode of b-arrestin. Ligand (L) binding to the extracellular orthosteric binding pocket leads to conformational changes within the GPCR transmembrane region to influence intracellular transducer binding. The phosphorylation (red circles) of the receptor C terminus by GPCR-kinase (GRK) initiates the recruitment of b-arrestin ( tarr). Conformational changes induced in barr ^arr*) as a result of binding to the

phosphorylated C terminus promotes coupling to the GPCR transmembrane core, which allosterically enhances ligand affinity.

[0038] Figure 2A shows non-phosphory!ated vAR interacts with Gs heterotrimer but not b- arrestml . Coomassie-stained gel show the co-imrmmopreeipitation of Gs heterotrimer (Gs) or b~ arrestin l ( arrl) with isoproterenol (ISO)-bound FLAG^AR. Loading controls represent 10% of input.

[0039] Figure 2B shows competition binding experiments using radiolabeled [ ¹²⁵ I]- cyanopmdolol (CYP). Gs increases ISO affinity for b ₂ AK HDLs (log IC50: -8.88 +/- 0.03) compared to no transducer (log IC50: -6.82 +/- 0.03), but arrl does not (log IC50: -6.81 +/- 0.02). Data shown are the mean of three independent experiments, with error bars representing standard error.

[0040] Figure 2C shows the fluorescence emission spectrum of bimane-labeled b?.AK HDLs rightward shift and decrease in fluorescence upon addition of ISO, indicative of receptor activation. The effects of ISO are enhanced by Gs but not arrl. Data shown are representative of three independent experiments.

[0041] Figure 3 A shows a cartoon schematic of the sortase ligation method. A synthetic phosphopeptide (pp) derived from the vasopressin-2-receptor (V ₂ R) with three N-terminal glycine residues (GGG-V ₂ Rpp) (SEQ ID NO: 16) is ligated onto receptors containing a C- termina! LPETGGH recognition motif. Phosphorylated serine and threonine residue are indicated with arrows.

[0042] Figure 3B shows Coomassie-stained gel showing the co-immunoprecipitation of heterotrimeric Gs and b-arrestinl (Parrl) with isoproterenol (ISO)-bound, phosphopeptide- ligated FLAG~p ₂ AR ((¾ARpp). Loading controls represent 10% of input.

[0043] Figure 3C shows competition binding experiments using radiolabeled [ ¹²⁵ I]- cyanopindoiol (CYP) with HDLs containing b ₂ ARpp. Gs increases the affinity of ISO for b ₂ AKrr (log IC50: -9 15 +/- 0.03) compared to no transducer (log IC50: -6.24 +/- 0.04), and |3arrl increases ISO affinity for b ₂ ARrr HDLs (log IC50: -7.14 +/- 0.07). Data shown are the mean of at least three independent experiments, with error bars representing standard error, and stars (*) indicate a log IC50 value significantly different from the control curve (P < 0.05, one way ANOVA).

[0044] Figure 3D shows competition binding experiments using radiolabeled [ ¹²⁵ I]- cyanopindolol (CYP) with HDLs containing b ₂ A ligated to a non-phosphorylated version of the V ₂ R peptide (bzAKhr). Gs increases the affinity of ISO for bcAKhr HDLs (log IC50: -9.02 +/- 0.04) compared to no transducer (log IC50: -6.42 +/- 0.09), but barrl does not increase ISO affinity for b ₂ AKhr HDLs (log IC50: -6.49 +/- 0.04). Data shown are the mean of at least three independent experiments, with error bars representing standard error, and stars (*) indicate a log XC50 value significantly different from the control curve (P < 0.05, one-way ANOVA). [0045] Figure 3E shows the effects of ISO on the HDL^ARpp-bimane fluorescence emission spectrum are enhanced by Gs and arrl. Data shown are representative of three independent experiments.

[0046] Figure 4A shows the allosteric interaction between phosphorylated b ₂ AE and b- arrestml requires the finger loop of b-arrestml. In competition radioligand binding with b?.AKrr HDDs, a finger loop deletion mutant of b-arrestinl ^arrl) (D62-77) has minimal effect on isoproterenol (ISO) binding (log IC50s: no transducer, -6.30 +/- 0.05: b3GG 1 , -7.25 +/- 0.07; parr ! D62-77, -6.48 +/- 0.03). Data shown in are the mean of at least three independent experiments, with error bars representing standard error, and stars (*) indicate a log IC50 value significantly different from the control curve (P < 0.05, one-way ANOVA).

[0047] Figure 4B shows bapΊD62~77 does not intensify the effects of ISO on the

fluorescence spectrum of p ₂ ARpp-bimane HDLs. Data shown are representative of three independent experiments.

[0048] Figure 4C shows competition radioligand binding with b ₂ AKrr HDLs containing a deletion of the third intracellular loop (D238-267). Both Gs (log IC50: -8.85 +/- 0.03) and arrl (log IC50: -7 63 +/- 0.06) retain their ability to increase isoproterenol (ISO) affinity (no transducer, log IC50: -6.91 +/- 0.04). Data shown are the mean of at least three independent experiments, with error bars representing standard error, and stars (*) indicate a log IC50 value significantly different from the control curve (P < 0.05, one-way ANOVA).

[0049] Figure 5A show's the allosteric enhancement of agonist binding induced by b- arrestinl at the M2 receptor. Competition binding experiments with sortase-iigated M ₂ .Rpp HDLs use [ ³ H]-N-Methyl-Scopolamine (NMS) as the tracer. Fleterotnmenc Gi (100 nM, log IC50: -7.51 +/- 0.06) and b-arrestinl (Parrl ) (1 mM, log IC50: -7.06 +/- 0.08) increase the affinity of the agonist carbachol to a similar extent (no transducer, log IC50: -5.31 +/- 0.09).

Data are the mean of three independent experiments, with error bars representing standard error, and stars (*) indicate a log 1C50 value significantly different from the control curve ( P < 0.05, one-way ANOVA).

[0050] Figure 5B shows the allosteric enhancement of agonist binding induced by b-arrestinl at the MOR receptor. Competition binding experiments with sortase-iigated MORpp HDLs use [¾]-Naloxone as the tracer. Gi (1 iiM, log IC50: -8.22 +/- 0.05) increases the affinity of the agonist DAMGO to a far greater extent than Parrl (1 mM, log IC50: -6.02 +/- 0.05) (no transducer, log IC50: -5.71 +/- 0.06). Data are the mean of three independent experiments, with error bars representing standard error, and stars (*) indicate a log IC50 value significantly different from the control curve (P < 0.05, one-way ANQVA).

[0051] Figure 5C shows a comparison of the difference in agonists’ log IC50 values m the presence of their cognate G proteins versus Parrl for sortase-ligated p ₂ ARpp, M ₂ Rpp, and MORpp HDLs.

[0052] Figure 6A shows the fluorescence spectra of b-arrestinl (Parr!) labeled with monobromobimane at residue 70 in the finger loop. Activation of H D L- p ₂ A R pp by the agonist isoproterenol (ISO) increases Parr 1 -bimane fluorescence, which is blocked by Nb80 binding to the receptor TM core. Data shown are representative of three independent experiments.

[0053] Figure 6B shows a comparison of Parr 1 -bimane fluorescence by agonist activation of p ₂ ARpp, M ₂ Rpp, and MORpp HDLs. The area under the fluorescence emission spectra were determined and normalized to M ₂ Rpp plus iperoxo (the maximum signal) in each experiment.

Ail three receptors are significantly different from one another ( *P < 0.05), and p ₂ ARpp and M ₂ Rpp are significantly different from their respective antagonist controls (not indicated, P < 0.05). Data are the mean of at least three independent experiments, with error bars representing standard error; P values were determined by one-way ANOVA.

[0054] Figure 6 C shows an in vitro GTPase assay measuring GTP hydrolysis as a readout of G protein activation. The basal level of GTP hydrolysis induced by G protein is robustly increased by HDL~p ₂ ARpp HDLs in the presence of ISO compared to no ligand (NL) (*P < 0.05), which is blocked (desensitization) by the addition of Nb80 (no significant difference between NL and ISO + Nb80). Data are the mean of at least three independent experiments, with error bars representing standard error; P values were determined by one-way ANOVA.

[0055] Figures 6D shows inhibition (% desensitization) of G protein activation by Parr! is strongest at M ₂ Rpp and significantly different from p?.ARpp and MORpp HDLs (*P < 0.05).

Data are the mean of at least three independent experiments, with error bars representing standard error; P values w¾re determined by one-way ANOVA. [0056] Figure 7A shows the generation and purification of phosphopeptide (pp)-ligated b?.AK (|½ARpp). Purified FLAG^AR in detergent containing the sortase consensus site LPETGHH after amino acid 365 and a C-terminal 6xHis tag (p ₂ AR-LPETGHH-FIis6) was modified using sortase-His6 and GGG-V2Rpp. Ligated ?.ARpp was subsequently purified from unligated receptor (|32AR-LPETGGH-His6), sortase-His6, and GGG-VbRpp by Talon metal affinity resin and size exclusion chromatography, as shown in the schematic.

[0057] Figure 7B shows protein (eoomassie) staining of ?.ARpp purification.

[0058] Figure 7C shows size exclusion chromatographic analysis of ₂ ARpp.

[0059] Figure 8A shows the increase in agonist affinity to the b ₂ AKrr induced by b-arrestinl is 100-fold less than that induced by G protein. Plot of changes in the log IC50 values of isoproterenol (ISO) derived from competition radioligand binding experiments using [ ^l25 I]~ cyanopmdolo! (CYP) and HDL-j¼ARpp in the absence or indicated concentration of transducer (Gs and b-arrestinl (|3arrl)). The star (*) indicates that curve fit maxima are significantly different (P < 0.05, /-test).

[0060] Figure 8B shows competition binding assays using HDL^LARpp, [ ¹²⁵ I]-CYP, and the indicated concentration of ICI-l 18,551 in the presence or absence of Gs (100 nM) or (3arrl (1 mM). Error bars represent standard error from at least three independent experiments. The stars indicate log IC50 values significantly different from the control curves ( P < 0 05, one-way

ANOVA).

[0061] Figure 8C shows the increase in agonist affinity to the p ₂ ARpp induced by b-arrestinl requires receptor phosphorylation. Figure 8C shows competition binding assays using HDL- bzAKrr, [ ¹²⁵ 1]-CYP, and the indicated concentration of ISO where HDL-fi ARpp was treated with calf-intestinal alkaline phosphatase (CIP) prior to assay setup. The stars indicate log IC50 values significantly different from the control curves (P < 0.05, one-way ANOVA).

[0062] Figure 9 A shows phosphopeptide-ligated (* *) MOR (MQRpp) and M2R (MzRpp) were generated by incubating receptors containing a C-terminal sortase recognition site (*) with GGG-V>Rpp and sortase. To determine ligation efficiency, a small fraction of the reaction was deglycosylated with PNGase to visualize changes in receptor molecular weight. Coomassie- stained protein gels are shown. [0063] Figure 9B shows competition binding assays using HDL-M?.Rpp performed as in Figure 5,4 except that HDLs were treated with calf-intestmal alkaline phosphatase (CIP) prior to assay setup.

[0064] Figure 9C shows competition binding assays using HDL-MORpp performed as in Figure 5 B except that HDLs w ^? ere treated with calf-intestinal alkaline phosphatase (CIP) prior to assay setup.

[0065] Figure 9D show's a graph of changes in the log IC50 values for DAMGO determined from competition radioligand binding experiments using [ ^J H]-Naloxone and HDL-MORpp in the absence or indicated concentration of transducer (Gi and b-arrestinl (Parrl)). Stars (*) indicate that the curve fit maxima are significantly different (P < 0.05, /-test).

[0066] Figure 9E show's competition binding assays using HDL-VkRpp performed as in Figure 5,4 using iperoxo as the competitor ligand in the presence or absence of Gi (100 nM) or arrl (1 mM). Stars (*) indicate log XC50 values significantly different from the control curve ( P < 0.05, one-way ANOVA).

[0067] Figure 9F shows a plot of changes in the log IC50 values of iperoxo obtained from competition binding experiments using [¾]-NMS and HDL-MaRpp in the absence or indicated concentration of transducer (Gi and [½rrl ). Stars (*) indicate that the curve fit maxima are significantly different (P < 0.05, /-test).

[0068] Figure 9G shows competition binding assays using HDL-IVhRpp performed as in Figure 5 A using a finger-loop deletion mutant of [½rrl (D62-77) (1 mM). Error bars represent standard error from at least three independent experiments. Stars (*) indicate log IC50 values significantly ⁷ different from the control curve ( P < 0.05, one-way ANOVA).

[0069] Figure 10A shows an analysis of b-arrestml coupling to the transmembrane core of bzAKrr by bimane fluorescence spectra of b-arrestinl ( arrl) labeled with monobromobimane at residue 70 in the presence of aARpp HDLs. Data are representative of three independent experiments.

[0070] Figure 10B shows an analysis of b-arrestinl coupling to the transmembrane core of M?.Rpp by bimane fluorescence spectra of b-arrestinl ( arrl) labeled with monobromobimane at residue 70 in the presence of JVhRpp HDLs. Data are representative of three independent experiments.

[0071] Figure 10C shows an analysis of b-arrestinl coupling to the transmembrane core of MORpp by bimane fluorescence spectra of b-arrestinl ( arri) labeled with monobromobimane at residue 70 m the presence of MORpp HDLs. Data are representative of three independent experiments.

[0072] Figure 11 shows the inhibition of G protein activation by b-arrestinl correlates with its coupling strength to the receptor transmembrane core. The GTPase activity (GTP hydrolysis) of purified G protein (Gs or Gi) was measured in vitro in the presence and absence of b ₂ AKrr (Gs), M ₂ Rpp (Gi), and MORpp (Gi) HDLs. Treatment with the agonists isoproterenol (ISO) (p ₂ ARpp) or DAMGO (MORpp) increases G protein activation, which is not significantly altered by the presence of b-arrestm! (parrl). Activation of M ₂ Rpp with the agonist iperoxo increases G protein activation, which is significantly blocked by b pΊ (*P < 0.05, one-way ANOVA). Error bars represent standard error from three independent experiments.

[0073] Figure 12 shows complexes of sortase-ligated p ₂ ARpp with parr! and Nb 25 survive selection conditions for DNA-encoded library screening. Sortase-ligated p ₂ ARpp was reconstituted with biotinylated ApoAl and complexed with parrl and the stabilizing nano body Nb25. Complexes were immobilized on NeutrAvidin beads, washed, and eluted as previously described (Aim et al, Mol Pharmacol 94, 850-861 , 2018. (1) HDL-p ₂ ARpp input; (2) parr i and Nb25 input, (3) NeutrAvidin bead flow-through (unbound proteins and dissociated complexes), (4) NeutrAvidin bead washes (unbound proteins and dissociated complexes; 5x more sample loaded than other lanes), (5) NeutrAvidin bead elution (specifically bound complexes).

[0074] Figure 13 shows complexes of HDL-p ₂ AR with heterotrimeric Gs protein and complexes of sortase-ligated p ₂ ARpp with Parrl can be used to efficiently screen DNA-encoded small molecule libraries. In two rounds of selection for binding to these complexes, the library size is reduced to <10 ⁸ molecules, enabling the selection output to be subjected to next- generation sequencing. This indicates that selection conditions are appropriately stringent to remove non-specifically bound molecules in the library. R0 = DNA-encoded librar input, R1 and R2 = selection for binding to immobilized receptor/transducer complexes, R3 = counter selection against His-tagged Gs and His-tagged Nb25 + Parrl bound to NiNTA beads.

[0075] FIG. 14A shows a cartoon for DNA-encoded small molecule screening of the 2AR reconstituted in high density lipoprotein (HDL) particles using a biotinylated version of the membrane scaffolding protein ApoAl, where the orthosteric site of the receptor is occupied by a high affinity b-agonist BI- 167107 to hold the receptor more in an active conformation.

[0076] FIG. 14B shows that the b2 receptor containing biotinylated-HDLs can be efficiently captured on NeutrAvdm beads.

[0077] FIG. 14C shows that the b2 receptor containing biotinylated-HDLs have comparable affinity for antagonist binding to that of b2AKe in membrane preparations.

[0078] FIG. 14D shows that 2ARs m HDL particles can functionally couple to

heterotrimeric Gs in competitive radioligand binding assays.

[0079] FIG. 14E shows the activity of 50 potential screening hits to increase binding of a radiolabeled agonist [’Hjb ^ty -methoxyfenoterol ( ³ H-FEN) to the 2AR expressing on cell membranes in the absence and presence of the transducer proteins G protein and b-arrestin.

[0080] FIG. 14F shows the concentration response curves for seven compounds identified from the screening as potential 2AR PAMs.

[0081] FIG I4G shows the structures of the seven compounds for which data is shown in FIG IF.

[0082] FIG I4H show's isothermal titration calorimetry (XTC) for Compound 6. The values summarize binding affinity (KD), stoichiometry (N), and thermodynamic parameters.

[0083] FIG ISA show's activity' of Compound 6 to increase the ability of an agonist, isoproterenol (ISO) to activate G protein -mediated cAMP production through the b2AK in a dose-dependent way.

[0084] FIG. 15B shows activity of Compound 43 to increase the ability of an agonist, isoproterenol (ISO) to activate G protein-mediated cAMP production through the b2AK. in a dose-dependent way.

[0085] FIG. 15C shows activity of Compound 6 to increase the ability of an agonist, isoproterenol (ISO) to recruit b-arrestin to the b2U2K., a chimeric receptor with a V2R tail at the C-terminus that displays stronger and more stable agonist-promoted b-arrestin binding than the native 2AR while retaining the pharmacological properties of the native b2AK

[0086] FIG. 15D shows activity of Compound 43 to increase the ability' of an agonist, isoproterenol (ISO) to recruit b-arrestin to the b2U2K.

[0087] FIG. 16A shows Compound 6-mediated dose-dependent left-shifts of the

isoproterenol competition curve in [ ^1/5 I]-cyanopindolol ( ^1/5 I-CYT) binding to the p2AR reconstituted m HDL particles.

[0088] FIG. 16B shows Compound 43-mediated dose-dependent left-shifts of the

125

isoproterenol competition curve in I-CYP binding to the (32 A R reconstituted in HDL particles.

[0089] FIG. 16C shows Compound 6-and Compound 43-mediated dose-dependent left-shifts of the isoproterenol competition curve in I-CYP binding with membranes prepared from 2AR-overexpressing ceils.

125

[0090] FIG. 16D shows the fold change in I-CYP binding to the p2AR for the data in FIG.

[0091] FIG. 16E shows Compound 6 and Compound 43-mediated dose-dependent increases

3

in 1-I-FEN binding to the b2A .

[0092] FIG. 17 shows the positive allosteric effects of Compound 6 to stabilize the active conformation of the p2AR, measured as the potentiation of ISO-induced decreases in the amount of fluorescence and increases in the maximum wavelength from the monobromobimane-labeled p2AR in HDL particles.

[0093] FIG. 18A shows Compound-6-induced shifts of the isoproterenol competition curve

125

to the left in I-C P binding to the P2AR obtained with membranes from cells expressing the P2AR or P2AR-Gs fusions.

[0094] FIG. 18B shows Compound-6- induced shifts of the isoproterenol competition curve

125

to the left in I-CYT binding to the b2AE obtainedwith membranes from ceils expressing the P2AR or b2n2K-bap- fusions.

[0095] FIG. ISC shows the effect of Compound 6 to increase 1H-FEN binding to the p2AR in the presence of Gs. [0096] FIG. I 8D shows the effect of Compound 6 to increase H-FEN binding to the p2AR in the presence of b-arrestm.

[0097] FIG. 19 shows the effect of ISO-induced cAMP production by the overexpressed b2 AR in the presence of the indicated concentrations of Compound-6.

[0098] FIG. 20A shows the effect of Compound 6 to increase 1H-FEN binding to the [32AR m the presence of the G-protein mimic Nb80.

[0099] FIG. 20B shows P2AR capture by Nbs (Nb80) using ELISA in the presence of the high-affinity agonist BI-167107, and compared to DMSO or the antagonist lCl-118551, a competing nanobody Nb-6B9, and saturating concentration of Compound-6.

[00100] FIG. 21 A shows the effect of Compound 6 on the ISO competition curve for binding to the Bl AR against the ^{1 25} I-CYP radiolabeled antagonist.

[00101] FIG. 21 B shows the effect of Compound 6 on the ISO concentration response at bIAK- mediated cAMP production.

[00102] FIG. 22A shows the effect of Compound 6 on the radioligand ( ^{J 25} I-CYP) competition binding dose-response curve (IC50 value) for ISO.

[00103] FIG. 22B shows the effect of Compound 6 on the radioligand ( ¹²³ I-CYP) competition binding dose-response curve (IC50 value) for epinephrine.

[00104] FIG. 22C shows the effect of Compound 6 on the radioligand ( ^!23 I-CYP) competition binding dose-response curve (IC50 value) for fenoterol.

[00105] FIG. 22D shows the effect of Compound 6 on the radioligand ( ^I25 I-CYP) competition binding dose-response curve (IC50 value) for c!enbutero!.

[00106] FIG. 22E shows the effect of Compound 6 on agonist-mediated cAMP accumulation dose-response by ISO.

[00107] FIG. 22F shows the effect of Compound 6 on agonist-mediated cAMP accumulation dose-response by epinephrine.

[00108] FIG. 22G shows the effect of Compound 6 on agonist-mediated cAMP accumulation dose-response by fenoterol.

[00109] FIG. 22H shows the effect of Compound 6 on agonist-mediated cAMP accumulation dose-response by clenbuterol. [00110] FIG 221 shows the effect of Compound 6 on agonist-mediated b-arrestin recruitment dose-response by ISO.

[00111] FIG 22J shows the effect of Compound 6 on agonist-mediated b-arrestin recruitment dose-response by epinephrine.

[00112] FIG 22K shows the effect of Compound 6 on agonist-mediated b-arrestin recruitment dose-response by fenoterol.

[00113] FIG 22L shows the effect of Compound 6 on agonist- mediated b-arrestin recruitment dose-response by clenbuterol.

[00114] FIG. 23 shows activity data for representative compounds of the invention.

DETAILED DESCRIPTION OF THE INVENTION

A, Homogeneous GPCR Phosphorylation

[00115] The present disclosure is predicated, at least in part, on the discovery that sortase- mediated ligation of a synthetic phosphopeptide to the C-terminus of GPCRs can be used to homogeneously phosphorylate GPCRs so that the effects of b-arrestin coupling to the transmembrane (TM) cores may be elucidated. The complex and methods disclosed herein allow for systematic comparison as to how agonists allostericafly influence the interactions of multiple transducers with multiple GPCRs, revealing unexpected diversity that may influence the balance of cellular signaling responses. In this regard, verifying the efficiency and pattern of

phosphorylation for GPCRs is known m the art to be technically challenging. As a result, it has been difficult to ascertain whether particular structural elements directly affect GPCRs’ interactions with arr or indirectly influence them at the level of GRK phosphorylation. The sortase-ligated chimeric receptor complexes described herein provide a tool to separate these variables, enabling independent manipulation of phosphorylation state and Parr binding to clarify the structural requirements of the GPCR- Parr interaction.

1. Chimeric GPCR Complex and In Vitro Production [00116] The disclosure provides a complex comprising (i) a chimeric G protein-coupled receptor (GPCR) comprising the amino acid sequence LPETGGG (SEQ ID NO: 1) located within the C-terminus of the GPCR and a synthetic phosphopeptide ligated to SEQ ID NO: 1; and (ii) a b-arrestin (Parr) protein bound to the C-terminus of the GPCR. The term“complex,” as used herein, refers to at least two molecules that are specifically associated with each other, such as, for example, by directly binding to each other. The associate between the at least two molecules of a complex may involve one or more chemical or physical bonds and/or chemical spacers providing such bond(s) (e.g., non-specific atachment via van der Waals forces, hydrogen bonding, covalent bonding electrostatic interactions, hydrophobic/hydrophilic interactions; etc.). The term“chimeric,” as used herein, refers to a substance or compound that is composed of parts of different origin.

[00117] As discussed above, G protein-coupled receptors (GPCRs) are the largest, most versatile, and most ubiquitous of the several families of plasma membrane receptors. GPCRs regulate virtually all known physiological processes in mammals. GPCRs are the largest family of membrane proteins and mediate most cellular responses to hormones and neurotransmitters, as well as being responsible for vision, olfaction, and taste. All GPCRs are characterized by the presence of seven membrane- spanning ot-helical segments separated by alternating intracellular and extracellular loop regions. GPCRs in vertebrates are commonly divided into five families on the basis of their sequence and structural similarity: rhodopsin (family A), secretin (family B), glutamate (family C), adhesion, and Frizzled/Taste2. The rhodopsin family is by far the largest and most diverse of these families, and members are characterized by conserved sequence motifs that imply shared structural features and activation mechanisms. Despite these similarities, individual GPCRs have unique combinations of signal-transduction activities involving multiple G-protein subtypes, as well as G-protein-independent signaling pathways and complex regulatory processes. Moreover, they are the most common targets of currently used therapeutic drugs, and it is estimated that nearly 36% of all FDA-approved drugs target at least one member of the GPCR gene family (Overington et al, Nat. Rev. Drug. Disc., 5: 993 (2006); and Rask- Andersen et al, Nat. Rev. Drug. Disc., 10: 579 (2011)). [00118] G protein-coupled receptor (GPCR) signaling begins when an agonist binds to and stabilizes an active receptor conformation. This agonist bound GPCR, acting through its transmembrane core, promotes interaction with heterotrimeric G proteins (Qabg), thus stimulating guanine nucleotide exchange and separation of the Ga subunit from the Obg subunits (Gilman, AG., Annu Rev. Biochem, 56: 615-649 (1987)). Once activated, GPCRs initiate a highly conserved signaling and regulatory cascade marked by interactions with: (i)

heterotrimeric G proteins, which mediate their actions largely by promoting second-messenger generation (Gilman, supra); (ii) GPCR kinases (GRKs), which phosphory!ate activated conformations of receptors (Moore et al, Annu Rev Physiol 69:451-482 (2007)); and (iii) b- arrestms (Parrs), which bind to the phosphorylated receptors to mediate desensitization of G protein signaling and receptor internalization (Goodman et al, Nature, 383(6599):447~450 (1996); and Laporte et al, Proc Natl Acad Sei USA 96(7):3712-3717). GPCR receptor complexes and signaling pathways are further described in, e.g., Kroeze et al, J. Cell Sei., 1 16: 4867-4869 (2003); Rosenbaum et al, Nature, 459(7245): 356-363 (2009); Cahill et al, Proc. Natl Acad Sci. USA, 114(10): 2562-2567 (2017); and Thomsen et al, Cell, 166(4): 907-919 (2016).

[00119] Nearly 800 GPCRs have been identified in humans, which are classified into numerous types and subtypes within the five families identified above based on sequence homology and functional similarity. In the context of the present disclosure, the chimeric GPCR can be obtained or derived from any GPCR family, group, subgroup, type, or subtype known in the art, such as those described m, e.g., (Bjarnadottir et al, Genomics, 88(3): 263-73 (2006); Attwood, T.K. and J.B. Findlay, Protein Engineering, 7(2): 195-203 (1994); Kolakowski, L.F., Receptors & Channels, 2(1): 1-7 (1994); and Foord et al, Pharmacological Reviews, 57(2): 279- 288 (2005 )). In one embodiment, the chimeric GPCR is a member of the adrenergic receptor family (e.g., alA, alB, all), a2A, a2B, a2C, bΐ, or b2 adrenergic receptor), a member of the dopamine receptor family (e.g., Dl , D2, D3, D4, or D5 dopamine receptor), a member of the opioid receptor family (e.g., delta, kappa, mu, nociceptin, or zeta opioid receptor), a member of the muscarinic acetylcholine receptor family (e.g., Mi, M2, M3, M ₄ , or M5 receptor), calcitonin receptor (CTR), a cannabinoid receptor (e.g., type 1 (CB1R) or type 2 (CB2R) receptor), a chemokine receptor (e.g., C-X-C chemokine receptor 4 (CXCR4)), a free fatty acid receptor (e.g., FFA1, FFA2, FFA3, or FFA4 receptor), G protein-coupled receptor 3 (GPR3), glucagon- like peptide 1 receptor (GLP-1R), a parathyroid hormone receptor (e.g., PTH1R or PTH2R), a somatostatin receptor (e.g., SSTR1, SSTR2, SSTR3, SSTR4, SSTR5), a sphingosine-1 phosphate receptor (e.g., S1P1R, S1P2R, S1P3R, S1P4R, or S1P5R), a vasopressin receptor, an angiotensin receptor, or thyroid stimulating hormone receptor (TSHR). For example, the chimeric GPCR may be P2-adrenergic receptor, angiotensin II type 1 A receptor, vasopressin V2 receptor, m opioid receptor (MOR), or muscarinic acetylcholine receptor 2 (M2R).

[00120] The GPCR is“chimeric” in that it comprises a non-native amino acid sequence located within the GPCR C-terminus and a synthetic (i.e., man-made and not naturally occurring) phosphopeptide ligated to the non-native amino acid sequence. The terms“C-terminus,” “carboxyl-terminus,”“carboxy-terrninus,”“C-termina l tail,”“C-terminal end,” and“COOH- terminus” are synonymous and used interchangeably herein to refer to the end of an ammo acid chain (protein or polypeptide), which is terminated by a free carboxyl group (-COOH). A“non- native” ammo acid sequence is any ammo acid that is not a naturally occurring ammo acid sequence of a GPCR m a naturally occurring position. Thus, the non-native amino acid sequence can be naturally found in a GPCR, but located at a non-native position within the GPCR protein. In one embodiment, the non-native ammo acid sequence facilitates introduction of the synthetic phosphopeptide at the C-terminus of the GPCR. The specific non-native amino acid sequence that is present in the chimeric GPCR wall depend upon the method by which the synthetic phosphopeptide is introduced into the GPCR C-terminus (discussed further herein). In one embodiment, the non-native amino acid sequence may comprise all or a portion of a recognition sequence (or recognition site) for an enzyme that catalyzes ligation of the phosphopeptide to the GPCR. A number of enzymes that catalyze protein or peptide ligation by acting on specific protein recognition sequences are knowai in the art and described herein. The chimeric GPCR may comprise any size portion of any suitable enzyme recognition sequence or site, including the entire recognition sequence. In one embodiment, the chimeric GPCR comprises the amino acid sequence LPETGGG (SEQ ID NO: I), which comprises a portion of the recognition sequence for the sortase enzyme (discussed further herein). [00121] A“phosphopeptide,” as used herein, is peptide or polypeptide that incorporates a phosphate group as a result of phosphorylation. Any suitable phosphopeptide may be introduced at the C-terminus of the GPCR. In one embodiment, the synthetic phosphopeptide is obtained or derived from a GPCR that differs from the GPCR that is present in the complex described herein. For example, if the complex comprises the b2 adrenergic receptor, then the synthetic

phosphopeptide may be obtained or derived from a GPCR other than the b2 adrenergic receptor. In certain embodiments, the synthetic phosphopeptide is derived from the C-terminus of a GPCR. In one embodiment, the synthetic phosphopeptide is derived from the C-terminus of a vasopressin-2-receptor (V ₂ Rpp). The V ₂ Rpp peptide has been shown to hind to b-arrestinl with high affinity and effectively prime b-arrestinl for interaction with the GPCR TM core (see, e.g., Shukla et al., Nature, 497(7447): 137-141 (2013); and Nobles et al., I Biol. Chenx, 282: 21370- 21381 (2007)). In one embodiment, the synthetic phosphopeptide comprises the amino acid sequence ARGRTPPSLGPQDESCTT A S S SL AKDTS S (SEQ ID NO: 2).

[00122] The synthetic phosphopeptide desirably is phosphorylated at at least one amino acid residue. In some embodiments, the synthetic phosphopeptide is phosphorylated at two or more amino acid residues (e.g., 2, 3, 4, 5, 8, 9, 10 or more). The synthetic phosphopeptide may be phosphorylated at any ammo acid residue, and it will be appreciated that phosphorylation of any given site on a protein can change the function or localization of that protein. In eukaryotes, protein phosphorylation is most common on serine, threonine, tyrosine, and histidine residues. Protein phosphorylation is described further in, e.g., Marks, F. (ed.), Protein Phosphorylation, John Wiley & Sons (2008); and Alberts et al. (eds.), Molecular Biology of the Cell, 6 ^th Ed., Garland Science (2014)). In embodiments where the synthetic phosphopeptide comprises the ammo acid sequence of SEQ ID NO: 2, the synthetic phosphopeptide is phosphorylated at residues 5 (threonine), 8 (serine), 15 (serine), 17 (threonine), 18 (threonine), 20 (serine),

21 (serine), and 22 (serine) of SEQ ID NO: 2.

[00123] The complex further comprises a b-arrestin (Parr) protein bound to the C-terminus of the GPCR. The term“arrestin,” as used herein, encompasses all types of naturally occurring and engineered variants of arrestin, including, but not limited to, visual arrestin (also referred to as Arrestin I), b-arrestin 1 (also referred to as Arrestin 2), b-arrestm 2 (also referred to as Arrestin 3), X arrestm (also referred to as arrestin 4). Visual arrestin is localized to retinal rods, whereas X arrestin, or arrestin 4, is found in retinal rods and cones b-arrestinl and P-arrestin2

(collectively referred to herein as“parrs”) are ubiquitously expressed multifunctional signaling adaptor proteins originally discovered for their role m desensitizing GPCRs (Lefkowitz and Shenoy, Science, 308: 512-517 (2005)). The complex may comprise b-arrestml or P-arrestin2. In one embodiment, the complex comprises b-arrestinl (b3pΊ).

[00124] b-arrestins regulate both GPCR and non-GPCR pathways under normal and pathological conditions, including cancer (Lefkowitz et al , MoI. Cell., 24: 643-652 (2006)). parrs also have been appreciated as independent signaling units by virtue of their crucial role as both adaptors and scaffolds for an increasing number of signaling pathways (see, e.g., Shuk!a et al., Trends Biochem Sci 36(9):457~469 (201 1 ); Shenoy, S.K. and R.J. Lefkowitz, Bioehem. J , 375(Pt 3): 503-515 (2003); Pierce et al., Nat Rev Mol Cell Biol 3(9):639-650 (2002); Reiter, E. and RJ. Lefkowitz, Trends Endocrinol Metab 17(4): 159-165 (2006); DeWire et al, Annu Rev Physiol 69:483-510 (2007); Peterson, Y.K and Luttrell, L.M., Pharmacol. Rev., 69(3): 256-297 (2017); and Cahill et al., supra).

[00125] With respect to binding, it is believed that arrestms make initial contact with phosphorylated receptors via adjacent lysines in the amino terminus (Vishmvetskiy et al, J. Biol. Chem., 275: 41049-41057 (2000)). Biochemical data suggests that this interaction perturbs the three-element interaction, guides phosphorylated receptors to the polar core, allows the negatively charged phosphate from the receptor to interact with positively charge Argl 69 (in b- arrestinl), and ultimately causes release of the C-terminal tail from the polar core (Palczewski et al., J. Biol. Chem., 266: 15334-15339 (1991 ); Gurevich et al., I Biol. Chem., 273: 15501-15506 (1998); Vishmvetskiy et al., J. Biol Chem., 275: 41049-41057 (2000); Gurevich and Gurevich, Trends Pharmacol. Sci., 25: 105-111 (2004)). This leads to the disruption of the basal state and subsequent conformational rearrangement of arrestin. Studies monitoring arrestin

conformational changes in live cells, along with other biochemical data, suggest that the arrestin amino terminus and C-terminal tail move closer upon binding to an activated receptor (Xiao et al., J. Biol. Chem., 279: 55744-55753 (2004); and Charest et al, EMBO Reports, 6: 334-340 (2005)). This conformational rearrangement enhances arrestm interaction with receptors and is also thought to expose binding motifs that interact with other proteins such as clathrin and AP2 (Moore et al., Annu Rev Physiol., 69: 451-482 (2007)).

[00126] In some embodiments, the complex further comprises an antigen-binding fragment of an antibody (Fab) that specifically binds to the complex. The inclusion of a Fab in the complex is believed to stabilize the interaction of b-arrestinl with the phosphorylated C terminus of the chimeric GPCR. Any suitable Fab may be used to stabilize the receptor complex conformation. In one embodiment, the Fab is conformationally-selective synthetic antibody fragment, referred to as Fab30, that recognizes the phosphopeptide-activated state of b-arrestinl (Shukia et al, Nature, 497(7441}. 137-141 (2013)). In some embodiments, the complex further comprises an antigen-binding fragment of a nanobody, a variable domain of a single chain antibody (e.g, from camelids) (Cahill TJ, 3rd, et al. (2017) Proc Natl Acad Set U SA H4(10):2562-2567).

[00127] The complex may be immobilized on a solid support. The solid support may be any suitable surface in planar or non-planar conformation, such as, for example, a surface of a microfluidic chip, an interior surface of a chamber, a bead, an exterior surface of a bead, an interior and/or exterior surface of a porous bead, a particle, a microparticle, an electrode, a slide (e.g., a glass slide), or a multi well (e.g., a 96-well) plate.

[00128] The disclosure also provides an in vitro method for producing the above-described complex. The method comprises (a) enzymatically ligating a synthetic phosphopeptide to the C- termmus of a purified GPCR to produce a phosphorylated chimeric GPCR comprising the amino acid sequence of SEQ ID NO: 1 located within the C-termmus of the GPCR, and (b) contacting the phosphorylated GPCR with purified b-arrestm (parr) protein, whereupon the purified b- arrestm (parr) protein binds to the C-terminus of the phosphorylated chimeric GPCR and forms a complex comprising the chimeric GPCR and the parr protein. Descriptions of the chimeric GPCR, synthetic phosphopeptide, and parr set forth above in connection with other embodiments of the complex also are applicable to those same aspects of the aforesaid method.

[00129] The GPCR and Parr are“purified” in that each is removed from its natural environment, along with any foreign or contaminating elements. Methods for protein isolation and purification are well known in the art, and any such method may be used to purify GPCRs and Parr in accordance with the present disclosure. Protein purification processes typically begin with protein extraction from cells, followed by precipitation, solubilization, and centrifugation. Downstream methods for protein purification include, but are not limited to, size exclusion chromatography, ion exchange chromatography, affinity chromatography (e.g., immunoaffinity chromatography), metal binding, high performance liquid chromatography, reversed phase chromatography, ultrafiltration, and electrophoresis. In one embodiment, the GPCR and/or arr may be solubilized using a detergent (e.g., dodecyl maltoside (DDM) or maltose neopentyl glycol (MNG)) and purified using affinity chromatography (see, e.g., Kobilka, B.K., Anal. Biochem., 231(1): 269-271 (1995)). Protein purification methods are further described in, e.g., GE Healthcare, Strategies for Protein Purification (available at

www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma -

Aldrich/General_Information/l/ge-strategies-for-protein-p urification.pdf); and Scopes, R.K. (ed ), Protein Purification: Principles and Practice, Springer Advanced Texts in Chemistry, 3 ^rd Ed., (1993); and Burgess, R.R., Deutscher M.P. (eds.), Guide to Protein Purification, 2 ^nd Ed., Methods in Enzymology, Volume 436, Academic Press (2009))

[00130] The synthetic phosphopeptide may be ligated to the C-terminus of the GPCR using any suitable method known in the art for protein tagging or conjugation. Such methods include, for example, enzyme-mediated ligation, chemical ligation, protein splicing, and expressed protein ligation (see, e.g., Berrade, L, and J.A. Camarero, Cell. Mol. Life. Sci., 66(24): 3909- 3922 (2009); Witte et al., J. Am Chem. Soc. 119(9): 21 14-21 18 (1997); and Muir et al, Proc. Natl. Acad. Sci. USA 95(12): 6705-6710 (1998)). In one embodiment, the synthetic phosphopeptide is added to the C-terminus of the GPCR by enzyme-mediated ligation.

Enzymatic strategies for coupling peptides or polypeptides are known in the art and include the use of natural ligases such as sortase, butelase, peptiligase, subtiligase, streptoligase, and omniligase (see, e.g., Schmidt et al., Curr Opm Chem Biol., 38: 1-7 (2017)). Enzymatic strategies using ligases such as sortase, butelase, peptiligase or omniligase generally exhibit greater chemoselectivity as compared to chemical ligation methods. Any suitable enzyme may be used to ligate the synthetic phosphopeptide to the GPCR C-terminus, including, but not limited to, sortase, butelase, or peptiligase. [00131] In one embodiment, ligation of the phosphopeptide to the C-terminus of the purified GPCR is catalyzed by a sortase enzyme (also referred to in the art as“sortagging”). In another embodiment, the sortase enzyme is obtained from a prokaryote. Sortase catalyzes a

transpeptidation reaction which typically involves pairing of sortase A from Staphylococcus aureus (SrtAstaph) with an LPXTG (SEQ ID NO: 4) -containing substrate. In the presence of Ca2+, the active site cysteine of SrtAstaph cleaves between threonine and glycine to generate a thioester-iinked acyl enzyme intermediate. This intermediate is then intercepted by an ammoglycine nucleophile, resulting in the site-specific ligation of the acyl donor and acceptor. Sortagging has been used has been used to introduce several types of common modifications into proteins, including lipids (Antos et al, J Am Chem Soc 130(48): 16338-16343 (2008)) and glycans (Samantaray et al., J Am Chem Soc 130(7): 2132-2133 (2008)). For example, sortagging has been used to generate of camelid-derived antibody fragment conjugates for the treatment of B-cell lymphoma, the installation of nonisotopica!ly labeled protein domains to facilitate NMR analysis of proteins with limited solubility, the construction of immuno-PET reagents for non-in vast ve cancer imaging, and the preparation of multifunctional protein nanoparticles (see, e.g., Fang et al, Angew Chem Int Ed Engl, 55: 2416-2420 (2016); Arner et al., I Biomol. NMR, 64: 197-205 (2016); Rashidian et al, ACS Cent Sci., 1 : 142-147 (2015); Chen et al, Chem. Commun., 51 : 12107-12110 (2015); and Antos et al, supra).

[00132] In one embodiment, the sortase enzyme may be a wild-type sortase enzyme obtained from any suitable species, including, for example, Staphylococcus aureus (SrtAstaph),

Streptococcous pyogenes (SrtAstrep), and Lactobacillus plantarum (SrtApiam). Sortase A from Streptococcus pyogenes (SrtAs _t rep), which is Ca2+-independent, can recognize an LPXTA (SEQ ID NO: 5) substrate in addition to LPXTG (SEQ ID NO: 4), and accommodates N-ternunal alanine residues as acyl acceptors. SrtApiam has been shown to catalyze transpeptidations involving non-amino acid primary amine nucleophiles and model proteins possessing

LAATGWM (SEQ ID NO: 6), LPKTGDD (SEQ ID NO: 7), and LPQTSEQ (SEQ ID NO: 8) sequences (Matsumoto et al, Biotechnol J., 7: 642-648 (2012); and Antos et al, supra). In one embodiment, the ligation is catalyzed by a wild-type SrtAstaph enzyme, and the GPCR comprises a suitable recognition sequence for SrtAstaph. For example, the purified GPCR may comprise the amino acid sequence LPETGGH (SEQ ID NO: 3).

[00133] While the majority of sortagging applications utilize the wilt-type SrtAstaph enzyme, in some applications SrtAstaph suffers from poor reaction rates and a dependency on a CA2+ eofactor. Thus, in some embodiments, the sortase enzyme may be a sortase enzyme that has engineered to improve performance. In this regard, a number of sortase variants with improved performance have been generated using directed evolution methods, including the pentamutant P94R/T) 160N/D 165 A/KI 90E/K196T (Chen et a!., Proa Nail Acad Sci. USA, 108 11399-1 1404 (2011) and heptamutants which add either E105K/E108A or E105K/E108Q mutation to the aforementioned pentamutant (see, e.g., Hirakawa et a ., Biotechnol., J., 10: 1487-1492 (2015); Witte et al, Nat. Protoe., 10: 508-516 (2015); and Wuethrich et al , PLoS ONE, 9: e!09883 (2014)). Sortase activity also may be improved by alternating the sortase recognition site on a particular substrate. Thus, the chimeric GPCR may comprise an amino acid sequence other than SEQ ID NO: 3 that is recognized by a wild-type or variant sortase enzyme. In this regard, for example, sortase activity has been observed with the alternate substrates IPKTG (SEQ ID NO:

9), MPXTG (SEQ ID NO: 10), LAETG (SEQ ID NO: 1 1 ), LPXAG (SEQ ID NO: 12), LPESG (SEQ ID NO: 13), LPELG (SEQ ID NO: 14), and LPEVG (SEQ ID NO: 15) (Beliucci et al, Angew Chetn Int Ed Engl, 52: 3703-3708 (2013); Piotukh et al, J Am Chem Soc, 133: 17536- 17539 (201 1 ); and Kruger et al., Biochemistry, 43: 1541-1551 (2004)). Sortase enzyme variants and alternate recognition sequences that may be used in the context of the present disclosure are further described m, e.g., Antes et al., supra.

[00134] The in vitro method of producing the aforementioned complex further comprises contacting the phosphorylated chimeric GPCR with purified b-arrestin (Parr) protein, whereupon the purified b-arrestin (bap·) protein binds to the C-terminus of the phosphorylated chimeric GPCR and forms a complex comprising the chimeric GPCR and the Parr protein. The term “contacting,” as used herein, refers to any type of combining action which brings the chimeric GPCR into sufficiently close proximity with the purified b-arrestin ^arr) such that a binding interaction will occur. Contacting may be achieved in a variety of different ways, including directly combining the chimeric GPCR with a purified Parr, exposing the chimeric GPCR to a purified Parr by introducing the purified Parr in close proximity to the GPCR, and the like.

[00135] In general, the GPCR would be reconstituted into an environment to mimic a cell membrane prior to complex formation, in order to assess activity of the complex. Thus, in some embodiments, the GPCR is reconstituted m high density lipoparticles (HDLs), as described in Staus et al., Nature, 535(7612): 448-452 (2016). Alternatively, the GPCR may be stabilized in other formats, such as, for example, detergent, bicelies, and/or vesicles (see e.g., Shen et al, Int.

J. Mol. Sci., 14: 1589-1607 (2013); Goddard et al, Methods Enzymo! , 556: 405-424 (2015); and Serebryany et al., Biochim. Biohphys Acta (BBA), 1818(2): 225-233 (2012)).

[00136] The present disclosure provides methods for using the above-described complex to identify ligands which bind to the chimeric GPCR and act as agonists or antagonists of G protein-mediated signaling, especially ligands that are biased for parr signal transduction. Thus, m certain aspects, the disclosure provides a method for selecting a modulator of a GPCR, such as a biased ligand for a GPCR, which comprises contacting the above-described complex with one or more compounds under conditions to allow for binding of the one or more compounds to the chimeric GPCR and measurement of an activity of the one or more compounds at the chimeric GPCR, and selecting at least one compound that displays the activity, or a change in an activity compared to a reference activity measurement, at the chimeric GPCR. Descriptions of the chimeric GPCR, synthetic phosphopeptide, and Parr set forth above in connection with other embodiments of the disclosure also are applicable to those same aspects of the aforesaid methods. In some embodiments, highly diverse DNA-encoded small molecule libraries may be screened for their ability to bind purified homogenously phosphorylated chimeric GPCRs in complex with b-arrestm using affinity based selection strategies, so as to enable identification of small molecules that bind to GPCRs and preferentially promote b-arrestin coupling over G- protein, or vice versa. Screening methods using GPCR G-protein complexes and/or GPCR alone may performed in parallel or in series to further isolate ligands with desired pharmacological properties, as described further herein.

[00137] The term“modulator,” as used herein, refers to any substance which alters or changes a characteristic of a GPCR receptor complex and/or a GPCR signaling pathway. For example, a modulator may be a substance winch changes the conformation of a GPCR itself, or the interaction of a GPCR with Parr, G protein, or GPCR kinase. Alternatively, a modulator may be a substance winch alters the signaling activity of a particular GPCR, either positively or negatively . In some embodiments, a modulator is a ligand for the chimeric GPCR. The term “ligand,” as used herein, refers to a substance that forms a complex with a larger biomolecule to exert a biological response. In the context of GPCRs, a ligand is any substance that binds to the GPCR and initiates or produces a signal, inhibits the initiation or production of a signal, or affects the binding of other molecules with these characteristics. A“biased ligand” is a ligand that selectively confers activity in one signaling pathway over another. For example, with regard to GPCRs specifically, a biased ligand may have a relative efficacy for activating a G-protein- coupled receptor function (e.g., signaling) that is greater than its relative efficacy for stimulating b-arrestin function (e.g., recruitment and/or signaling), or vice versa. This type of ligand selectivity also is referred to as“biased agonism” or“functional selectivity” (see, e.g., Rankovic et al, Bioorg. Med. Chem. Let., 26(2): 241-250 (2016)). Biased ligands for GPCRs are further described in, e.g., International Patent Application Publication WO 2008/021552.

[00138] The one or more compounds (e.g., modulators or ligands) may be any suitable compound that is capable of binding to a receptor (e.g., a GPCR). For example, the compound may be a small molecule, a protein, a peptide, or a nucleic acid molecule. The term“small molecule,” as used herein, refers to a low molecular weight (generally less than 900 daltons) non-peptide organic compound that may regulate a biological process. Small molecules include, but are not limited to, lipids, monosaccharides, and secondary messengers (e.g., cyclic AMP, cyclic GMP) Small molecules have been in a variety of applications, such as, for example, agonists and antagonists of GPCRs, enzyme inhibitors, nuclear receptor ligands, and ion channel modulators. Indeed, many commonly used drugs are small molecules because, unlike peptides and proteins, they can be designed to be metabolically stable and orally active. As such, the small molecule may be a therapeutic agent, or a compound from which a therapeutic agent is derived (e.g., a“lead” compound). By“therapeutic agent” is meant any agent (e.g., drug), regimen, or strategy that may ameliorate a particular disease condition.

[00139] A“peptide” is a compound comprised of two or more ammo acids linked by one or more peptide bonds, which link the amino group of one amino acid to a carboxyl group of the other. A“polypeptide” is a polymer comprised of several (e.g., at least 10) amino acids joined by peptide bonds. A“protein” is a compound composed of one or more polypeptide chains with a mass of at least about 10 kilodaltons (kD). The terms“nucleic acid sequence” and“nucleic acid molecule” are synonymous and refer to a polymer of DNA or RNA, i.e , a polynucleotide, which can be single-stranded or double-stranded and which can contain non-natural or altered nucleotides. The terms“nucleic acid” and“polynucleotide” as used herein refer to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecule, and thus include double- and single- stranded DNA, and double- and single-stranded RNA. The terms include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides such as, though not limited to, methylated and/or capped polynucleotides.

[00140] In one embodiment, the disclosure provides a method for selecting a modulator of a G protein- coupled receptor (GPCR), which method comprises (i) contacting the above-described complex with one or more compounds under conditions to allow for measurement of an activity of the one or more compounds at the chimeric GPCR, (ii) measuring the presence or absence of activity of the one or more compounds, and (in) selecting at least one compound that displays the activity at the chimenc GPCR. The activity measured by the aforementioned method may be any suitable activity associated with ligand-receptor binding, but typically is a binding activity (e.g., the degree to which a compound binds or does not bind to the chimeric GPCR) or functional activity.

[00141] Binding activity of a compound or ligand to a receptor (e.g., as GPCR) may be measured using a variety of suitable methods known in the art. Such methods include, for example, radioactive techniques (e.g., radioligand binding assays, filtration techniques combined with radioactivity counting, scintillation proximity analysis and autoradiography for radioactive ligands, and time-resolved fluorescence resonance energy transfer), and non-radioactive techniques (e.g., fluorescence polarization, fluorescence resonance energy transfer, and surface plasmon resonance). Receptor binding assays are described in detail in, for example, de Jong et al., Journal of Chromatography B, 829(1-2): 1-25 (2005); and Davenport, A.R. (ed.), Receptor Binding Techniques, 3 ^rd Ed., Methods in Molecular Biology, Book 897, Human Press (2012).

[00142] One of ordinary skill in the art will appreciate that GPCRs regulate complex and multifaceted signaling networks m cells. Indeed, as the receptors for hormones,

neurotransmitters, ions, photons and other stimuli, GPCRs are among the essential nodes of communication between the internal and external environments of cells. The classical role of GPCRs is to couple the binding of agonists to the activation of specific heterotrimeric G proteins, leading to the modulation of downstream effector proteins. Thus, a“functional activity” of a GPCR encompasses a wide variety of signaling outcomes which may result from a compound or ligand binding to the chimeric GPCR of the complex described herein.

[00143] In one embodiment, the methods described herein may be used to identify an orthosteric GPCR ligand. The term“orthosteric ligand,” as used herein, refers to a molecule or compound that binds to the extracellular site of a GPCR where an endogenous ligand binds (e.g., epinephrine for adrenergic receptors) (also referred to as an“orthosteric-binding pocket”).

Orthosteric ligands may be identified by performing the methods described herein in the presence of the above-described complex and in the absence of a GPCR agonist. Orthosteric ligands identified by the disclosed methods may exhibit different types of GPCR signaling activity, including, but not limited to, antagonism, balanced antagonism, b-arrestm biased agonism, G-protein biased agonism, inverse agonism, and neutral antagonism. The term “antagonist” encompasses small molecules that bind to an orthosteric site but stabilize an inactive receptor state. Antagonistic ligands may compete with agonist binding and inhibit activation of G protein and b-arrestin in cellular assays. The term“balanced agonist” encompasses small molecules that bind to an orthosteric site and stabilize an unbiased active receptor state(s). Balanced agonistic ligands may proportionally induce activation of G protein and b-arrestin in cellular assays to a similar extent as an endogenous ligand. The term“b- arrestin biased agonist” encompasses small molecules that bind to an orthosteric site and stabilize specific active receptor states that preferentially induce coupling to b-arrestin over G- protein. b-arrestin biased agonist ligands may disproportionaliy induce b-arrestin and G-protem activation m cellular assays, where the former is greater than the latter. The term“G biased agonist” encompasses small molecules that bind to an orthosteric site and stabilize specific active receptor states that preferentially induce coupling to G-protein over b-arrestin. Such ligands may disproportionaliy induce G-protein and b-arrestin in cellular assays, where the former is greater than the latter. The term“inverse agonist” encompasses small molecules that binds to the same site as an agonist but induce a pharmacological response opposite to that agonist. The term “neutral antagonist” encompasses small molecules that exhibit no activity' in the absence of an agonist or inverse agonist but can block the activity of either.

[00144] In other embodiments, the methods described herein may be used to identify an allosteric GPCR ligand. The term“allosteric ligand,” as used herein, refers to a molecule or compound that binds to a site of a GPCR other than the endogenous ligand binding site.

Allosteric ligands themselves rarefy induce effects on GPCR function, but rather modify the properties of an orthosteric ligand, such as by increasing or decreasing its potency or efficacy. Allosteric ligands may be identified by performing the methods described herein in the presence of the above-described complex and a GPCR agonist. Allosteric ligands identified by the disclosed methods may exhibit different types of GPCR signaling activity, including, but not limited to negative allosteric modulation (NAM), balanced positive allosteric modulations (PAM), b-arrestin biased positive allosteric modulation, and G biased positive allosteric

modulation. The term“negative allosteric modulator” encompasses small molecules that bind to an allosteric site that stabilizes an inactive receptor state. NAM ligands may decrease the affinity of an orthosteric ligand and reduce activation of G protein and b-arrestin in cellular assays proportionally (a balanced NAM) or disproportionaliy (a biased NAM). The term“balanced positive allosteric modulator (PAM)” encompasses small molecules that bind to an allosteric binding site and enhance orthosteric ligand affinity, while the balanced signaling properties of the orthosteric ligand are maintained. PAM ligands may proportionally potentiate activation of G protein or b-arrestin by the orthosteric ligand in cellular assays. The term“b-arrestin biased positive allosteric modulator” encompasses small molecules that bind to an allosteric binding site which convert a balanced orthosteric agonist into a b-arrestin biased ligand. Such allosteric ligands may change the balanced signaling properties of an orthosteric ligand in cellular assays, such that b-arrestin activation is greater than G-protem activation. The term“G biased positive allosteric modulator” encompasses small molecules that bind to an allosteric binding site which convert a balanced orthosteric agonist into a G-biased ligand. Such allosteric ligands may change the balanced signaling properties of the orthosteric ligand in cellular assays, such that G- protein activation is greater than b-arrestin activation.

[00145] Following identification of at least one compound that displays an activity at the chimeric GPCR, the method for selecting a modulator of a GPCR may further comprise measuring a reference activity at (a) an equivalent chimeric GPCR without C-terminal phosphorylation, (b) an equivalent chimeric GPCR in the absence of b-arrestin, (c) an equivalent chimeric GPCR in the presence of G protein, and/or (d) an equivalent GPCRwith a native C~ terminus; and selecting a compound that exhibits a difference in the activity at the chimeric GPCR compared to the reference activity. The“reference activity” may be any suitable GPCR activity described herein, e.g., binding activity or functional activity (e.g., signaling activity). In embodiments where the reference activity is binding activity, the binding activity may be measured using any of the methods disclosed herein or known in the art. When the reference activity is a functional activity, the functional activity of the chimeric GPCR itself, b-arrestin, and/or G protein may be assessed, depending on the configuration of the complex (i.e., absence of b-arrestin or presence of G protein). In this regard, G protein activity mediated by a GPCR can be measured using any of a wide variety of assays, including those well known in the art.

For example, G protein activity can be assay ed by determining the level of calcium, cAMP, diacylglycerol, or inositol triphosphate in the presence and absence of a candidate modulator or ligand. G protein activity can also be assayed, for example, by determining phosphatidyhnositol turnover, GTP-y-S loading, adenylate cyclase activity, GTP hydrolysis, and the like, in the presence and absence of a candidate modulator or ligand (see, e.g., Kostenis, Curr. Pharm. Res. 12(14): 1703- 1715 (2006)). Similarly, b-arrestin function mediated by a GPCR response to a candidate modulator or ligand can be measured using any of a variety of assays. For example, b- arrestin function recruitment to the GPCR or GPCR internalization can be assayed in the presence and absence of a candidate modulator or ligand. In other embodiments, the b-arrestin function in the presence and absence of a candidate modulator or ligand may be measured using by resonance energy transfer, bimolecular fluorescence, enzyme complementation, visual translocation, co-immunoprecipitation, cell fractionation, or assaying interaction of b-arrestin with a naturally occurring binding partner (see, e.g., Violin et al, Trends Pharmacol. Sei.

28(8):416-427 (2007); and Carter et al, J. Pharm. Exp. Ther. 2:839- 848 (2005)).

[00146] In accordance with the disclosed method for selecting a modulator of a GPCR, the difference in activity may be an enhancement (e.g., an increase or an improvement) in the activity' at the chimeric GPCR compared to the reference activity or a decrease in the activity at the chimeric GPCR compared to the reference activity'. When the difference in activity is an enhancement in the activity, the enhancement may be greater than or less than the enhancement in the activity measured for a reference ligand. The term“reference ligand,” as used herein, refers to a ligand against which the acti vity of a candidate modulator compound or ligand is measured. In one embodiment, the reference ligand may be an endogenous ligand for the GPCR (wherein more than one endogenous ligand for the GPCR exists, the reference ligand may be the endogenous ligand of highest potency) or an exogenous ligand for the GPCR. For example, a reference ligand for the angiotensin II type 1 receptor is the endogenous ligand, angiotensin IT, while a reference ligand for the 2AR may be the exogenous ligand isoproterenol. When the difference in the activity is a decrease in the activity at the chimeric GPCR compared to the reference activity, the decrease may be greater or less than the decrease in the activity measured for a reference ligand.

[00147] Enhanced activity for a test compound at the chimeric GPCR compared to an equivalent chimeric GPCR without C-terminal phosphorylation or an equivalent chimeric GPCR in the absence of b-arrestin indicates that the test compound couples through b-arrestin. A greater relative enhancement in activity for the test compound compared to the b-arrestin enhancement of a reference ligand (e.g., endogenous ligand) may indicate that the test compound has b-arrestin biased activity relative to the reference ligand. A lesser relative enhancement in activity for the test compound compared to the b-arrestin enhancement of a reference ligand may

J .) indicate the absence of b-arrestin biased activity relative to the reference ligand. Such a test compound may be G biased rather than b-arrestin biased.

[00148] Decreased activity for a test compound at the chimeric GPCR compared to an equivalent chimeric GPCR without C -terminal phosphorylation or an equivalent chimeric GPCR in the absence of b-arrestin indicates that the test compound negatively couples through b- arrestin.

[00149] Comparison of the relative activity of a test compound and reference ligand in the presence and absence of G protein may indicate the degree of b-arrestin biased activity or G biased activity.

[00150] The activity may be enhanced or decreased to any suitable degree as compared to the referenced activity. When the enhancement or decrease in activity is greater than the enhancement or decrease in the activity measured for a reference ligand, the enhancement or decrease in activity may be at least about 5% greater than the enhancement or decrease in the activity measured for a reference ligand (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or higher). In one embodiment, the enhancement or decrease in activity is at least about 25% greater than the enhancement or decrease in the activity measured for a reference ligand (e.g., 35%, 45%, 55%, 65%, 75%, 85%, 95%, 125% or higher). In other embodiments, the enhancement or decrease in activity is at least about 50% greater than the enhancement or decrease in the activity measured for a reference ligand, or at least about 100% greater than the enhancement or decrease in the activity measured for a reference ligand.

[00151] Similarly, when the enhancement or decrease in activity is less than the enhancement or decrease in the activity measured for a reference ligand, the enhancement or decrease m activity may be at least about 5% less than the enhancement or decrease in the activity measured for a reference ligand (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or higher). In one embodiment, the enhancement or decrease in activity is at least about 25% less than the enhancement or decrease in the activity measured for a reference ligand (e.g., 35%,

45%, 55%, 65%, 75%, 85%, 95%, 125% or higher) in other embodiments, the enhancement or decrease m activity is at least about 50% less than the enhancement or decrease in the activity measured for a reference ligand, or at least about 100% less than the enhancement or decrease in the activity measured for a reference ligand.

[00152] In another embodiment, following identification of at least one compound that displays an activity at the chimeric GPCR, the method for selecting a modulator of a GPCR may further comprise measuring a reference activity at (a) an equivalent chimeric GPCR without C- terminal phosphorylation, (b) an equivalent chimeric GPCR in the absence of b-arrestin, (c) an equivalent chimeric GPCR in the presence of G protein, and/or (d) an equivalent GPCRwith a native C-terminus, and selecting a compound that exhibits substantially no difference in the activity' at the chimeric GPCR compared to its reference activity. In this manner, the selected compound would display an activity that is independent of b-arrestin binding to the chimeric GPCR. The compound exhibits“substantially no difference” in activity compared to a reference activity if the activity of the selected compound is essentially the same as the reference activity, or if the activity' of the selected compound is enhanced or decreased as compared to the reference activity by no more than about 5% (e.g., 4%, 3%, 2%, 1% or less). Substantially no difference in activity for a test compound at the chimeric GPCR compared to an equivalent chimeric GPCR without C-terminal phosphorylation or an equivalent chimeric GPCR in the absence of b-arrestin indicates that the test compound does not couple through b-arrestin.

[00153] In embodiments where the disclosed method identifies a biased ligand for a GPCR, the method comprises selecting at least one compound that binds to the chimeric GPCR and activates at least one signaling pathway over one or more other signaling pathways mediated by the chimeric GPCR. In this regard, for example, the compound may preferentially activate a parr-dependent signaling pathway over a G protein-dependent signaling pathway. Alternatively, the compound may preferentially activate a G protein-dependent signaling pathway over a Parr- dependent signaling pathway over a G protein-dependent signaling pathway. Suitable Parr- dependent signaling pathways include, but are not limited to, Mitogen-Activated Protein Kinase (MAPK) signaling, receptor transactivation, receptor trafficking, protein ubiquiti nation, transcriptional regulation, GPCR desensitization, and GPCR internalization. In another embodiment, the selected compound may activate a signaling pathway that is different than the signaling pathway activated by a reference ligand (e.g., an endogenous GPCR ligand). Alternatively, the selected compound may activate one of a plurality of signaling pathways activated by a reference ligand (e.g., an endogenous GPCR ligand).

[00154] The disclosure further provides a method of identifying a biased ligand for a G protein-coupled receptor (GPCR) which comprises contacting the aforementioned GPCR complex with one or more compound s under conditions to allow for binding of the one or more compounds to the GPCR, and selecting a compound that binds to the GPCR and either (i) exhibits a change in an activity measurement compared to a reference activity measurement for the compound or (ii) exhibits substantially no change in an activity' measurement compared to a reference activity measurement for the compound at (a) an equivalent GPCR without C-terminal phosphorylation; (b) an equivalent GPCR in the absence of b-arrestin, (c) an equivalent GPCR in the presence of G protein, and/or (d) an equivalent GPCRwith a native ( -terminus Descriptions of the complex, compounds, activity measurements, and reference activity' set forth above in connection with other embodiments of the disclosure also are applicable to those same aspects of the aforesaid method of method of identifying a biased ligand for a GPCR. In one embodiment, the change in the activity measurement is an enhancement in the activity measurement, such as an enhancement in the activity measurement for the compound that is greater than the enhancement in the activity measurement for a reference ligand for the GPCR, as described herein. Alternatively, the enhancement in the activity measurement for the compound is less than the enhancement in the activity measurement for a reference ligand for the GPCR, as described herein. In another embodiment, the change in the activity measurement is a decrease m the activity measurement, as described herein. As discussed above, any suitable activity may be measured when identifying a biased ligand for GPCR, such as, for example binding affinity, functional potency, and/or efficacy. In one aspect, the enhancement in the activity measurement may be an allosteric enhancement in the activity measurement. Alternatively, the activity measurement may be an antagonistic activity, such that the compound essentially blocks G protein-mediated signal transduction.

B. b2 Positive Allosteric Modulators

1. Definitions [00155] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below; although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[00156] The terms“comprise(s),”“include(s),”“having,” ‘has,”“can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms“a,” “an” and“the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments“comprising,”“consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[00157] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6 0-7.0, the numbers 6.0, 6.1, 6 2, 6.3, 6.4, 6.5, 6.6, 6 7, 6.8, 6.9 and 7.0 are explicitly contemplated

[00158] The modifier“about” used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (for example, it includes at least the degree of error associated with the measurement of the particular quantity). The modifier“about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the expression“from about 2 to about 4” also discloses the range“from 2 to 4.” The term“about” may refer to plus or minus 10% of the indicated number. For example,“about 10%” may indicate a range of 9% to 11%, and“about 1” may mean from 0.9-1.1. Other meanings of“about” may be apparent from the context, such as rounding off, so, for example “about 1” may also mean from 0.5 to 1.4. [00159] The terms“administer”,“administering”,“administered” or“administration” refer to any manner of providing a compound or a pharmaceutical composition (e.g., one described herein), to a subject or patient. Routes of administration can be accomplished through any means known by those skilled in the art. Such means include, but are not limited to, oral, buccal, intravenous, subcutaneous, intramuscular, transdermal, by inhalation and the like.

[00160] “Contacting” as used herein, e.g., as in“contacting a sample” refers to contacting a sample directly or indirectly in vitro, ex vivo, or m vivo (i.e. within a subject as defined herein). Contacting a sample may include addition of a compound to a sample, or administration to a subject. Contacting encompasses administration to a solution, cell, tissue, mammal, subject, patient, or human. Further, contacting a cell includes adding an agent to a cell culture.

[00161] “Effective amount,” as used herein, refers to a dosage or an amount of a compound or a composition effective for eliciting a desired effect. This term as used herein may also refer to an amount effective at bringing about a desired in vivo effect in an animal, e.g., a mammal, e.g., a human. For example, in methods of treating cancer, an effective amount may be an amount sufficient to treat the disorder.

[00162] As used herein, the term“subject” is intended to include human and non-human animals. Exemplary human subjects include a human patient having a disorder, e.g., cancer, or a normal subject. The term“non-human animals” includes all vertebrates, e.g., non-mammals (such as chickens, amphibians, reptiles) and mammals, such as non-human primates,

domesticated and/or agriculturally useful animals (such as sheep, dogs, cats, cows, pigs, etc.), and rodents (such as mice, rats, hamsters, guinea pigs, etc.).

[00163] As used herein, the term“treat” or“treating” a subject having a disorder refers to administering a compound or a composition described herein to the subject, such that at least one symptom of the disorder is cured, healed, alleviated, relieved, altered, remedied, ameliorated, or improved. Treating includes administering an amount effective to alleviate, relieve, alter, remedy, ameliorate, cure, improve or affect the disorder or the symptoms of the disorder. The treatment may inhibit deterioration or worsening of a symptom of a disorder.

[00164] As described herein, compounds of the invention can optionally be substituted with one or more substituents, such as are illustrated generally above, or as exemplified by particular classes, subclasses, and species of the invention. As described herein, the variables in formula I encompass specific groups, such as, for example, alkyl and cycloalkyl. As one of ordinaiy skill in the art will recognize, combinations of substituents envisioned by this invention are those combinations that result m the formation of stable or chemically feasible compounds.

[00165] The term "stable," as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and preferably their recovery, purification, and use for one or more of the purposes disclosed herein. In some embodiments, a stable compound or chemically feasible compound is one that is not

substantially altered when kept at a temperature of 40°C or less, in the absence of moisture or other chemically reactive conditions, for at least a week.

[00166] The term "alkyl" as used herein, means a straight or branched chain saturated hydrocarbon. Representative examples of alkyl include, but are not limited to, methyl, ethyl, npropyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n- hexy!, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-oety!, n-nonyl, and n~ decyl.

[00167] The term "alkylene" or "alkylenyl,"or as used herein, means a divalent group derived from a straight or branched chain saturated hydrocarbon. Representative examples of alkylene/alkylenyl include, but are not limited to, -CTL·-, -CH2CH2-, -CH2CH2CH2-, - CH2CH(CH3)CH2-, and CH2CH(CH3)CH(CH3)CH ₂ -.

[00168] The term "aryl," as used herein, means phenyl or a hicyclic aryl. The bicyclic aryl is naphthyl, dihydronaphthalenyl, tetrahydronaphthalenyl, mdanyl, or indenyl. The phenyl and bicyclic aryls are attached to the parent molecular moiety through any carbon atom contained within the phenyl or bicyclic aryl.

[00169] The term "halogen” means a chlorine, bromine, iodine, or fluorine atom.

[00170] The term "haloalkyl," as used herein, means an alkyl, as defined herein, in which one, two, three, four, five, six, or seven hydrogen atoms are replaced by halogen. For example, representative examples of haloalkyl include, but are not limited to, 2-fluoroethyl,

difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 2,2,2-trifluoro-l, 1-dimethylethyl, and the like. [00171] The term "cycloalkyl" as used herein, means a monocyclic all-carbon ring containing zero heteroatoms as ring atoms, and zero double bonds. Examples of cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

The cycloalkyl groups described herein can be appended to the parent molecular moiety through any substitutable carbon atom.

[00172] The terms "heterocycle" or "heterocyclic" refer generally to ring systems containing at least one heteroatom as a ring atom where the heteroatom is selected from oxygen, nitrogen, and sulfur. In some embodiments, a nitrogen or sulfur atom of the heterocycle is optionally substituted with oxo Heteroeycles may be a monocyclic heterocycle, a fused hicydic heterocycle, or a spiro heterocycle. The monocyclic heterocycle is generally a 4, 5, 6, 7, or 8- membered non-aromatic ring containing at least one heteroatom selected from O, N, or S. The 4- membered ring contains one heteroatom and optionally one double bond. The 5-membered ring contains zero or one double bond and one, two or three heteroatoms. The 6, 7, or 8-membered ring contains zero, one, or two double bonds, and one, two, or three heteroatoms. Representative examples of monocyclic heterocycle include, but are not limited to, azetidmyl, azepanyl, diazepanyl, 1 ,3-dioxanyl, 1 ,4-dioxanyl, 1 ,3-dioxolanyl , 4,5-dihydroisoxazol-5-yl, 3,4- dihydropyranyl, 1 ,3-dithiolanyl, 1 ,3-dithianyl, imidazolmyl, imidazolidinyl, isothiazolinyl, isothiazolidmyl, isoxazolinyl, isoxazolidmyl, rnorpholinyl, oxadiazolinyl, oxadiazolidmyl, oxazolmyl, oxazolidinyl, oxetanyl, piperazmyl, piperidinyl, pyranyl, pyrazolmyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothienyl, thiadiazolmyl, thiadiazolidinyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidothiomorpholinyl, thiopyranyl, and tritluanyl. The fused bicyclic heterocycle is a 7-12-membered ring system having a monocyclic heterocycle fused to a phenyl, to a saturated or partially saturated carbocyclic ring, or to another monocyclic heterocyclic ring, or to a monocyclic heteroaryl ring. Representative examples of fused bicyclic heterocycle include, but are not limited to, 1,3- benzodioxol-4-yl, 1,3-benzodithiolyl, 3-azabicyclo[3.1.Ojhexanyl, hexahydro-lH-furo[3,4- c]pyrrolyl, 2, 3-dihydro- 1 ,4-benzodioxinyl, 2, 3-dihydro- 1 -benzofuranyl, 2,3 -dihydro- 1 - benzothienyl, 2,3-dihydro-lH-indolyl, 5,6,7,8-tetrahydroimidazo[l,2-a]pyrazinyl, and 1 ,2,3,4- tetrahydroqumolinyl. Spiro heterocycle means a 4-, 5-, 6-, 7-, or 8-membered monocyclic heterocycle ring wherein two of the substituents on the same carbon atom form a second ring having 3, 4, 5, 6, 7, or 8 members. Examples of a spiro heterocycle include, but are not limited to, l,4-dioxa-8-azaspiro[4.5]decanyl, 2-oxa-7-azaspiro[3.5]nonanyl, 2-oxa-6- azaspiro[3.3]heptanyl, and 8-azaspiro[4.5] decane. The monocyclic heterocycle groups of the present invention may contain an alkylene bridge of 1, 2, or 3 carbon atoms, linking two nonadjacent atoms of the group. Examples of such a bridged heterocycle include, but are not limited to, 2,5-diazabicyclo[2.2. l]heptanyl, 2-azabicyclo[2.2.1]heptanyl, 2- azabicyclo[2.2.2]octanyl, and oxabicyclo[2.2.1]heptanyl. The monocyclic, fused bicyclic, and spiro heterocycle groups are connected to the parent molecular moiety through any substitutable carbon atom or any substitutable nitrogen atom contained within the group.

[00173] The term "oxo" as used herein refers to an oxygen atom bonded to the parent molecular moiety. An oxo may he attached to a carbon atom or a sulfur atom by a double bond. Alternatively, an oxo may be attached to a nitrogen atom by a single bond, i.e., an N-oxide.

[00174] Terms such as "alkyl," "cycloalkyl," "alkylene," etc. may be preceded by a designation indicating the number of atoms present in the group in a particular instance ( e.g ,

Ciaalkyi, " " C3-6cycloalky 1 " "Ci-ralkylene"). These designations are used as generally understood by those skilled in the art. For example, the representation "C" followed by a subscripted number indicates the number of carbon atoms present in the group that follows.

Thus, "Csalkyl" is an alkyl group with three carbon atoms (i.e. , n-propyl, isopropyl). Where a range is given, as in "Ci-4," the members of the group that follows may have any number of carbon atoms falling within the recited range. A "Ci-ralkyl," for example, is an alkyl group having from 1 to 4 carbon atoms, however arranged (i.e., straight chain or branched).

[00175] The above substituents may be abbreviated herein. For example, the abbreviations Me, Et, Ph and Bn represent methyl, ethyl, phenyl and benzyl, respectively. A more

comprehensive list of standard abbreviations used by organic chemists appears in a table entitled Standard List of Abbreviations of the Journal of Organic Chemistry . The abbreviations contained in said list are hereby incorporated by reference.

[00176] For compounds described herein, groups and substituents thereof may be selected m accordance with permitted valence of the atoms and the substituents, and such that the selections and substitutions result m a stable compound, e.g., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.

[00177] In accordance with a convention used in the art, the group:

[00178] is used in structural formulae herein to depict the bond that is the point of attachment of the moiety or substituent to the core or backbone structure.

[00179] In one aspect, the present invention provides a compound according to Formula (I), or pharmaceutically acceptable salts thereof, wherein R ^f , R ² , R ³ , R ⁴ , R ³ , and X ¹ are as defined herein.

[00180] In some embodiments, R ¹ is phenyl optionally substituted with 1-3 substituents independently selected from the group consisting of Ci-ealkyl, Ci-ehaloalkyl, C3-7cycloalkyl, halogen, cyano, -OH, -OCi-ealkyl, -OCi-ehaloaikyl, -NIL·, -NHCi-balkyl, -N(Ci-6alkyl) ₂ , -OC3- 6cycloalkyl, -MKb-ecycloalkyl, -N(Ci-6alkyl)(C ₃ -6cycloalkyl), and -N(C ₃ -6cyeloalkyl)2, wherein optionally two substituents join to form a 5- to 7-membered non-aromatic fused ring containing 1-2 heteroatom groups selected from NR ¹³ and O. In certain embodiments, R ¹ is phenyl optionally substituted with 1 -3 substituents independently selected from the group consisting of Ci-ealkyl, Ci-ehaloalkyl, C3-7cycloalkyl, halogen, -OCi-saikyl, -OCi-shaloaikyl, or -OC3- 6cycloalkyl, wherein optionally two substituents join to form a 5- to 7-membered non-aromatic fused ring containing 1-2 oxygen atoms. More particularly, in some embodiments, R ¹ is

In any of the embodiments described herein are particular embodiments wherein X ¹ is

S

[00182] In any of the embodiments described herein, where R ² and R ³ together do not form a ring, are particular embodiments wherein R ² is hydrogen or Ci-6alkyl. [00183] In any of the embodiments described herein, where R and R’ together do not form a ring, are particular embodiments wherein R ^" is CXalkyl, Cii-Tcycloalkyl, or aryl, the aryl being optionally substituted with 1-5 substituents independently selected from the group consisting of Ci-ealkyl, Ci-ehaloalkyl, Cii-Tcycloalkyl, halogen, cyano, -OH, -OCi-ealkyl, -OCi-ehaloalkyl, -

NEb, -NHCi-ealkyl, -N(Ci-6alkyl)2, -OC3-6cycloalkyl, -NHC3-6cycloalkyl, -N(Ci-6alkyl)(C3- ecycloalkyl), and -N(C3-6cycloalkyl)2. In further embodiments, the aryl is optionally substituted with 1-3 substituents independently selected from the group consisting of Ci-6alkyl, halogen, and halogen

halogen

C -shaloalkyi. In still further embodiments, the aryl is selected from

[00184] In other embodiments, R ² and R ⁵ together with the nitrogen to which they are attached form a 4- to 8-membered heterocyclic ring optionally containing one additional heteroatom selected from N, Q, and S, and being optionally substituted with 1-4 substituents independently selected from the group consisting of Ci-ealkyl, Ci-fhaloalkyl, halogen, cyano, - OH, oxo, -OCi-ealkyl, -NH2, -NHCi-6alkyl, and -N(Ci-6alkyl)2.

[00185] In any of the embodiments described herein, where R ⁴ and R ⁵ together do not form a ring, are particular embodiments wherein R ⁴ is hydrogen.

[00186] In any of the embodiments described herein, where R ⁴ and R ⁵ together do not form a ring, are particular embodiments wherein R ⁵ is -CHR ^5a R ⁵ °. In some embodiments, R ⁵ has the

R ^5b

following stereochemical configuration: R ^a X ^H

[00187] In any of the embodiments described herein, where R ⁴ and R together do not form a ring, R ^5a may be aryl or -Ci-3alkyiene---aryi, the aryl in R ^sa being optionally substituted with 1 -5 substituents independently selected from the group consisting of Ci-6alkyl, Ci-ehaloalkyl, Cs- 7cycloalkyl, halogen, cyano, -OH, -OCnealkyl, -OCi-ohaloalkyl, -NH?„ -NHCnealkyl, -NfCi- 6alkyl) ₂ , -OCs-ecycloalkyl, -MHCi-eeycloalkyl, -N(C _l-6 alkyl)(C3-6cycloalkyl), and -N(C3- 6cycloalkyl)2. In some embodiments, R ^3a may be -Ciaalkylene-aryl, wherein the aryl is optionally substituted with the foregoing list of optional substituents. More particularly, R ^3a may be -CHa-phenyl, the phenyl being optionally substituted with 1-3 substituents independently selected from the group consisting of Ci-6alkyl, Ci-ehaloalkyl, CV/eyeioaikyl, halogen, cyano, - OH, -OCi-ealkyl, -OCi-ehaloalkyl, -NH2, -MHCnealkyl, -N(Ci-6alkyl) ₂ , -OC3-6cycloalkyl, - NHCs-ecycloalkyl, -N(Ci -6alkyl)(C3-6cycloalkyl), and -N(C3-6cycloalkyi) ₂ . Still more particularly, the phenyl in R ^5a may be optionally substituted with 1 -3 substituents independently selected from the group consisting of Ci -ealkyl, -OH, and Ci -ehaloalkyl. Still more particularly,

R ^5a may

[00188] In any of the embodiments described herein where R ⁴ and R ³ together do not form a ring, R/* may be X ² or -Ci-saikylene-X ² . More particularly, R ^5b may be X ² or -CH2-X ² . Still more particularly, R ^5b may be -CH2-X ² .

[00189] In other embodiments, R ⁴ and R ⁵ together with the nitrogen to which they are attached form a 4- to 8-membered heterocyclic ring optionally containing one additional heteroatom selected from N, O, and S, and being optionally substituted with 1-4 substituents independently selected from the group consisting of Ci-ealkyl, Ci-ehaloalkyl, halogen, cyano, - OH, oxo, -OCi-ealkyl, -NH2, -NHC -ealkyl, and -N(Ci-6alkyl)2.

[0019Q] In any of the embodiments described herein, X may be -CM, -C(0)0H, -C(0)0Ci-

4alkyl, -C(0)NH ₂ , -C(0)NHCi-4alkyl, C(0)\(Ci ia!kvl)u -SO2NH2, SO2M !( ^' i ra!kyl or - S0 ₂ N(Ci-4alkyl)2. In particular embodiments, X ² is -C(0)OH or -C(0)NH2.

[00191] In certain embodiments of the invention R ⁴ is hydrogen; R ⁵ is -CHR ^3a R ^5b ; R ^5a is -

CH2-phenyl, the phenyl being optionally substituted with 1-5 substituents independently selected from the group consisting of Ci-ealkyl, Ci-6haloalkyl, C3-7cycloalkyl, halogen, cyano, -OH, - OCi-ealkyl, -OCi-ehaloalkyl, M l;. -NHCi-eaikyl, -N(Ci-6alkyl) ₂ , -OCs-ecydoalkyl, M IC 6cycloaikyl, -N(Ci-6alkyl)(C3-6cycloalkyl), and -N(C3-6cycloalkyl)2; R ^5b is -CH2-X ² ; and X ² is - C(0)OH or -C(0)NH2. In further embodiments according to the foregoing the phenyl at R ^5a is optionally substituted with 1-3 of the foregoing optional substituents. These embodiments

include still further embodiments wherein yet further embodiments included in the foregoing, R ³ has the following stereochemistry . For

example, R ^a may

C(0)NH ₂ , with the following s

[00192] In some embodiments, the compound of formula (I) is selected from

[00193] (R)-N-(4-amino-l-(4-(tert-butyl)phenyl)-4-oxobutan-2-yl)-5-( N-isopropylsulfamoyl)-

2-((4-methoxyphenyl)thio)benzamide;

[00194] (R)-N-(4-amino-l-(4-(tert-butyl)phenyl)-4-oxobutan-2-yl)-5-( N- cyclopentylsulfamoyl)-2-((4-methoxyphenyl)thio)benzamide;

[00195] (R)-N-(4-amino-l-(4-(tert-butyl)phenyl)-4-oxobutan-2-yl)-5-( N-isopropyl-N- methylsulfamoyl)-2-((4-methoxyphenyl)thio)benzamide;

[00196] (R)-N-(4-amino-l-(4-(tert-butyl)phenyl)-4-oxobutan-2-yl)-2-( (4- methoxyphenyl)thio)-5-(N-(m-tolyl)sulfamoyl)benzamide;

[00197] (R)-N-(4-amino-l-(4-(tert-butyl)phenyl)-4-oxobutan-2-yl)-5-( N-(3- bromophenyl)sulfamoyl)-2-((4-methoxyphenyl)thio)benzamide;

[00198] (R)-N-(4-amino-l-(4-(tert-butyl)phenyl)-4-oxobutan-2-yl)-5-( N-(4- fluorophenyl)sulfamoyl)-2-((4-methoxyphenyl)thio)benzamide; [00199] (R)-N-(4-amino-l-(4-(tert-butyl)phenyl)-4-oxobutan-2-yl)-5-( N-(3- fluorophenyl)suifamoyl)-2-((4-methoxyphenyl)thio)benzamide;

[00200] N-isopropyl-4-((4-methoxyphenyl)thio)-N-methyl-3-(piperidine -l - carbonyl)benzenesulfonamide

[00201] (R)-N-(4-amino-l-(4-(tert-butyl)phenyl)-4-oxobutan-2-yl)-5-( N-isopropyl-N- methylsulfamoyl)-2-((4-(trifluoromethoxy)phenyl)thio)benzami de;

[00202] (R)-N-(4-amino-l-(4-(tert-butyl)phenyl)-4-oxobutan-2-yi)-5-( N-isopropyl-N- methylsulfamoyl)-2-((3-methoxyphenyl)thio)benzamide;

[00203] (5-(N-isopropyl-N-methylsulfamoyl)-2-((4-methoxyphenyl)thio) benzoyl)-D-tyrosine; and

[00204] (R)-N-(l-amino-I-oxo-3-phenylpropan-2-yl)-5-(N-isopropyl-N-m ethylsulfamoyl)-2-

((4-methoxyphenyl)thio)benzamide; or

[00205] a pharmaceutically acceptable salt thereof

[002Q6] Certain compounds may exist in one or more particular geometric, optical, enantiomeric, diastereomeric, epimeric, atropic, stereoisomer, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, and r- forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d- and 1 -forms; (+) and (-) forms; keto-, end-, and enolate-forms; syn- and anti-forms; synclinal- and anticlinal-forms; a- and b-forrns; axial and equatorial forms; boat-, chair-, twist-, envelope-, and half chair-forms; and combinations thereof, hereinafter collectively referred to as "isomers" (or "isomeric forms").

[00207] in one embodiment, a compound described herein may be an enantiomerically enriched isomer of a stereoisomer described herein. For example, the compound may have an enantiomeric excess of at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% Enantiomer, when used herein, refers to either of a pair of chemical compounds whose molecular structures have a mirror-image relationship to each other.

[00208] in one embodiment, a preparation of a compound disclosed herein is enriched for an isomer of the compound having a selected stereochemistry', e.g., R or S, corresponding to a selected stereocenter. For example, the compound has a purity corresponding to a compound having a selected stereochemistry of a selected stereocenter of at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.

[00209] In one embodiment, a composition described herein includes a preparation of a compound disclosed herein that is enriched for a structure or structures having a selected stereochemistry, e.g., R or S, at a selected stereocenter. Exemplary R/ ^' S configurations can be those provided in an example described herein.

[00210] An "enriched preparation," as used herein, is enriched for a selected

stereoconfiguration of one, two, three or more selected stereocenters within the subject compound. Exemplary selected stereocenters and exemplary' stereoconfigurations thereof can be selected from those provided herein, e.g., in an example described herein. By enriched is meant at least 60%, e.g., of the molecules of compound m the preparation have a selected

stereochemistry' of a selected stereocenter. In an embodiment it is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%. Enriched refers to the level of a subject moiecule(s) and does not connote a process limitation unless specified.

[00211] Compounds may be prepared in racemic form or as individual enantiomers or diastereomers by either stereospecific synthesis or by resolution. The compounds may, for example, be resolved into their component enantiomers or diastereomers by standard techniques, such as the formation of stereoisomenc pairs by salt formation with an optically active base, followed by fractional crystallization and regeneration of the free acid. The compounds may also be resolved by formation of stereoisomers e esters or amides, followed by chromatographic separation and removal of the chiral auxiliary. Alternatively, the compounds may be resolved using a chiral HPLC column. The enantiomers also may' be obtained from kinetic resolution of the racemate of corresponding esters using lipase enzymes.

[00212] Examplary tautomeric forms include, for example, the following tautomeric pairs: keto/enol and imine/enamine.

[00213] Note that specifically included in the term "isomer" are compounds with one or more isotopic substitutions. Examples of isotopes suitable for inclusion in the compounds of the invention are hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, such as, but not limited to H, ¾, ^fJ C, ^l4 C, ^l5 N, ^{ϊ 8} 0, ^{1 ;} 0, ^{i J} P, ³² P, ^"3 S, ^1S F, and ⁶ Cl, respectively. Substitution with heavier isotopes such as deuterium, i.e. ² H, can afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances. The compound may incorporate positron-emitting isotopes for medical imaging and positron-emitting tomography (PET) studies for determining the distribution of receptors. Suitable positron- emitting isotopes that can be incorporated in compounds of formula (I) are ^{1 ]} C, ⁿ N, ^l3 0, and ^lS F. isotopically-iabeled compounds of formula (I) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described m the accompanying Examples using appropriate isotopically-iabeled reagent in place of non- isotopical!y-labeled reagent. In some embodiments, m compounds of formula (I), any hydrogen atom may be deuterium.

[00214] A compound described herein can be in the form of a salt, e.g., a pharmaceutically acceptable salt. As used herein, the term "pharmaceutically acceptable salt" refers to those salts which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M Berge, et al. describe pharmaceutically acceptable salts in detail in J Pharmaceutical Sciences, 1977, 66, 1 -19, incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauiyl sulfate, malate, maieate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N(Ci-4alkyl)4 salts. This invention also envisions the quaternization of any basic nitrogen-containing groups of the compounds disclosed herein. Water or oil-soluble or dispersable products may be obtained by such quaternization. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl (e.g., phenyl/substituted phenyl) sulfonate.

[00215] It may be convenient or desirable to prepare, purify, and/or handle an active compound in a chemically protected form. The term "chemically protected form" is used herein in the conventional chemical sense and pertains to a compound in which one or more reactive functional groups are protected from undesirable chemical reactions under specified conditions (e.g., pH, temperature, radiation, solvent, and the like). In practice, well known chemical methods are employed to reversibly render unreactive a functional group, which otherwise would be reactive, under specified conditions. In a chemically protected form, one or more reactive functional groups are in the form of a protected or protecting group (also known as a masked or masking group or a blocked or blocking group). By protecting a reactive functional group, reactions involving other unprotected reactive functional groups can be performed, without affecting the protected group; the protecting group may be removed, usually m a subsequent step, without substantially affecting the remainder of the molecule. See, for example, Protective Groups in Organic Synthesis (T. Green and P. Wuts; 3rd Edition; John Wiley and Sons, 1999). Unless otherwise specified, a reference to a particular compound also includes chemically protected forms thereof.

[00216] A wide variety- of such "protecting," "blocking," or "masking” methods are widely used and well known in organic synthesis. For example, a compound which has two nonequivalent reactive functional groups, both of which would be reactive under specified conditions, may be derivatized to render one of the functional groups "protected," and therefore unreactive, under the specified conditions; so protected, the compound may be used as a reactant which has effectively only one reactive functional group. After the desired reaction (involving the other functional group) is complete, the protected group may be "deprotected" to return it to its original functionality.

[00217] A hydroxy group may be protected as an ether (-OR) or an ester (-OC(O)R), for example, as: a t-butyl ether; a benzyl, benzhydryl (diphenylmethyl), or trityl (triphenylmethyl) ether; a trimethy!silyl or t-butyldimethylsilyl ether; or an acetyl ester (-0C(0)CH3, -OAc).

[00218] An aldehyde or ketone group may be protected as an acetal (RCH(OR)2) or ketal (R ₂ C(OR) ₂ ), respectively, in which the carbonyl group (R ₂ C=0) is converted to a diether (R ₂ C(0R) ₂ ), by reaction with, for example, a primary alcohol. The aldehyde or ketone group is readily regenerated by hydrolysis using a large excess of water in the presence of acid.

[00219] An amine group may be protected, for example, as an amide (-NRC(O)R) or a urethane (-NRC(O)OR), for example, as: a methyl amide (-NHC(0)CH3); a benzyloxy amide (- NHC(0)0CH ₂ C6H ₅ , -NH-Cbz); as a t-butoxy amide (~M !C(0)( ⁾ C(n l·) ^, . -NH-Boc); a 2- biphenyl-2-propoxy amide (-NHCO(())C(G¾) ₂ C6H4C6H5, -NH-Bpoc), as a 9-fluorenyimethoxy amide (-NH-Fmoc), as a 6-nitroveratryloxy amide (-NH-Nvoc), as a 2-trimethylsilylethyloxy amide (-NH-Teoc), as a 2,2,2-trichloroethyloxy amide (-NH-Troc), as an allyloxy amide (-NH- Ailoc), as a 2(-phenylsuiphonyl)ethyioxy amide (-NH-Psec); or, in suitable cases (e.g., cyclic amines), as a mtroxide radical (>N-0«).

[00220] A carboxylic acid group may be protected as an ester, for example, as: an alkyl ester (e.g., a methyl ester; a t-butyl ester); a haloalkyi ester (e.g., a haloalkyi ester); a tnalkylsilylalkyl ester; or an arylalkyl ester (e.g., a benzyl ester; a mtrobenzyl ester); or as an amide, for example, as a methyl amide.

[00221] A thiol group may be protected as a tluoether (-SR), for example, as: a benzyl thioether; an acetamidomethyl ether (-S-CH ₂ NHC(0)CH3). 3. Methods of Use

[00222] Also disclosed are methods of using the disclosed compounds and compositions to treat a disease or condition ameliorated by b2 receptor activation comprising administering to a subject m need thereof a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt or pharmaceutical composition thereof.

[00223] Compounds of the invention have activity as positive allosteric modulators (PAMs) of the b2 adrenergic receptor and display positive cooperativity with orthosteric agonists, thus enhancing their binding to the receptor and ability to stabilize its active state. The compounds of the invention also exhibit positive cooperativity with G protein and b-arrestin, thus potentiating their stabilization of high-affinity agonist-bound states of the receptor, as well as downstream cAMP production and b-arrestin recruitment to the activated receptor. The positive allosteric activity is specific for the b ₂ AK compared to its closely related sub-type, the biAK

[00224] Compounds of the invention have several potential benefits as therapeutic drugs to increase specificity as well as to decrease adverse effects for some p2AR-related diseases such as asthma. For example, the compounds have strong specificity for the b2Aϋ over the most closely related subtype bΐ AR. The compounds also show a ceiling level of activity, which can reduce risks from target-based overdose. As allosteric modulators, compounds of the invention only exert their modulating activity when a b2^ohί8ί of the b2AK is available. Altogether, PAMs of the invention may accomplish fine-tuning of the activity of the b2A to provide better therapeutic treatments for diseases like asthma, for which p2AR agonists are clinically used. PAMs are expected to allow therapeutic administration of b2AK agonists at lower doses, thereby reducing potential toxicity and/or other off-target effects. In some embodiments, coadministration of a PAM of the invention with a b2AK agonist may provide enhanced therapeutic effect compared to the therapeutic effect achieved by administration of the b2AK agonist alone.

[00225] b2 adrenergic receptor agonists are useful for treating diseases or conditions such as obstructive airway disease or bronchospasms (e.g.,COPD and asthma), and pre-term labor.

[00226] Diseases or conditions that may be treated with compounds and compositions of the invention include an obstructive airway disease, bronchospasms, and pre-term labor. [00227] Particular diseases or conditions include asthma of whatever type, etiology, or pathogenesis; or asthma that is a member selected from the group consisting of atopic asthma; non-atopic asthma; allergic asthma; atopic, bronchial, IgE-mediated asthma; bronchial asthma; essential asthma; true asthma; intrinsic asthma caused by pathophysiologic disturbances;

extrinsic asthma caused by environmental factors; essential asthma of unknown or non-apparent cause; nonatopic asthma; bronchitic asthma; emphysematous asthma; exercise-induced asthma; occupational asthma; infective asthma caused by bacterial, fungal, protozoal, or viral infection; non-allergic asthma; incipient asthma; wheezy infant syndrome;

[00228] Other diseases or conditions include chronic or acute bronchoconstriction; chronic bronchitis; small airways obstruction; and emphysema.

[00229] Still further diseases or conditions include obstructive or inflammatory airways diseases of whatever type, etiology, or pathogenesis; or an obstructive or inflammatory airways disease that is a member selected from the group consisting of chronic obstructive pulmonary' disease (COPD); COPD that includes chronic bronchitis, pulmonary emphysema or dyspnea associated therewith; COPD that is characterized by irreversible, progressive airways obstruction; and exacerbation of airways hyper-reactivity consequent to other drug therapy.

[00230] In some embodiments, compounds of the invention bind to an allosteric site of the b2 receptor in a subject. In further embodiments, the binding of the compound or its salt to the allosteric site stabilizes an active conformation of the b2 receptor. In still further embodiments, the binding of the compound or its salt potentiates the activity of a [52 agonist, which may be administered m combination with a compound or composition of the invention. In certain embodiments, the compounds of the in vention may potentiate tome receptor acti vity or receptor activity mediated by endogenous levels of agonist present. In certain embodiments, the compound, its pharmaceutical salt, or composition is administered in combination therapy with a b2 agonist wherein the amount of the compound, pharmaceutical salt, or composition may reduce tolerance to the effects of the b2 receptor agonist compared to the tolerance in a reference subject receiving treatment with the b2 receptor agonist alone.

[00231] in another aspect of the invention, pharmaceutically acceptable compositions are provided, wherein these compositions comprise any of the compounds as described herein, and optionally comprise a pharmaceutically acceptable carrier, adjuvant or vehicle. In certain embodiments, these compositions optionally further comprise one or more additional therapeutic agents. In one embodiment, the pharmaceutical composition comprises a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers or vehicles.

[00232] In one aspect, provided is a pharmaceutical composition comprising a compound according to Formula (I), or a tautomer or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[00233] Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[00234] The pharmaceutical compositions may include a“therapeutically effective amount” or a“prophylactically effective amount” of the agent. A“therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary', to achieve the desired therapeutic result. A therapeutically effective amount of the composition may be determined by a person skilled in the art and may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the composition to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of a compound of the invention (e.g., a compound of formula (I)) are outweighed by the therapeutically beneficial effects. A“prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[00235] For example, a therapeutically effective amount of a compound of formula (I), may be about 1 mg/kg to about 1000 mg/kg, about 5 mg/kg to about 950 mg/kg, about 10 mg/kg to about 900 mg/kg, about 15 mg/kg to about 850 mg/kg, about 20 mg/kg to about 800 mg/kg, about 25 mg/kg to about 750 mg/kg, about 30 mg/kg to about 700 mg/kg, about 35 mg/kg to about 650 mg/kg, about 40 mg/kg to about 600 mg/kg, about 45 mg/kg to about 550 mg/kg, about 50 mg/kg to about 500 mg/kg, about 55 mg/kg to about 450 mg/kg, about 60 mg/kg to about 400 mg/kg, about 65 mg/kg to about 350 mg/kg, about 70 mg/kg to about 300 mg/kg, about 75 mg/kg to about 250 mg/kg, about 80 mg/kg to about 200 mg/kg, about 85 mg/kg to about 150 mg/kg, and about 90 mg/kg to about 100 mg/kg.

[00236] As described herein, the pharmaceutically acceptable compositions of the invention additionally comprise a pharmaceutically acceptable carrier, adjuvant, or vehicle, which, as used herein, includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's Pharmaceutical Sciences, Sixteenth Edition, E.W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used m formulating pharmaceutically acceptable compositions and known techniques for the preparation thereof. Except insofar as any conventional carrier medium is incompatible with the compounds of the invention, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutically acceptable composition, its use is contemplated to be within the scope of this invention. Some examples of materials which can serve as

pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, or potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes,

polyethylenepolyoxypropylene-block polymers, wool fat, sugars such as lactose, glucose and sucrose; starches such as com starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; com oil and soybean oil; glycols; such a propylene glycol or polyethylene glycol: esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen- free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

[00237] The pharmaceutically acceptable compositions of this invention can be administered to humans and other animals orally, rectally, parenteral!y, mtracistemally, intravagmally, intraperitoneally, topically (as by powders, ointments, or drops), bucally, as an oral or nasal spray, or the like, depending on the seventy of the disease being treated.

[00238] Thus, the compounds and their pharmaceutically acceptable salts may be formulated for administration by, for example, solid dosing, eyedrop, in a topical oil-based formulation, injection, inhalation (either through the mouth or the nose), implants, or oral, buccal, parenteral, or rectal administration. Techniques and formulations may generally be found in“Remington's Pharmaceutical Sciences,” (Meade Publishing Co., Easton, Pa.). Therapeutic compositions must typically be sterile and stable under the conditions of manufacture and storage.

[00239] The route by which the disclosed compounds are administered and the form of the composition will dictate the type of carrier to be used. The composition may be in a variety of forms, suitable, for example, for systemic administration (e.g., oral, rectal, nasal, sublingual, buccal, implants, or parenteral) or topical administration (e.g., dermal, pulmonary, nasal, aural, ocular, liposome delivery systems, or iontophoresis).

[00240] The term“parenterally,” as used herein, refers to modes of administration which include intravenous, intramuscular, intraperitoneal, mtrasternal, subcutaneous and mtraartieu!ar injection and infusion.

[00241] Carriers for systemic administration may include one or more of diluents, lubricants, binders, dismtegrants, colorants, flavors, sweeteners, antioxidants, preservatives, glidants, solvents, suspending agents, wetting agents, surfactants, emulsifying agents and dispersing agents, combinations thereof, and others. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of the injectable

pharmaceutical form can be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

[00242] Suitable diluents include sugars such as glucose, lactose, dextrose, and sucrose; diols such as propylene glycol; calcium carbonate; sodium carbonate; sugar alcohols, such as glycerin; mannitol; and sorbitol. The amount of diluent(s) in a systemic or topical composition is typically about 50 to about 90%.

[00243] Suitable lubricants include silica, talc, stearic acid and its magnesium salts and calcium salts, calcium sulfate; and liquid lubricants such as polyethylene glycol and vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, com oil and oil of theobroma. The amount of lubricant(s) in a systemic or topical composition is typically about 5 to about 10%.

[00244] Suitable binders include polyvinyl pyrrolidone; magnesium aluminum silicate;

starches such as corn starch and potato starch; gelatin; tragacanth; and cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, methylcellulose, microcrystalline cellulose, and sodium carboxymethylceliulose. The amount of bmder(s) in a systemic composition is typically about 5 to about 50%.

[00245] Suitable dismtegrants include agar, aiginic acid and the sodium salt thereof, effervescent mixtures, croscarmelose, crospovidone, sodium carboxymethyl starch, sodium starch g!ycolate, clays, and ion exchange resms. The amount of disintegrant(s) in a sy stemic or topical composition is typically about 0.1 to about 10%.

[00246] Suitable colorants include a colorant such as an FD&C dye. When used, the amount of colorant in a systemic or topical composition is typically about 0.005 to about 0.1%.

[00247] Suitable flavors include menthol, peppermint, and fruit flavors. The amount of flavor(s), when used, in a systemic or topical composition is typically about 0.1 to about 1.0%.

[00248] Suitable sweeteners include aspartame and saccharin. The amount of sweetener(s) in a systemic or topical composition is typically about 0.001 to about 1%. [00249] Suitable antioxidants include butylated hydroxyanisole (“BHA”), butylated hydroxytoluene (“BHT”), and vitamin E. The amount of antioxidant(s) m a systemic or topical composition is typically about 0.1 to about 5%.

[00250] Suitable preservatives include benzalkonium chloride, methyl paraben and sodium benzoate. The amount of preservative(s) m a systemic or topical composition is typically about 0.01 to about 5%.

[00251] Suitable glidants include silicon dioxide. The amount of glidant(s) in a systemic or topical composition is typically about 1 to about 5%.

[00252] Suitable solvents include water, isotonic saline, ethyl oleate, glycerine, hydroxylated castor oils, alcohols such as ethanol, and phosphate buffer solutions. The amount of solvent(s) in a systemic or topical composition is typically from about 0 to about 100%.

[00253] Suitable suspending agents include AVICEL RC-591 (from EMC Corporation of Philadelphia, PA) and sodium alginate. The amount of suspending agent(s) in a systemic or topical composition is typically about 1 to about 8%.

[00254] Suitable surfactants include lecithin, Polysorbate 80, and sodium lauryl sulfate, and the TWEENS from Atlas Powder Company of Wilmington, Delaware. Suitable surfactants include those disclosed in the C.T.F.A. Cosmetic Ingredient Handbook, 1992, pp. 87-592; Remington's Pharmaceutical Sciences, 15th Ed. 1975, pp. 335-337; and McCutcheon's Volume 1, Emulsifiers & Detergents, 1994, North American Edition, pp. 236-239. The amount of surfactant(s) in the systemic or topical composition is typically about 0.1% to about 5%.

[00255] Although the amounts of components in the systemic compositions may vary depending on the type of systemic composition prepared, in general, systemic compositions include 0.01% to 50% of an active compound (e.g., a compound of formula (I)) and 50% to 99.99% of one or more carriers. Compositions for parenteral administration typically include 0.1% to 10% of actives and 90% to 99.9% of a carrier including a diluent and a solvent.

[00256] Compositions for oral administration can have various dosage forms. For example, solid forms include tablets, capsules, pills, granules, and bulk powders. These oral dosage forms include a safe and effective amount, usually at least about 5%, and more particularly from about 25% to about 50% of actives. The oral dosage compositions include about 50% to about 95% of carriers, and more particularly, from about 50% to about 75%.

[00257] Tablets can be compressed, tablet triturates, enteric-coated, sugar-coated, film-coated, or multiple-compressed. Tablets typically include an active component, and a carrier comprising ingredients selected from diluents, lubricants, binders, disintegrants, colorants, flavors, sweeteners, glidants, and combinations thereof. Specific diluents include calcium carbonate, sodium carbonate, mannitol, lactose and cellulose. Specific binders include starch, gelatin, and sucrose. Specific disintegrants include alginic acid and croscarme!ose. Specific lubricants include magnesium stearate, stearic acid, and talc. Specific colorants are the FD&C dyes, which can be added for appearance. Chewable tablets preferably contain sweeteners such as aspartame and saccharin, or flavors such as menthol, peppermint, fruit flavors, or a combination thereof.

[00258] Capsules (including implants, time release and sustained release formulations) typically include an active compound (e.g., a compound of formula (I)), and a carrier including one or more diluents disclosed above in a capsule comprising gelatin. Granules typically comprise a disclosed compound, and preferably glidants such as silicon dioxide to improve flow characteristics. Implants can be of the biodegradable or the n on-biodegradable type.

[00259] The selection of ingredients in the carrier for oral compositions depends on secondary considerations like taste, cost, and shelf stability, which are not critical for the purposes of this invention.

[00260] Solid compositions may be coated by conventional methods, typically wath pH or time-dependent coatings, such that a disclosed compound is released in the gastrointestinal tract in the vicinity of the desired application, or at various points and times to extend the desired action. The coatings typically include one or more components selected from the group consisting of cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropy! methyl cellulose phthalate, ethyl cellulose, EUDRAGIT® coatings (available from Evonik industries of Essen, Germany), waxes and shellac.

[00261] Compositions for oral administration can have liquid forms. For example, suitable liquid forms include aqueous solutions, emulsions, suspensions, solutions reconstituted from non-effervescent granules, suspensions reconstituted from non-effervescent granules, effervescent preparations reconstituted from effervescent granules, elixirs, tinctures, syrups, and the like. Liquid orally administered compositions typically include a disclosed compound and a carrier, namely, a carrier selected from diluents, colorants, flavors, sweeteners, preservatives, solvents, suspending agents, and surfactants. Peroral liquid compositions preferably include one or more ingredients selected from colorants, flavors, and sweeteners.

[00262] Other compositions useful for attaining systemic delivery of the subject compounds include sublingual, buccal and nasal dosage forms. Such compositions typically include one or more of soluble filler substances such as diluents including sucrose, sorbitol and mannitol; and binders such as acacia, microcrystal line cellulose, carboxymethyl cellulose, and hydroxypropyl methylcellu!ose. Such compositions may further include lubricants, colorants, flavors, sweeteners, antioxidants, and glidants.

[00263] The pharmaceutical compositions of the present invention may also be administered by nasal aerosol or inhalation through the use of a nebulizer, a dry powder inhaler or a metered dose inhaler. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, hydrofluorocarbons, and/or other conventional solubilizing or dispersing agents.

[00264] Aerosol propellants are required where the pharmaceutical composition is to be delivered as an aerosol under significant pressure. Such propellants include, e.g., acceptable fluoroch!orohydrocarbons such as dichlorodifluoromethane, dichlorotetrafluoroethane, and trichloromonofluoromethane; nitrogen; or a volatile hydrocarbon such as butane, propane, isobutane or mixtures thereof.

[00265] The disclosed compounds can be topically administered. Topical compositions that can be applied locally to the skm may be in any form including solids, solutions, oils, creams, ointments, gels, lotions, shampoos, leave-on and rinse-out hair conditioners, milks, cleansers, moisturizers, sprays, skin patches, and the like. Topical compositions include: a disclosed compound (e.g., a compound of formula (I)), and a carrier. The carrier of the topical composition preferably aids penetration of the compounds into the skin. The carrier may further include one or more optional components. [00266] The amount of the carrier employed in conjunction with a disclosed compound is sufficient to provide a practical quantity of composition for administration per unit dose of the compound. Techniques and compositions for making dosage forms useful in the methods of this invention are described in the following references: Modern Pharmaceutics, Chapters 9 and 10, Banker & Rhodes, eds. (1979); Lieberman et al, Pharmaceutical Dosage Forms: Tablets (1981); and Ansel, Introduction to Pharmaceutical Dosage Forms, 2nd Ed., (1976).

[00267] A carrier may include a single ingredient or a combination of two or more ingredients. In the topical compositions, the carrier includes a topical carrier. Suitable topical carriers include one or more ingredients selected from phosphate buffered saline, isotonic water, deionized water, monofunctional alcohols, symmetrical alcohols, aloe vera gel, allantoin, glycerin, vitamin A and E oils, mineral oil, propylene glycol, PPG-2 myristy! propionate, dimethyl isosorbide, castor oil, combinations thereof, and the like. More particularly, carriers for skin applications include propylene glycol, dimethyl isosorbide, and water, and even more particularly, phosphate buffered saline, isotonic water, deionized water, monofunctional alcohols, and symmetrical alcohols.

[00268] The carrier of a topical composition may further include one or more ingredients selected from emollients, propellants, solvents, humectants, thickeners, powders, fragrances, pigments, and preservatives, ail of which are optional.

[00269] Suitable emollients include stearyl alcohol, glyceryl monoricinoleate, glyceryl monostearate, propane- 1 ,2-diol, butane- 1 ,3-diol, mink oil, cetyl alcohol, isopropyl isostearate, stearic acid, isobutyl palmitate, isocetyl stearate, oleyl alcohol, isopropyl laurate, hexyl laurate, deey! oleate, octadecan-2-ol, isocetyl alcohol, cetyl palmitate, di-n-butyl sebacate, isopropyl myristate, isopropyl palmitate, isopropyl stearate, butyl stearate, polyethylene glycol, triethylene glycol, lanolin, sesame oil, coconut oil, arachis oil, castor oil, acetylated lanolin alcohols, petroleum, mineral oil, butyl myristate, isostearic acid, palmitic acid, isopropyl linoleate, lauryl lactate, mynstyl lactate, decyl oleate, mynslyl myristate, and combinations thereof. Specific emollients for skin include stearyl alcohol and polydimethylsiloxane. The amount of emol!ient(s) in a skin-based topical composition is typically about 5% to about 95%. [00270] Suitable propellants include propane, butane, isobutane, dimethyl ether, carbon dioxide, nitrous oxide, and combinations thereof. The amount of propellant(s) m a topical composition is typically about 0% to about 95%.

[00271] Suitable solvents include water, ethyl alcohol, methylene chloride, isopropanol, castor oil, ethylene glycol monoethyl ether, diethylene glycol monobutyl ether, diethylene glycol monoethyl ether, dimethylsulfoxide, dimethyl formamide, tetrahydrofuran, and combinations thereof. Specific solvents include ethyl alcohol and homotopic alcohols. The amount of solvent(s) in a topical composition is typically about 0% to about 95%.

[00272] Suitable humectants include glycerin, sorbitol, sodium 2-pyrrolidone-5-earboxylate, soluble collagen, dibutyl phthalate, gelatin, and combinations thereof. Specific humectants include glycerin. The amount of humectant(s) m a topical composition is typically 0% to 95%.

[00273] The amount of thickener(s) in a topical composition is typically about 0% to about 95%.

[00274] Suitable powders include beta-cyclodextrins, hydroxypropyl cyclodextrins, chalk, talc, fullers earth, kaolin, starch, gums, colloidal silicon dioxide, sodium polyacrylate, tetra alkyl ammonium smectites, trialkyl aryl ammonium smectites, chemically-modified magnesium aluminum silicate, organically-modified Montmonllonite clay, hydrated aluminum silicate, fumed silica, carboxy vinyl polymer, sodmm carboxymethyl cellulose, ethylene glycol monostearate, and combinations thereof. The amount of powder(s) in a topical composition is typically 0% to 95%.

[00275] The amount of fragrance in a topical composition is typically about 0% to about 0.5%, particularly, about 0.001% to about 0.1%.

[00276] Suitable pH adjusting additives include HC1 or NaOH in amounts sufficient to adjust the pH of a topical pharmaceutical composition.

[00277] it will be appreciated that appropriate dosages of the compounds, and compositions comprising the compounds, can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects of the treatments of the present invention. The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds, and/or materials used m combination, and the age, sex, weight, condition, general health, and prior med ical history of the patient. The amount of compound and route of administration will ultimately be at the discretion of the physician, although generally the dosage will be to achieve local concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious side-effects.

[00278] Administration in vivo can be effected in one dose, continuously or intermittently (e.g , in divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. In general, a suitable dose of the compound is in the range of about 100 pg to about 250 mg per kilogram body weight of the subject per day.

[00279] The composition may be administered once, on a continuous basis (e.g. by an intravenous drip), or on a periodic/intermittent basis, including about once per hour, about once per two hours, about once per four hours, about once per eight hours, about once per twelve hours, about once per day, about once per two days, about once per three days, about twice per week, about once per week, and about once per month. The composition may be administered until a desired reduction of symptoms is achieved.

[00280] The present compounds, compositions, and methods may be administered as part of a therapeutic regimen along with other treatments appropriate for the particular injury or disease being treated.

[00281] in one aspect, the disclosed compounds can be used in combination with one or more other drugs in the treatment, prevention, control, amelioration, or reduction of risk of diseases or conditions for which disclosed compounds or the other drugs can have utility, where the combination of the drugs together are safer or more effective than either drug alone. Such other drug(s) can be administered, by a route and in an amount commonly used therefor, contemporaneously or sequentially with a compound of the present disclosure. When a compound of the present disclosure is used contemporaneously with one or more other drugs, a pharmaceutical composition in unit dosage form containing such other drugs and a disclosed compound is preferred. However, the combination therapy can also include therapies m which a disclosed compound and one or more other drugs are administered on different overlapping schedules. It is also contemplated that when used in combination with one or more other active ingredients, the disclosed compounds and the other active ingredients can be used in lower doses than when each is used singly.

[00282] Combination therapy includes administration of a single pharmaceutical dosage formulation containing one or more of the compounds described herein and one or more additional pharmaceutical agents, as well as administration of the compounds and each additional pharmaceutical agent, in its own separate pharmaceutical dosage formulation. For example, a compound described herein and one or more additional pharmaceutical agents, can be administered to the patient together, in a single oral dosage composition having a fixed ratio of each active ingredient, such as a tablet or capsule; or each agent can be administered in separate oral dosage formulations. Where separate dosage formulations are used, the present compounds and one or more additional pharmaceutical agents can be administered at essentially the same time (e.g., concurrently) or at separately staggered times (e.g , sequentially).

[00283] The above combinations include combinations of a disclosed compound not only with one other active compound, but also with two or more other active compounds. Likewise, disclosed compounds can be used in combination with other drugs that are used in the prevention, treatment, control, amelioration, or reduction of risk of the diseases or conditions for which disclosed compounds are useful. Such other drugs can be administered, by a route and in an amount commonly used therefor, contemporaneously or sequentially with a compound of the present disclosure. When a compound of the present disclosure is used contemporaneously with one or more other drugs, a pharmaceutical composition containing such other drugs in addition to a disclosed compound is preferred. Accordingly, the pharmaceutical compositions include those that also contain one or more other active ingredients, in addition to a compound of the present disclosure. [00284] Additional pharmaceutical agents include PDE3 and/or PDE4 inhibitors, 5- lipoxygenase (5-LO) inhibitors; 5-lipoxygenase activating protein (FLAP) antagonists; dual inhibitors of 5-lipoxygenase (5-LO) and antagonists of platelet activating factor (PAF);

leukotriene antagonists (LTRAs) including antagonists of LTB4 , LTC4 , LTD4, and LTE4; b2- adrenoceptor agonists; cromolyn sodium, theophylline and aminophylhne; inhaled

glucocorticoids, interleukin- 5 (IL-5) inhibiting monoclonal antibodies; and antibodies that inhibit binding of IgE to the high-affinity IgE receptor (e.g., omalizumab).

[00285] PDE4 inhibitors include roflumilast.

[00286] Dual PDE3/PDE4 inhibitors include RPL-554 [9,i0-dimethoxy-2(2,4,6- trimethylphenylimino)-3-(N-carbamoyl-2-aminoethyl)-3,4,6,7-t etrahydro-2H-pyrimido[6,l- a]isoquinolin-4-one] and RPL565 [6,7-dihydro-2-(2,6-diisopropylphenoxy)-9, 10-dimethoxy-4H- pyrimido [6, 1 -a] isoquinolin-4-one]

[00287] 5-LO inhibitors include N-hydroxyureas such as zileuton, ABT-761, fenleuton, Abbott-79175, Abbott-85761 , and SB-210661 , methoxytetrahydropyrans such as ZD-2138, 2- cyanonaphthlanes/2-cyanoqinolines such as L-739,010 and L-746-530.

[00288] FLAP inhibitors include indole-quinolines such as MK-591 , MK-886, and BAY xl 005.

[00289] LTRAs include ablukast, monteluast, ontazolast, and zafirluast.

[00290] Representative 5-LO inhibitors, FLAP inhibitors, and LTRAs are disclosed in U.S. 6,894,041 , which disclosure is incorporated herein by reference.

[00291] p2-adrenoceptor agonists including albuterol, levalbuterol, arformoterol, salbutamol, formoterol, indacaterol, olodaterol, terbutaline, ritodrme, hexoprenaline, metaproterenol, nylidrin, orciprenaline, and salmeterol.

[00292] Inhaled glucocorticoids include flunisolide, triamcinolone acetonide, bee!omethasone dipropionate, budesonide, ciclesonide, fluticasone furoate, fluticasone propionate, mometasone, and mometasone furoate.

[00293] Anticholinergics include glycopyrrolate (e.g., glycopyrronium bromide), ipratropium bromide, aclidinium bromide, tiotropium, and umeclidi um. [00294] Monoclonal antibodies that inhibit IL-5 include benralizumab (an IL-5 receptor inhibitor), mepolizumab (binds IL-5), and reslizumab (binds IL-5).

[00295] In another aspect, the disclosure provides a kit. A kit will include a compound of formula (I) as described herein. A kit may also include instructions for use of the compound of formula (I) or at least one active agent. Instructions included in kits can be affixed to packaging material or can be included as a package insert. While the instructions are typically written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD, DVD), and the like. As used herein, the term "instructions" can include the address of an internet site that provides the instructions.

[00296] In some embodiments, the at least one disclosed compound and the at least one active agent are co-formulated. In some embodiments, the at least one disclosed compound and the at least one active agent are co-packaged. The kits can also comprise compounds and/or products co-packaged, co-formulated, and/or co-delivered with other components. For example, a drug manufacturer, a drug reseller, a physician, a compounding shop, or a pharmacist can provide a kit comprising a disclosed compound and/or product and another component for delivery to a patient.

[00297] That the disclosed kits can be employed in connection with disclosed methods of use.

[00298] The kits may include information, instructions, or both that use of the kit will provide treatment for medical conditions in mammals (particularly humans). The information and instructions may be in the form of words, pictures, or both, and the like. In addition or in the alternative, the kit may include the compound, a composition, or both; and information, instructions, or both, regarding methods of application of compound, or of composition, preferably with the benefit of treating or preventing medical conditions in mammals (e.g., humans).

[00299] It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the compounds and methods of the present disclosure described herein are readily applicable and appreciable, and may be made using suitable equivalents without departing from the scope of the present disclosure or the aspects and embodiments disclosed herein. Having now described the present disclosure in detail, the same will be more clearly understood by reference to the following examples which are merely intended only to illustrate some aspects and embodiments of the disclosure, and should not be viewed as limiting to the scope of the disclosure. The disclosures of all journal references, U.S. patents and publications referred to herein are hereby incorporated by reference in their entireties. tion may be prepared as illustrated in the following schemes and examples.

Abbreviations:

Calcd calculated

DCM dichlorometliane

DMF N,N-dimethyiformamide

DMSO dimethylsulfoxide

EDC 1 -(3 dimethylamiiiopropyl)-3-ethylcarbodiimide methiodide

Et ethyl

ESI-TOF electrospray ionization time-of-flight

EtOAc ethyl acetate

F moc fluorenylmethyloxy carbonyl

h hour

HATU 1 -[Bis(dimethylamino)methylene]-lH- 1 ,2,3-triazolo[4,5-b]pyridinium 3- oxi de hexafluorophosphate

HO At 1 -hydroxy-7-azabenzotriazole

HOBt 1 -hydroxybenzotriazole

HRMS high resolution mass spectrometry

Me methyl

MeCN acetonitrile NHS N-hydroxysuccinimide

Ph phenyl

ppm parts per million psig pounds per square inch pyr pyridine

rt or r.t. room temperature TFA trifluoroacetic acid

Synthesis and characterization of compound 6 and its derivatives

Activated intermediate

(X - activating moiety)

Cmpd-6 and related analogs may be prepared using the procedure shown in Scheme 1 and following general conditions. Reagents and conditions: (a) NHS (X = activating moiety), EDC, DMF, r.t, 12 h (b) M¾, MeCN, r.t, 6 h, 82% (c) Piperidine, DMF, r.t, 3 h, 99% (d) K2CO3, DM, reflux, 42 h, 63%. (e) HOAt, EDC, DCMZDMF, r.t , 24 h, 68%. [00327] Synthesis of p2AR PAMs, as demonstrated for compound 6 (Cmpd-6).

Alternatively, Cmpd-6 and related analogs may be prepared using the procedure shown in Scheme 1 and following general conditions. Reagents and conditions: (a) HOBt (X ^::: activating moiety), EDC, DCM, r.t, 6 h (b) NEb solution (7N methanolic ammonia), DCM, r.t, 6 h, 85%

(c) Piperidine, DMF, r.t, 3 h, 99%. (d) Ullmann-type reaction i.e., synthesis of symmetric biaryls via copper-catalyzed coupling herein using Cul, L-proline, K2CO3, DMF, reflux, 24 h, 85%. fe) HATU/ Hunig’s base (N,N-diisopropylethylamine, DIEA)-assisted amidation: HATH, D1EA, DMF, r.t, 18 h, 75%.

[00328] General chemistry: Starting materials for Cmpd-6 and analog synthesis were purchased from Sigma-Aldrich; St. Louis, MO, Thermo Fisher Scientific; Waltham, MA, Enamine; Monmouth Jet., NJ, Chem-Xmpex; Wood Dale, IL, Combi-Blocks; San Diego, CA, Santa Cruz Biotechnology: Dallas, TX, Toronto Research Chemicals; Toronto, Canada, and TCI America; Portland, OR; and used without further purification.

[00329] Silica gel coated with F254 fluorescent indicator on aluminum plates was used for analytical thin layer chromatography (TLC). The course of reactions was followed by visualization under UV (254 nm or 366 nm) and/or using standard staining procedures such as mnhydrm and KMnCG Compounds may be purified by recrystallization, by manual flash column chromatography (FCC) system using silica gel 60 (Si02; 230-400 mesh, Merck), or

Reveleris® X2 flash chromatography system for purification and Biichi R-300 rotary evaporator

[00330] Chemical and structural characterization of compounds

• ^! H NMR and ^l3 C NMR spectra may be acquired on a FT-NMR Bruker Avance Ultra Shield Spectrometer at 400.13, commonly in deuterated solvents such as DMSO-de and CDCb.

High-resolution time-of -flight mass spectra (HRMS ESI-TOF) may be performed on a W ⁷ aters LCT Premier XE (TOF) using electrospray ionization.

[00331] Synthesis of (9H-Fluoren-9-yl)niethyl(R)-(4-amino-l-(4-(tert-butyl)phenyl )-4- oxobutan~2-y!)~carbamate (2). To an ice-cold stirred solution of Fmoc-(R)-3-amino-4-(4-tert- butylphenyl)butyne acid (250 mg, 0.55 mmol) and N-hydroxysuccinimide (82 mg, 0.71 mmol) in dry DMF ^' (10 mL) are added EDC*HC1 (137 mg, 0.71 mmol) under nitrogen atmosphere. The mixture is allowed to reach room temperature and stirred overnight. The reaction mixture is then concentrated under reduced pressure, and then the residue was diluted with EtOAc (150 mL) and washed with water (3 ^c 50 mL). The organic phase is dried and concentrated. The crude product la is then used for the next step without further purification. Aqueous ammonia solution at 28% (0.77 mL, 1 1 mmol) is added to a solution of la obtained above (305 mg, -0.55 mmol) m MeCN (7 mL) at room temperature. After stirring at room temperature for 2h, the solvent and volatiles are removed in vacuo, and the solid residue is suspended in FLO (80 mL). The resulting mixture is extracted with DCM (3x100 mL), and the combined organic layers are washed with brine (200 mL), dried over Na?.S04 and concentrated m vacuo. The crude product is purified by flash chromatography (EtOAc/petroleum ether: 1 : 1) to give 2 (typical yield: 195 mg, 82% yield over two steps) as a white solid. HRMS (ESI, positive) for C29H33N2Q3+ [M+H] + calcd 457.2486, found 457.2487.

[00332] Synthesis of (R)-3-amino-4-(4-(tert-butyl)phenyl)butanamide (3), To a solution of 2 (167 mg, 0.38 mmol) in DMF (4 mL) is added piperidine (0.8 mL) at rt. After being stirred at rt for 6 h, the mixture is concentrated in vacuo. The crude product is purified by flash

chromatography (eluting with 10: 1 CFECh/MeOH and 10: 1 :0.1 CH?.Cl2/MeOH' ^' Et3N) to afford 3 (typical yield: 89 mg, 99% yield) as white solid. HRMS (ESI, positive) for C14H23N2O+ [M+H]

+ calcd 235.1805, found 235.1806.

[00333] Synthesis of 5-(N-Isopropy!-N-methylsuIfamoyl)-2-((4- methoxyphenyl)thio)benzoic add (5), To a solution of 2-fluoro-5-[[methyl(l -methylethyl) amino]sulfonyl]-benzoic acid (550 mg, 2 mmol) in DMF (20 mL) is added K2CO3 (662 mg, 4.8 mmol), and followed by 4-methoxybenzenethiol (0.25 mL, 2 mmol), and then the mixture is heated at reflux for 42 h. After the reaction is complete, the solvents are removed in vacuo. The crude product is poured into H2O (200 mL), and acidified with aqueous 2 M HC1 to adjust pH to 3-4. The resulting mixture was extracted with DCM (3x 150 mL), and the combined organic layers are washed with brine (2x100 mL), dried over Na2SC>4, and concentrated in vacuo. The crude product is purified via flash chromatography (CtLCk/MeOH: 10: 1) to afford 5 (typical yield: 500 mg, 63% yield) as a white solid. HRMS (ESI, negative) for C18H20NO5S2- [M~H]+ caicd 394.0788, found 394.0787. [00334] Synthesis of (R)-N-(4-Amino-l-(4-(tert-butyl)phenyl)-4-oxobutan-2-yl)-5-( N- isopropyl-N-methylsuIfamoyl)-2-((4-methoxyphenyl)thio)benzam ide (Compound 6). To a stirred mixture of 3 (77 mg, 0.33 mmol) and 5 (130 mg, 0.33 mmol) in DCM/DMF (10: 1 v/v, 10 mL) is added EDC*HC1 (62 mg, 0.33 mmol) and HO At (44 mg, 0.33 mmol), and the mixture is stirred at rt for 24 h. After the solvent is removed in vacuo, the crude product is purified by flash chromatography (EtOAc/petroleum ether, 1 : 1) to afford 6 (typical yield: 136 mg, 68% yield) as white solid. HRMS (ESI, positive) for C32H4iN3NaOsS2+ [M+Na] + calcd 634.2380, found 634.2381

[00335] Synthesis of N-isopropyl-4-((4-methoxyphenyI)thio)-N-methyl-3-(piperidine - 1 carbonyl)benzenesulfonamide (Compound A3). The title compound is typically prepared in a manner analogous to compound 6 at step 5. Except in this step in scheme 1, the secondary amine-piperidine (65 mg, 0.6 mmol) with 5 (200 mg, 0.5 mmol) is used instead of 3. The compound is purified as white solid (typical yield: 80%). ESI-MS (positive mode): m/z 463.1729 [M + H] + and m/z 485 1544 [M + Na] +

[00336] Synthesis of (R)-N-(4-amino-l-(4-(tert-butyl)phenyl)-4-oxobutan-2-yl)-5-( N- isopropyI-N-methylsulfamoyl)-2-((4-(trifluoromethoxy)phenyl) thio)benzainide (Compound A4). The title compound, bearing a 4-OCF ₃ group instead of a 4-OCH _{3 i} s prepared in an analogous manner, except in the fourth step in scheme 1, the intermediate 4- (trifluoromethoxy)benzenethiol (351 mg, 1.8 mmol) with 4 (500 mg, 1.8 mmol) is used instead of 4-methoxybenzenethiol. The compound is purified as white solid (typical yield: 70%). ESI- MS (positive mode): m/z 666.2299 [M + H] + and m/z 688.2115 [M + Na] +.

[00337] Synthesis of (R)-N-(4-amino-l-(4-(tert-butyl)phenyl)-4-oxobutan-2-yl)-5-( N- isopropyl-N-methylsulfamoyl)-2-((3-methoxyphenyl)thio)benzam ide (Compound A5). The title compound, bearing a meta-OCl-fi instead of a para-OCHs is prepared in an analogous manner, except in the fourth step in scheme 1, the intermediate 3-methoxybenzenethio! (254 mg, 1.8 mmol) with 4 (500 mg, 1.8 mmol) is used instead of 4-methoxybenzenethiol. The compound is purified as white solid (typical yield: 75%). ESI-MS (positive mode): m/z 612.2562 [M + H] + and m/z 634.2375 [M + Na] +. [00338] Synthesis of (5-(N-isopropyl-N-methylsulfanioyl)-2-((4- methoxyphenyl)thio)benzoyl)-D-tyrosine (Compound A6). The title compound, bearing a 4 OH on the benzene ring instead of tert-butyl with a carboxyl group on the right end of the molecule was prepared in a manner analogous to Compound 6, except m step 5 of scheme 1, the intermediate D-Tyrosme tert-butyl ester (72 mg, 0.3 mmol) with 5 (100 mg, 0.3 mmol) was used instead of 3. Following trifluoroacetic acid (TF A) -mediated removal of the tert-butyl group (DCM, 2 mL; TFA, 1 mL; 12 h; RT), the product was concentrated in vacuo and purified by flash chromatography using a gradient and mixture of solvents (EtOAc-DCM and DCM- MeOH), to afford the product as white solid (60% yield). ESI-MS (positive mode): m/z 559. 1572 [M + H] + and m/z 581.1375 [M + Na] +.

[00339] Synthesis of (R)-N-( 1-amino- l-oxo-3-pheny!propan-2-yl)-5-(N-isopropyl-N- methylsulfamoyl)-2-((4-methoxyphenyl)thio)benzamide (Compound A7), The title compound, primarily lacking a tert-butyl group was prepared in a manner analogous to

Compound 6, except in step 5 of scheme 1, the intermediate (R)~2~amino-3-phenylpropanamide (78 rng, 0.3 mmol) with 5 (100 mg, 0.3 mmol) was used instead of 3. The compound was purified as white solid (80 % yield). ESI-MS (positive mode): m/z 542 1774 [M + H] + and m/z 564.1581 [M + Na] +.

[0034Q] Synthesis of (R)-N~(4~Amino-l~(4~(tert~foutyi)phenyI)-4-oxofoutan~ 2-yl)-5-(N-(3- fluorophenyI)sulfamoyl)-2-((4-methoxyphenyl)thio)benzamide (Compound 43) The title compound, bearing the fluorobenzene moiety instead of isopropyl at the sulfonamide linkage, is prepared in a manner analogous to Compound 6, except step 5 of scheme 1, the intermediate 5- (N-(3-Fiuorophenyl)suifamoyi)-2-((4-methoxyphenyi) thio) benzoic acid (52 mg, 0.12 mmol) with 3 (28 mg, 0.12 mmol) was used instead of 5. The compound is purified as white solid (typical yield: 44 mg, 56%). FIRMS (ESI, negative) for C34H37FN3O5S2T [M-HJ+ calcd

650.2153, found 650.2153.

[00341] The compounds and intermediates may be isolated and purified by methods well- known to those skilled in the art of organic synthesis. Examples of conventional methods for isolating and purifying compounds can include, but are not limited to, chromatography on solid supports such as silica gel, alumina, or silica derivatized with alkylsilane groups, by recrystallization at high or low temperature with an optional pretreatment with activated carbon, thin-layer chromatography, distillation at various pressures, sublimation under vacuum, and trituration, as described for instance in“ Vogel's Textbook of Practical Organic Chemistry,” 5th edition (1989), by Fumiss, Hannaford, Smith, and Tatchell, pub. Longman Scientific &

Technical, Essex CM20 2JE, England.

[00342] A disclosed compound may have at least one basic nitrogen whereby the compound can be treated with an acid to form a desired salt. For example, a compound may be reacted with an acid at or above room temperature to provide the desired salt, which is deposited, and collected by filtration after cooling. Examples of acids suitable for the reaction include, but are not limited to tartaric acid, lactic acid, succinic acid, as well as mandelic, atrolactic,

methanesulfonic, ethanesulfonic, toluenesulfonic, naphthalenesulfonic, benzenesulfonic, carbonic, fumaric, maleic, gluconic, acetic, propionic, salicylic, hydrochloric, hydrobromic, phosphoric, sulfuric, citric, hydroxybutyric, camphorsuifome, malic, phenylacetic, aspartic, or glutamic acid, and the like.

[00343] Reaction conditions and reaction times for each individual step can vary depending on the particular reactants employed and substituents present in the reactants used. Specific procedures are provided in the Examples section. Reactions can be worked up in the

conventional manner, e.g. by eliminating the solvent from the residue and further purified according to methodologies generally known m the art such as, but not limited to, crystallization, distillation, extraction, trituration and chromatography . Unless otherwise described, the starting materials and reagents are either commercially available or can be prepared by one skilled in the art from commercially available materials using methods described in the chemical literature. Starting materials, if not commercially available, can be prepared by procedures selected from standard organic chemical techniques, techniques that are analogous to the synthesis of known, structurally similar compounds, or techniques that are analogous to the above described schemes or the procedures described in the synthetic examples section.

[00344] Routine experimentations, including appropriate manipulation of the reaction conditions, reagents and sequence of the synthetic route, protection of any chemical functionality that cannot be compatible with the reaction conditions, and deprotection at a suitable point in the

/ .} reaction sequence of the method are included m the scope of the invention. Suitable protecting groups and the methods for protecting and deprotectmg different substituents using such suitable protecting groups are well known to those skilled m the art: examples of which can be found m PGM Writs and TW Greene, in Greene’s book titled Protective Groups in Organic Synthesis (4 ^th ed.), John Wiley & Sons, NY (2006), which is incorporated herein by reference in its entirety'. Synthesis of the compounds of the invention can be accomplished by methods analogous to those described in the synthetic schemes described hereinabove and in specific examples.

[00345] When an optically active form of a disclosed compound is required, it can be obtained by carrying out one of the procedures described herein using an optically active starting material (prepared, for example, by asymmetric induction of a suitable reaction step), or by resolution of a mixture of the stereoisomers of the compound or intermediates using a standard procedure (such as chromatographic separation, recrystallization or enzymatic resolution).

[00346] Similarly, when a pure geometric isomer of a compound is required, it can be obtained by carrying out one of the above procedures using a pure geometri c isomer as a starting material, or by resolution of a mixture of the geometric isomers of the compound or

intermediates using a standard procedure such as chromatographic separation.

[00347] Using procedures analogous to the foregoing schemes and examples, additional compounds of formula (I) may be prepared, including those in FIG. 14G. As can be appreciated by the skilled artisan, further methods of synthesizing the compounds of the formulae herein will be evident to those of ordinary skill in the art. Additionally, the various synthetic steps may be performed m an alternate sequence or order to give the desired compounds. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the compounds described herein are known in the art and include, for example, those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and P.G.M. Wuts, Protective Groups m Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents or Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof. [00348] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

[00349] Molecular Biology For construction of pcDNA-Zeo-tetO, an Ndel-Xhol fragment from pACMV-tetO (Reeves PJ, et al. (2002) P roc Natl Acad Sci U SA 99(21): 13413-13418.), which contained tet operator sequences inserted into the CMV promoter, was ligated into the Ndel/XhoI-digested pcDNA3. l-Zeo backbone. All p2-adrenergic receptor (b2A11) constructs were of human origin and contained an N-terminal FLAG-tag and a C-terminal 6xHis-tag. For P2AR constructs used in sortase ligations, the sortase consensus site (LPETGHH) was inserted after ammo acid 365 (p2AR-LPETGHH). The minimal cysteine 62 A R (b2AKD4) used m bimane fluorescence experiments and the minimal ICL3 mutant (p2ARA238-267) were designed as previously described (Yao XJ, et al. (2009) Proc Natl Acad Sci U SA 106(23):9501-9506., Kumari P, et al. (2016) Nat Commun 7: 13416.). All p2AR constructs expressed in Sf9 insect ceils contained the N187E glycosyiation mutation. Human Muscarinic-2 Receptor (M2R) and Mu (p)-Opioid Receptor (MOR) were cloned into pcDNA-Zeo-tetO with an N-terminal FLAG- tag and C-terminal sortase consensus site followed by a 6xHis-tag. To enhance stability and expression, a minimal cysteine (C59A, C125S, CHOI, Cl 50V, C242V, C251V, and C269S) and truncated (after amino acid 393) variant of rat bapΊ (Parrl-MC-393) in pGEX4T was generated. The D62-77 finger loop deletion and the V70C mutation, corresponding to V74C of visual arrestin (Hanson SM, et al. (2006) Proc Natl Acad Sci U SA l03(!3):4900-4905.), were introduced in pGEX4T- arrl~MC-393.

[0035Q] p2AR Expression and Purification: With the exception of b2AKD238-267, all 2AR constructs were expressed in Sf9 insect cells using the BestBae Baculovirus Expression System (Expression Systems). Cells were infected at a density of 3 ^c 106 cells/mL and harvested 60 h thereafter. Receptor was solubilized in n-Dodecyl-fi-D-Maitopyranoside (DDM) (Anatrace) and purified using FLAG-MI and alprenoloi-affimty chromatography as previously- described (Kobilka BK (1995) Anal Biochern 231 (1 ):269-271.). b2A D238-267 in pcDNA-Zeo- tetO was transfected into Expi293F cells (Invitrogen) stably integrated with the plasmid pcDNA/TR (Invitrogen) to express the tetracycline repressor (Expi293F-TR). Cells were transfected using Expifectamine (Invitrogen) as described m the manufacturer’s protocol with receptor expression being induced 48 h post-transfection with 4 pg/niL doxycycline, 5 mM sodium butyrate, and 1 mM of the b2AK antagonist alprenolol. Cultures were harvested 30-36 h thereafter, and all purification steps conducted at 4 °C with protease inhibitors (benzamidine and leupeptm) unless stated otherwise. Cell pellets were resuspended (10 mL/g wet cell pellet mass) in room temperature lysis buffer (10 mM TRIS, pH 7.4, 2 mM EDTA, 10 mM MgC12, 5 units/mL benzonase (Sigma) and 2 mg/ml iodoacetamide) with 1 mM alprenolol for 20 min. Membrane was pelleted at 30,000 x g for 20 min and resuspended in 10 mL/g original cell pellet mass of solubilization buffer (20 mM HEPES, pH 7.4, 100 rnM NaCl, 1% DDM, 0.05% cholesterol hemisuccinate (CHS), 10 mM MgC!2, 5 units/mL benzonase, and 2 mg/mL iodoacetamide) with 1 mM alprenolol. After extensive dounce homogenization, solubilizing membrane was sequentially stirred at room temperature and then 4 °C for 1 h each. Insoluble material was removed by centrifugation at 30,000 x g for 30 min, supernatant loaded onto Ml- FLAG resin with 2 rnM CaC32 at 1-3 mL/'min, and resin washed with 20 column volumes of wash buffer (20 rnM HEPES, pH 7.4, lOOtnM NaCl, 0.3% DDM, 0 01 % CHS, and 2 mM CaC12). Receptor was eluted in elution buffer (20 rnM HEPES, pH 7.4, 100 mM NaCl, 0.1% DDM, 0.01 % CHS, 0.2 mg/ml, FLAG-peptide, and 5 mM EDTA), and monomeric receptor was collected by size exclusion chromatography on a Superdex 200 Increase column (GE Healthcare Life Sciences).

[00351] M2R and MOR Expression and Purification: The M2R and the MOR were expressed in and purified from Expi293F-TR cells as described above with the following modifications. The antagonists atropine (1 mM) and naloxone (1 mM) were included during expression and purification for the M2R and the MOR, respectively. For M2R solubilization, 10% glycerol was added, and NaCl was increased to 750 mM based on previous studies (Kruse AC, et ai (2013) Nature 504(7478): 101 -106). Additionally, Ml -FLAG resin was washed with 5 column volumes of high (750 mM) and low (100 mM) NaCl-containing wash buffer at ratios of 4:0, 3: 1, 2:2, 1 :3, and 0:4, respectively. In addition to DDM, MOR solubilization buffer contained 0.3% 3-[(3-cholamidopropyl) dimethylammonioj-l-propanesulfonate (CHAPS) (Manglik A, et ah (2012) Nature 485(7398): 321 -326).

[00352] G protein, parrl, and Nb80 Purification: Heterotrimeric G protein was purified as previously described (Gregorio GG, et al (2017) Nature 547(7661):68-73). In brief,

Trichoplusia ni HigliFive insect cells were infected with two viruses made from BestBac baculovirus system, the first expressing both human G l --His6 and Gy2, and the second God or the short Gas splice variant. Cells were harvested 48 h post-infection. Heterotrimeric Gs or Gi was purified from solubilized cell membranes using Ni-NTA chromatography and HiTrap Q sepharose anion exchange (GE Healthcare Life Sciences). Purification of parrl was conducted as previously described (Nobles KN, et al. (2007) J Biol Chem 282(29): 21370-21381.). In short, GST- arrl -MC-393 was expressed in BL21(DE3) bacteria, lysed using a microfluidizer, and captured using glutathione sepharose. bApT was removed from GST by thrombin digestion and further purified using HiTrap Q sepharose anion exchange. Nb80 was purified as previously described (Staus DP, et al. (2016) Nature 535(7612):448-452).

[00353] High-Density Lipoprotein (HDL) Reconstitution: Receptor reconstitution into HDL particles was conducted as described elsewhere (Staus DP, et ah (2016) Nature

535(7612):448-452). In short, DDM-solubilized receptor (2 mM) was incubated for 1 h at 4 °C with 80 mM Apolipoprotein Al (MSPlDl) and a 3:2 molar ratio of 8 niM phosphatidylcholine (POPC) with phosphatidylglycerol (POPG). Bio-beads (Bio-Rad) were added (0.5 mg/pL reconstitution volume) thereafter and rotated overnight at 4 °C. HDL~reeeptor was isolated from non-receptor-containing HDL particles using Ml -FLAG chromatography.

[00354] Sortase Ligation Reactions: All sortase reactions were conducted in buffer containing 20 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% DDM, 0.01% CHS, and 5 rnM CaC12. Detergent-solubilized receptor (10 mM) was incubated with GGG-V2Rpp (GGG- ARGRpTPPpSLGPQDEpSCpTpTApSpSpSLAKDTSS) (50 mM) (lower-case

“p” ^:=: phosphorylation) or non-phosphorylated GGG-V2R, and 2 m.M SortaseA containing five mutations that increase ligation efficiency, as described previously (Chen I, et al. (201 1 ) Proc Nad Acad Set USA 108(28): 1 1399-1 1404.). Ligations were incubated overnight at 4 °C, and unligated receptor (containing C-terminal 6xHis-tag) was removed using Talon resin (Invitrogen). Size exclusion chromatography was utilized to specifically isolate monomeric ligated receptor.

[00355] Bimaee Fluorescence: For bimane labeling of proteins, alprenoloi-pure b2 A R, P2AR-LPETGGH or ion-exchange-pure Parrl -MC- 393 V70C was incubated with 100 mM TCEP at 4 °C for 15 mm, then with a 3 -fold excess of monobromobimane (Sigma) at 4 °C overnight.

An additional 3-fold molar excess of monobromobimane was added the next day, and the reaction was allowed to continue for 1 h at room temperature before quenching with excess L- cysteme. Excess label was removed by size exclusion chromatography. Bimane-labeled j3arrl was concentrated and flash frozen with 15% glycerol. For bimane- labeled receptors,

phosphopeptide ligation (P2AR-LPETGGH-bimane) and HDL reconstitution (p2AR-himane and p2ARpp-bimane) were carried out as described above.

[00356] Bimane-labeled P2AR or [32ARpp HDLs were incubated with or without

isoproterenol for 15 min at room temperature before the addition of excess transducer. Final concentrations were 250 nM HDL, 10 mM isoproterenol, 500 nM Gs (+5 mM MgCl2), 1 mM parr 1 -393 minimal cysteine or Parr 1-393 minimal cysteine D62-77 in buffer comprised of 20 mM HEPES, pH 7.4, 100 mM NaCl. The reactions were equilibrated for at least 30 min in black, solid-bottom 96-well microplates before fluorescence emission spectra were collected on a CLARIOstar plate reader (BMG LABTECH) in top-read mode, with excitation at 370 nm (16 nm bandpass) and emission scanning from 400 nm to 600 nm (10 nrn bandpass) in 1 nm increments. Reactions were set up in duplicate in each experiment, and wells for background subtraction contained all components except the bimane-labeled HDLs. Experiments were repeated at least three times.

[00357] For experiments with Parrl -bimane, HDL-receptors were incubated with ligands (and for p2ARpp, Nb80) for 15 min at room temperature. bApΊ -bimane was added to each well, and the reactions were equilibrated for at least 30 min before being read as described above. Final concentrations were 375 nM HDLs, 10 mM ligand, 500 nM Nb80, and 250 nM Parrl -bimane. Reactions were set up in duplicate in each experiment, and wells for background subtraction contained ail components except Parrl -bimane. To normalize data, the area under each averaged, background-subtracted curve between 425 nm and 600 nm was calculated (GraphPad Prism), and areas were normalized to the maximum value (M2Rpp plus iperoxo) in each experiment. Experiments were repeated at least three times.

[00358] Receptor-Transducer Co-immunoprecipitation: Since b2AK and |3arrl are of similar molecular weight, we used a b2AK construct with T4-lysozyme (T4L) fused to the receptor’s N terminus to obtain separation by SDS-PAGE (Zou Y, et al. (2012) PLoS One 7(10):e46Q39.). Assay and wash buffer consisted of 20 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% DDM, and 2 mM CaC12. Unligated or phosphopeptide-ligated FLAG-T4L-p2AR in DDM were mixed with 10 mM isoproterenol and stoichiometric amounts of heterotrimeric G protein or arrl -MC-393. The antibody fragment Fab30 was added to J3arrl to stabilize the interaction with the phosphorylated receptor C terminus (Shukla A_K, et al. (2013) Nature 497(7447): 137- 141.). After reactions were incubated at room temperature for 1 h, FLAG-T4L- 2AR was immunoprecipitated using FLAG-MI resin, and transducers were eluted by the addition of 1 mg/mL FLAG peptide and 10 mM EDTA.

[00359] Radioligand Binding: All equilibrium competition radioligand binding studies were conducted in a final volume of 200 pL containing HDL-receptor, radioligand, a titration of unlabeled competitor, and the presence or absence of the indicated transducer protein. All components were diluted in assay buffer containing 20 rnM HEPES, pH 7.4, 100 mM NaCl, and 1 mg/mL bovine serum albumin (BSA). Radioligands used in [52AR, M2R, and MOR competition binding experiments were [125I]-Cyanopindolol (GYP; 60 pM), [3H]-N-Methyl- Scopolamine (NMS; 1 nM), and [3H]-Naloxone (2.5 nM), respectively. G protein or arrl were used at a final concentration of 100 nM or 1 mM, respectively, unless specified otherwise.

Binding reactions proceeded at room temperature and w¾re harvested onto glass-fiber filters (GFB) with 0.3% polyethyleneimine (PEI) using a 96- well Brandel harvester. Binding data w¾re analyzed in GraphPad Prism using a sigmoidal dose-response curve fit, and differences m log IC50 values were analyzed by one-way ANOVA.

[00360] G protein GTPase Assay: The GTPase activity of Gas or God was measured in vitro using the GTPase Glo Assay (Promega) with the following modifications. Final reaction buffer consisted of 20 mM HEPES, pH 7.4, 100 mM NaCl, 10 mM MgC12, and 1 mg/mL BSA. HDL- 2AR (4 nM), -M2R (100 nM), or MOR (100 nM) was incubated in the absence or presence of the indicated agonist (5 mM) and parrl (1 mM) for 15 min at room temperature. The antibody fragment Fab30 (1 mM) was included with Parrl to stabilize its interaction with the ligated receptor C terminus (Shukla AK, et al. (2013) Nature 497(7447): 137-141.). G protein (500 nM) and GTP (2.5 mM) were subsequently added, and reactions proceeded for 1 h at room

temperature before addition of GTPase Glo reagent and ADP, as described in the manufacturer’s protocol.

EXAMPLE 1

[00361 This example demonstrates that the binding interaction of arr with the p2-adrenergic receptor (b2AK).

[00362] The binding of Parr to GPCRs is mainly initiated through an interaction with the phosphorylated receptor C terminus, and conformational changes induced in Parr by this interaction promote coupling to the receptor I'M core, as shown in FIG.1. Co- immunoprecipitation experiments confirmed that heterotrimeric Gs protein, but not parrl, can interact with purified non-phosphorylated P2-adrenergic receptor (P2AR), as shown in FIG. 2A.

[00363] To verify that this apparent lack of interaction with Parr is not simply due to poor complex stability', two assays capable of detecting complex formation in situ were performed. First, competition radioligand binding was used to measure the allosteric effects of transducers on ligand binding to the receptor. As described by the ternary complex model, first for G proteins and later for parrs, ligand-induced changes in receptor conformation enhance the binding and affinity of transducers, which reciprocally increase ligand affinity by stabilizing an active receptor state (De Lean A, et al. (1980) J Biol Chem 255(15): 7108-71 17., Gurevich W, et al. (1997) J Biol Chem 272(46):28849-28852.). When wild-type (WT) p2AR was reconstituted in high-density lipoprotein (HDL) particles to mimic a cellular membrane environment (Denisov IG & Sligar SG (2016) Nat Struct Mol Biol 23(6):48! -486.), G protein enhanced the affinity of the full agonist isoproterenol for non-phosphorylated HDL-p2AR by nearly 1000-fold, as expected, but Parrl had no effect even at micromolar concentrations, as shown in FIG. 2B.

[00364] Second, to directly monitor (32AR conformational changes associated with activation, the C265 at the cytoplasmic end of TM6 was labeled with monobromobimane, an environmentally sensitive fluorophore. Receptor activation leads to an outward movement of TM6 that places the bimane label in a more solvent-exposed position, causing a decrease in fluorescence and a shift in Amax (Yao XJ, et al (2009) P roc Nat! Acad Sci USA 106(23):9501- 9506.). Indeed, isoproterenol reduced P2AR-bimane fluorescence compared to control (DMSO), and addition of Gs but not b3pΊ further attenuated fluorescence, as shown in FIG. 2C.

[00365] The results of this example demonstrate that non-phosphorylated b2AP fails to form a productive interaction with arr.

EXAMPLE 2

[00366J This example describes the preparation of a complex comprising a chimeric GPCR and parrl in accordance with the present disclosure.

100367] Phosphory lation of the (fcAR was induced using the prokaryotic enzyme sortase to ligate a synthetic phosphorylated peptide onto the receptor C terminus (Fig. 3A and 7A). This strategy quantitatively yielded receptor with a defined, homogeneous phosphorylation pattern, which is difficult to achieve or validate with either in cellulo or in vitro GRK phosphorylation. Briefly, a phosphopeptide (pp) derived from the C terminus of the vasopressm-2-receptor (V ₂ R) was ligated to the C -terminus of b2AP, based on previous crystallographic and biophysical data indicating that VaRpp binds to arr with high affinity and effectively primes it for interaction with GPCRs’ TM core (Shukla AK, et al. (2013) Nature 497(7447): 137-141.). In contrast to wild-type (WT) b ₂ AE (Fig. 2A), phosphorylated fuAR (b2AErr) immunopreclpitated both Gs and P rr 1 (Fig. 3B). bApΊ enhanced isoproterenol affinity for the p ₂ ARpp by 9-fold compared to 800-fold by Gs (Fig. 3C and 8A). However, as for Gs, b pT did not increase the binding of the antagonist ICI-118,551 (Fig. 8B). The parr 1 -mediated increase in agonist affinity required phosphorylation of pcARpp since ligation of a non-phosphorylated V2R peptide or phosphatase treatment abrogated Parri’s allosteric effect (Fig. 3D and 8C).

[00368] While parrl augmented isoproterenol’s decrease in the fluorescence of p ₂ ARpp- bimane, its effects were less profound than those of G protein (Fig. 3E), consistent with the ~l 00-fold difference in the cooperativity between G protein and parrl observed by radioligand binding (Fig. 3C). These findings suggest that despite binding to a similar pocket, G protein and arr differ substantially in the strength of then allosteric interactions with the b2AK TM core.

[00369] The above system allowed for rigorous assessment of the contributions of specific regions within each protein that have been implicated in mediating the TM eore/parr interaction. For example, the“finger loop” region of parrl is extended upon parr’s binding to phosphoiylated receptors and is believed to insert into the TM core. This region has been reported to be essential for an engaged conformation of Parrl with the TM core of in cellulo phosphorylated P?.AR, as assessed by negative stain electron microscopy using a parrl finger loop-deleted mutant (Cahill TJ, 3rd, et ai. (2017) Proc Natl Acad Sci USA 1 14( 10): 2562-2567). This same mutant,

Parrl D62-77, failed to stabilize an active state of p ₂ ARpp by competition radioligand binding (Fig. 4A) and bimane fluorescence (Fig. 4B), consistent with previous findings.

[00370] It has been suggested that intracellular loop three (ICL3) of the pvAR is critical for engagement of parrl with the TM core (Kumari P, et al. (2016) Nat Commun 7: 13416.). The phosphopeptide-ligated version of a previously reported deletion mutant, p2ARppA238-267, retained a normal affinity for the agonist isoproterenol when reconstituted in HDL particles (Fig. 4C). Surprisingly, agonist affinity increased approximately 10-fold in the presence of parrl (Fig 4C), which was comparable to parrl’s effect on WT paARpp (Fig. 3C).

[00371] The results of this example demonstrate the generation of a receptor complex that exhibits a homogeneous phosphorylation pattern, and that the finger loop of Parr, but not the p2AR ICL3, is required for the TM core interaction.

EXAMPLE 3

[00372] This example describes the preparation of a complex comprising a chimeric muscarinic acetylcholine receptor 2 (M2R) or m-opioid receptor (MOR) and parrl in accordance with the present disclosure.

[00373] For the paAR, the TM core’s allosteric communication with G protein is substantially stronger than it is with parr. To determine if this is a conserved phenomenon among other GPCRs, the allosteric coupling of G protein and parr at the muscarinic acetylcholine receptor 2 (M2R) and m-opioid receptor (MOR) was investigated. Using the sortase ligation strategy described in Example 2 for the p ₂ AR, the V ₂ Rpp was ligated onto the C termini of purified M ₂ R (M2Rpp) and MOR (MORpp) (Fig. 9A), the receptors were reconstituted into HDL particles, and the allosteric coupling of their cognate G protein (Gi heterotrimer) and Parr was measured using competition radioligand binding. Competitor ligands that were full agonists and exhibited similar affinities for their respective receptors as isoproterenol does for the p ₂ AR were selected. As observed for the b ₂ AK, G protein induced more than a 100-fold increase in agonist affinity for both M?Rpp (earbachoi, Fig 5A) and MORpp (DAMGO, Fig. 5B), consistent with previous reports (Kruse AC, et al (2013) Nature 504(7478): 101 -106, Huang W, et al. (2015) Nature 524(7565):315-321). bApΊ enhanced agonist affinity for both M ₂ Rpp and MORpp in a phosphorylation-dependent manner (Fig. 5 A, 5B, 9B, and 9C), but {3arrl increased earbachoi affinity for M ₂ Rpp by 57-fold compared to only 2- and 9-fold for MORpp and B.-ARpp.

respectively (Fig. 5 A, 5B, and 9D). A summary of transducer allosteric binding at each receptor is shown in Figure 5C, which shows a 100-fold difference between G protein and parr effects on agonist affinity for the p ₂ ARpp and MORpp but less than a 3 -fold difference with M ₂ Rpp. The comparable effects of G protein and parr at the M ₂ Rpp were not carbachol-specific but were also observed with the agonist iperoxo (Fig. 9E and 9F). pArrl’s effects at the M ₂ Rpp also appeared to be dependent on the transmembrane core interaction, as deletion of the finger loop eliminated its allosteric coupling (Fig 9G).

[00374] The results of the example demonstrate that, even for GPCRs which preferentially couple to the same G protein isoform as the p ₂ AR, such as the M2R and the MOR, allosteric communication with G protein does not always vary proportionally to allosteric communication with Parr.

EXAMPLE 4

[00375] This example demonstrates the function and stability of the chimeric receptor complexes described herein

[00376] The ternary complex model posits that the observed enhancement of agonist affinity in the presence of parr must be reciprocated by an equivalent increase in Parr’s affinity for the receptor transmembrane core (De Lean A, et al. (1980) J Biol Chem 255(15): 7108-7117) Thus, the strength of arr engagement with the receptor core would be expected to follow the same rank order of allosteric cooperativity among the three receptors tested. The degree of Parrl engagement was assessed by site-specifically labeling its finger loop with monobromobimane (Parr 1 -bimane), as coupling to a receptor’s TM core results in an increase in fluorescence due to reduced solvent exposure of the label (Sommer ME, et al (2007) J Biol Chem 282(35):25560- 25568, Hanson SM et al. (2006) Proc Natl Acad Sci USA l03(13):4900-4905). As expected, Parr 1 -bimane fluorescence increased for p ₂ ARpp stimulated with isoproterenol compared to the antagonist 10-118,551 (Fig. 6A and Fig. 10A) Importantly, the single domain antibody Nb80, which binds to agonist-activated p ₂ AR in the same region as G protein, competitively blocked the agonist-induced increase in fluorescence (Fig. 6A). These results confirm that the agonist effects on parrl -bimane are indeed mediated through interaction with p ₂ ARpp’s TM core.

Comparison of parr 1 -bimane’s response to agonist stimulation of p ₂ ARpp, M ₂ Rpp, and MORpp showed that M ₂ Rpp displayed the highest level of agonist-induced Parr! -bimane engagement (Fig. 6B and Fig. 10A-C), consistent with the observed allosteric cooperativities of these receptors.

[00377] One mechanism by which parr desensitizes receptors’ activation of G protein signaling is steric occlusion of the TM receptor core. It was hypothesized that Parr might more efficiently desensitize GPCRs such as the M ₂ R - those for which parr has similar allosteric binding properties as G protein - compared to GPCRs with very divergent transducer coupling, such as the p ₂ AR and the MOR. To test this, an in vitro GTPase activity assay was performed that can quantitatively measure agonist-induced receptor activation of G protein. As shown in Figure 6C, addition of isoproterenol to the p ₂ AR enhanced G protein activation as measured by an increase in GTP hydrolysis, which was blocked by competitive binding of Nb80. A similar agonist-induced increase in GTPase activity was observed for both M ₂ Rpp and MORpp (Fig.

11). Desensitization, or inhibition of GTP hydrolysis, by parr was significantly elevated for M2Rpp compared to MORpp and b ₂ A1Trr (Fig. 6D and Fig. 1 1).

[00378] The results of this example demonstrate that that the efficiency of Parr-mediated receptor desensitization in vitro correlates with the strength of the receptor’s allosteric interaction with Parr relative to G protein. EXAMPLE 5

[00379] Sortase-ligated bcAHrr for DNA-encoded Library Screen. Purified human B?AR or p ₂ ARpp was reconstituted in detergent-free high density lipoprotein (HDL) particles. HDL reconstitution was performed using a biotinylated version of the membrane scaffolding protein ApoAl . In addition to providing the receptor with a native-like membrane environment, the biotinylated HDL particles provide an excellent immobilization scheme that avoids any perturbations at the receptor during the screening process. Receptors containing biotinylated- HDLs can be efficiently captured on NeutrAvdm beads. For positive selections (rounds 1 and 2), HDLs were incubated with a molar excess of appropriate transducer proteins, BI-167107, and Neutravidin beads to initiate complex formation and immobilize the complexes. For complexes with Gs, p2.AR HDLs were incubated with His-tagged heterotrimeric Gs and Nb35 (Rasmussen et al, Nature 201 1 , 477, 549-555). For complexes with b-arrestin, ?.ARpp HDLs were incubated with b-arrestinl and His-tagged Nb25. The beads were transferred to a column and washed to remove unbound proteins. Stability results are shown in Fig. 12.

[00380] DNA-encoded Library Test Screen. A test library of approximately 10 ¹⁴ DNA- tagged small molecules was applied to the beads, incubated at room temperature, and then washed extensively. Specifically bound molecules w¾re eluted twice by applying buffer containing 10% Fos-choline to the beads and incubating at 37°C and then 72.5°C. The number of recovered compounds w¾s determined by qPCR on an aliquot of the combined eluates.

Compounds w¾re purified from the eiuate using a Qiagen Nucleotide Removal Kit before beginning the next round of selection. For counter selection (round 3), complexes were formed in the presence of NiNTA beads, and unbound molecules were recovered and analyzed. Results of the test screen are shown in Fig. 13.

EXAMPLE 6

[00381] Screening and identification of primary b2 PAM hits. Using a recently developed approach for screening DNA-encoded small molecule libraries (DELs) against GPCRs, over 500 million unique DNA-encoded small molecules were screened to obtain positive allosteric modulators (PAMs) at the 2-adrenergic receptor (b2AK.). In order to increase chances to get PAMs, the orthosteric site of the receptor was occupied by a high affinity b-agonist BI- 167107, which shifted the b?.AK population toward active conformations (Fig. 14A). Further, purified human b ₂ A1¾5 were reconstituted in detergent-free high density lipoprotein (HDL) particles (FIGs. 14A and 14B). HDL reconstitution was performed using a biotinylated version of the membrane scaffolding protein ApoAl. In addition to providing the receptor with a native-like membrane environment, the biotinylated HDL particles provide an excellent immobilization scheme that avoids any perturbations at the receptor during the screening process. Receptor containing biotinylated-HDLs can be efficiently captured on NeutrAvdin beads (FIG. 14B), and have a comparable affinity for antagonist binding to that of b2AKd in membrane preparations (FIG. 14C). By competitive radioligand binding assays, the b2AK8 in HDL particles can functionally couple to heterotrimeric Gs (FIG. 14D).

[00382] Using the BI-167l07-occupied p ₂ AR in HDL particles, four different DLLs were screened, each of which comprised more than 100 million unique compounds, to isolate molecules specifically binding to the active state of the receptor. The total number of molecules in each library was 0.5-lxl Q ^l4 . Three rounds of iterated selection (FIG. 14 A) with each of the libraries were performed until the molecule number was decreased to around 1x10 ⁶ , which was monitored by quantitative polymerase chain reaction (qPCR). Following amplification of preserved DNA barcodes by PCR, the samples were subjected to Next-generation sequencing (NGS) to identify compounds that outlasted the entire selection procedure. Sequences having significant copy numbers (i.e high signal-to-noise ratio) were deconvoluted to their

corresponding chemical structures from the database. Through this analysis, 50 compounds were determined as primary candidates that possibly bind to the bzAK (Tablel) and named them Compounds 1-50.

[00383] These compounds were synthesized without their code DNA m a small scale to evaluate their activity as PAMs.

Table 1, DNA-encoded libraries used in screening

* For more detail on the encoding libraries, see Kontijevskis, {2017} J Chem Inf Model

57:680-699

** Number of encoding positions in DNA-encoded combinatorial library

*** Number of fragments in each encoding position.

[00384] PAMs are expected to potentiate binding of orthosteric agonists to GPCRs and coupling of transducer proteins, G protein and b-arrestin to receptors. Accordingly, these 50 potential hits from the selection were tested for their ability to increase binding of a radiolabeled agonist, Tl-fenoterol (Tl-FEN) to the b2AK in membrane preparations, both in the absence and presence of transducers (FIGs. 14E and 14F). Through this test, seven structurally-related compounds were identified as shown in FIG. 14G, including compound-6 (Crnpd-6) that showed the strongest activity among the compounds, as potential |32AR PAMs. Compounds 6 and 43 were resynthesized in a large scale for further characterization of their PAM activity. To assess direct molecular interaction between Cmpd-6 and the agonist-bound, active ?.AR, isothermal titration calorimetry (ITC) was employed. The values summarizing binding affinity (KD), stoichiometry (N), and thermodynamic parameters are shown in FIG 14H.

EXAMPLE 7

[00385] PAM activity of Cmpd-6 and -43 in p2AR-mediate down-stream functions. To further evaluate the PAM activity of Cmpd-6 and -43 in 2AR-mediated down-stream functions, [32AR agonist-induced G protein cAMP production and b-arrestin recruitment to the receptor was determined in the presence of these compounds using cellular assays. It is well known that the second messenger generation process down-stream of GPCR activation has high signal amplification compared to stoichiometric reactions such as b-arrestin recruitment to the receptor. Accordingly, endogenously expressed b2AK was used in the assay cells to measure cAMP production, while b-arrestin recruitment was measured using stably overexpressed b2U2K to achieve similar extents of signals between these two assays. The b2U2K, a chimeric receptor with a V2R tail at the C -terminus, displays stronger and more stable agonist-promoted b-arrestin binding than the native 2AR wdule retaining the pharmacological properties of the native b2AK. Both Cmpd-6 and -43 increased the ability of an agonist, isoproterenol (ISO) to activate G protein-mediated cAMP production through the b2AK in a dose-dependent way (FIGs. ISA and 15B). Cmpd-6 and -43 increased the maximal response induced by ISO as well as potentiated the EC50 value of the ISO dose-response, which was apparent in its left-shifted dose-response curve. In this assay, Cmpd-6 shows stronger activity than Cmpd-43, which is consistent with the preliminary data showing the extent of doses-dependent increases in ³ H-FEN binding to the p2AR by these compounds shown in FIG. 14F. A comparable pattern of the results was obtained with Cmpd-6 and -43 in the assay monitoring agonist- induced b-arrestin recruitment to the b2U2R (FIGs 15C and 15D).

[00386] Increases in the ISO-induced maximal response by Cmpd-6 and -43 in both assays suggests that ISO may act as a partial agonist and does not reach the full response available in this system, allowing Cmpd-6 and -43 to further increase the maximal response by ISO. To verify this, cAMP production by the overexpressed p2AR was monitored in the presence of Cmpd-6, the system that has much higher amplification (FIG. 19). Cmpd-6 promoted leftward shits of the ISO dose response EC50 value, with increases in the basal activity in a dose- dependent way, but not increases in the ISO-stimulated maximal response. This shows that even a full agonist such as ISO can act as a partial agonist depending on the assay system, which would not have been suspected without the cooperativity displayed by these new PAMs. Overall, the results demonstrate that Cmpd-6 and -43 have PAM activity for p2AR-mediated down stream functions, and that Cmpd-6 has stronger PAM activity than Cmpd-43. EXAMPLE 8

[00387] Cmpd-6 and -43 potentiate the binding affinity of agonists for the b2AM. A hallmark of GPCR PAM molecules is that they ailostericaliy stabilize the active conformation of the agonist-bound receptor, as do transducer proteins, G protein and b-arrestin, as illustrated in the GPCR ternary complex model. Since P AM-mediated stabilization of the GPCR active conformation leads to potentiation of agonist binding affinity for the receptor, Cmpd-6 and -43 were tested for their effects on the binding of an agonist to the b2AK For this, orthosteric agonist ISO competition binding to the b2AK reconstituted in HDL particles was assayed against a radiolabeled antagonist ^!23 I-cyanopindolol (CYP) in the presence of Cmpd-6 and -43 (FIGs. 16A and 16B). As expected for PAMs, both compounds potentiated the binding of ISO to the 2AR, as evidenced by the robust shift of the dose-dependent competition curve of ISO to the left in the presence of these compounds at various concentrations. Cmpd-6 potentiated the IC50 value of ISO close to 50-fold, which was substantially more than the ~30-fold change elicited by Cmpd-43. Comparable extents of the ISO dose-response curve shift induced by Cmpd-6 and -43 in a radioligand competition binding experiment were also obtained with membranes prepared from b2AE-oneG6crG658Ϊ¾ cells (FIGs. 16C and 16D).

[00388] The result shown in FIG. 16E further confirms the PAM activity of Cmpd-6 and -43 for increasing the binding of an orthosteric agonist to the b2Ab. Cmpd-6 and -43 dose- dependently increase the binding of the radiolabeled orthosteric agonist ³ H-FEN to the b2A expressed in cell membranes, consistent with the result in the preliminary experiment with these compounds synthesized in a small scale (FIGs. 14E and 14F). Again Cmpd-6 is more efficacious than Cmpd-43 in increasing Ή-FEN bindmg to the ]32AR. Further, the low micro- molar affinity (EC50) value of Cmpd-6 obtained in this assay is comparable to its KD value measured for its direct interaction with the bzAK by ITC analyses (FIG 14H). Another feature of allosteric molecules observed in both binding experiments is the“ceiling” effect. The increases in the binding of both agonists, ISO (FIGs. 16A and 16B) and FEN (FIG. 16B) w¾re saturated over increasing concentrations of these allosteric compounds. EXAMPLE 9

[00389] Cmpd-6 stabilizes the agonist-induced active conformation of the b2AM.

Agonist-induced activation of the b2AK causes the outward movement of transmembrane helix 6 (TM6), which can be detected by labeling of cysteine-265 at the intracellular base of TM6 with monobromobimane, an environmentally sensitive fluorescent label. Following receptor activation, the outward movement of TM6 leads to decreases in fluorescence intensity and increases in the maximum wavelength for emission. Cmpd-6 alone induced decreases in overall fluorescence intensity, but not increases in the maximum wavelength from the bimane-labeled 2AR in HDL particles (FIG. 17). On the other hand, ISO decreased fluorescence to a similar extent but also increased the maximum wavelength. This suggests that the conformational ensemble of the b2AK when bound to Cmpd-6 alone is similar to, but distinct from, that induced by orthosteric agonists. Interestingly, Cmpd-6 further potentiates ISO-induced decreases m the amount of fluorescence and increases in the maximum wavelength from the bimane-labeled b2AK. The Cmpd-6-mediated potentiation of ISO effects is similar in magnitude to that observed with an allosteric nanobodySO (Nb80) that mimics the G protein-stabilized active conformation of the agonist-bound b2AK. These data demonstrate that Cmpd-6 stabilizes the active conformation of the agonist-bound b2AK, engaging the outward movement of TM6 to an extent comparable to that mediated by transducers like G protein.

EXAMPLE 10

from the cellular assays (FIGs 15A-15D) strongly supports the PAM activity of Cmpd-6 and suggests a functional cooperativity between the compound and the transducers Gs and b-arrestm. To confirm this co-operative property of Cmpd-6, competition radioligand binding was performed on membrane preparations expressing 2AR C-terminal fusions with Gsa or b- arrestinl (FIGs. 18A and 18B). Compared to |32AR alone, both transducer fusions revealed the expected high-affinity coupling to the receptor with a left-shift in ISO dose response curves. Importantly, addition of Cmpd-6 at b2AK fusions, and compared to uncoupled receptor, enhanced both Gsa- and b-arrestinl -mediated high-affinity coupling to the receptor and also resulted in a significant potentiation of ISO affinity. The PAM activity of Cmpd-6 was also assessed by dose response binding of the radiolabeled orthosteric agonist ³ H-FEN aimed at saturating high-affinity sites on the b2AK (FIGs. 18C and 18D). Compared to no transducer controls, addition of Cmpd-6 or the exogenous transducers, heterotrimeric Gs (at b2AK membranes; FIG. 18C) and b-arrestinl (at phosphorylated b2n2K membranes; FIG. 18D), robustly increased the high-affinity Ή-FEN binding to the receptor. Interestingly, addition of Cmpd-6 together with Gs or b-arrestinl further enhanced the maximal high-affinity Ή-FEN binding. While there was noticeable cooperatively between Cmpd-6 and Gs, this potentiation in ³ H-FEN binding was prominent in the presence of the G-protein mimic Nb80 (FIG. 20A).

Together with the findings from cellular assays, these binding studies demonstrate a positive cooperativity between Cmpd-6 and transducers to modulate high-affinity state agonist binding to the b2LK.

[00391] Of note, the data in FIG. 20 A also suggest that Cmpd-6 does not occlude transducer coupling to b2AK and likely binds to a potentially unique allosteric site in the receptor.

Accordingly, to test whether Cmpd-6 physically competes for binding to the intracellular transducer binding pocket, an ELISA was performed to capture b2AK with the G-protein mimic Nb80 that recognizes agonist-bound active state of the receptor (FIG. 20B). In the presence of the high -affinity agonist BI- 167107, and compared to DMSO or the antagonist I Cl- 118551 , there was a marked increase in receptor capture by NbBO. This receptor capture was robustly inhibited in the presence of saturating amounts of a competing nanobody Nb6B9, which is an affinity matured version of Nb80 and thus competes for a common binding epitope on the b2AK.

Interestingly, and in contrast to Nh6B9, the addition of a saturating concentration of Cmpd-6 did not alter the capture of b2AK by Nb80. These data suggest that presence of Cmpd-6 does not interfere with transducer-coupling to the paAR, which further establishes the positive cooperativity between transducers and the compound.

EXAMPLE 11

[00392] The PAM activity of Cmpd-6 is specific for the b2AK. The specificity of Cmpd-6 for the b2AK was evaluated through in vitro agonist competition radioligand binding to the bΐ AR, the most closely related subtype of adrenergic receptors. In this assay, Cmpd-6 induces a minimal shift of the ISO competition curve for binding to the bΐ AR against the ^{1 5} I-CYP radiolabeled antagonist (FIG. 21 A) unlike the robust ISO curve shift by Cmpd-6 observed with the b2AK. This displays that Cmpd-6 specifically induces the high affinity binding of the orthosteric agonist ISO to the b2AB. but not to the bΐ AR. Marginal changes were promoted by Cmpd-6 in the ISO dose response pattern of bΐ AR-mediated cAMP production (FIG. 21B), which is markedly different from that of the b2AR-medlated response. These findings clearly demonstrate that the PAM activity' of Cmpd-6 is specific for the 2AR.

EXAMPLE 12

[00393] PAM activity of Cmpd-6 when the p2AR is stimulated with a range of different agonists. Some allosteric modulators show differential activity depending on orthosteric agonists stimulating the receptor, a phenomenon known as probe-dependence. Cmpd-6 was examined for such differential activity when the orthosteric site of the P2AR is occupied with a range of agonists. These are epinephrine (EPI) and fenoterol (FEN), which are very strong partial, almost full, agonists compared to ISO, and clenbuterol (CLEN), which is a weak partial agonist. The extent of the dose-response curve (IC50 value) shift induced by Cmpd-6 in radioligand ( ^!23 I-CYP) competition binding to the purified 2AR was evaluated with each of these agonists (Figs. 22A-22D). This permits testing of the allosteric activity of Cmpd-6 solely for binding of an agonist in the absence of transducer-coupling to the receptor. The extent of the curve shift m the presence of Cmpd-6 in this assay essentially followed the efficacy of the tested agonists to induce down-stream signaling.

[00394] The PAM activity of Cmpd-6 for down-stream signaling of the (32AR when stimulated with each of these four agonists in a dose-dependent way was compared using cell- based functional assays, monitoring cAMP accumulation (FIGs. 22E-22H) and b-arrestin recruitment to the receptor (FIGs. 22I-22L). In general, full and strong partial agonists show- greater affinity (EC50 value) shift by Cmpd-6 compared to that observed with the weak partial agonist CLEN. However, CLEN displayed a substantially greater Cmpd-6-mediated increase in the maximal response than did the full agonists. Interestingly, no direct relationship between the extent of the EC50 shift by Cmpd-6 and the efficacy of ISO, EPI and FEN was seen. In functional assays, Cmpd-6 induced a noticeably greater shift with EPI than with ISO and FEN while fold-increases by Cmpd-6 in the maximal response induced by these agonists were comparable. Thus unique probe dependence of Cmpd-6 with this small panel of agonists was not observed.

EXAMPLE 13

[00395] Structure-activity relationships of | 2AR PAMs. FIG. 23 illustrates activity data for representative b2AK PAMs. The allosteric effect of Cmpd-6 derivatives on orthosteric agonist ³ H-FEN binding to the p2AR was tested m the absence and presence of transducers, either trimeric Gs protein or b-arrestinl. Their allosteric activity was also evaluated in ISO- stimulated 2AR downstream signaling, that is G protein-mediated cAMP production and b- arrestin recruitment to the activated receptor. Changes in the Vmax value by Cmpd-6 or each analog at 32 mM are expressed as percentages of the maximal level of the ISO-induced activity in the vehicle (DMSO) control m each assay. Changes in the EC50 value are expressed as fold- shifts compared to the control value obtained the vehicle (DMSO)-treated curve in each assay. Every value represents mean ± SEM obtained from four independent experiments done in duplicate. Statistical analyses were performed using‘one-way ANOVA’ with‘Dumieif post- tests compared to the control (Cmpd-6-treated) value in each assay. *P<0.05, **P<0.01, ***P<0.00l . Rc, receptor.

[00396] For reasons of completeness, various aspects of the invention are set out in the following numbered clauses:

[00397] Clause 1 A. A complex comprising:

(i) a chimeric G protein-coupled receptor (GPCR) comprising the ammo acid sequence LPETGGG (SEQ ID NO: 1) located within the C-terminus of the GPCR and a synthetic phosphopeptide ligated to SEQ ID NO: 1 ; and

(ii) a b-arrestin (parr) protein bound to the C-terminus of the GPCR. [00398] Clause 2A. The complex of clause 1 A, which further comprises an antigen- binding fragment of an antibody (Fab) that specifically binds to the complex.

[00399] Clause 3 A. The complex of clause 1 A or clause 2A, wherein the chimeric

GPCR is a member of the adrenergic receptor family, a member of the dopamine receptor family, a member of the opioid receptor family, a member of the muscarinic acetylcholine receptor family, calcitonin receptor (CTR), a cannabmoid receptor, a chemokine receptor, a free fatty acid receptor, G protein-coupled receptor 3, glucagon-like peptide 1 receptor (GLP-1R), a parathyroid hormone receptor, a somatostatin receptor, a sphingosine- 1 phosphate receptor, a vasopressin receptor, an angiotensin receptor, or thyroid stimulating hormone receptor (TSHR).

[00400] Clause 4 A. The complex of clause 3 A, wherein the chimeric GPCR is a b:2- adrenergic receptor, angiotensin II type 1 A receptor, vasopressin V2 receptor, m opioid receptor (MOR), or muscarinic acetylcholine receptor 2 (M2R).

[00401] Clause 5A. The complex of any one of clauses 1A-4A, wherein the synthetic phosphopeptide is derived from the C-termmus of a vasopressin-2-receptor (V2R).

[00402] Clause 6A. The complex of clause 5A, wherein the synthetic phosphopeptide comprises the ammo acid sequence ARGRTPPSLGPQDESCTTASSSLAKDTSS (SEQ ID NO:

2).

[00403] Clause 7 A. The complex of clause 6A, wherein the synthetic phosphopeptide is phosphoryiated at residues 5, 8, 15, 17, 18, 20, 21, and 22 of SEQ ID NO: 2. [00404] Clause 8A. An in vitro method for producing the complex of any one of clauses 1A-7A, which method comprises:

(a) enzymatically ligating a synthetic phosphopeptide to the C-terminus of a purified GPCR to produce a phosphorylated chimeric GPCR comprising the amino acid sequence of SEQ ID NO: 1 located within the C-terminus of the GPCR, and

(b) contacting the phosphorylated chimeric GPCR with purified b-arrestin (Parr) protein, whereupon the purified b-arrestin (Parr) protein binds to the C-terminus of the phosphorylated chimeric GPCR and forms a complex comprising the chimeric GPCR and the parr protein.

[00405] Clause 9A. The method of clause 8A, wherein the purified GPCR comprises the amino acid sequence LPETGGH (SEQ ID NO: 3).

[00406] Clause 10 A. The method of clause 8A or clause 9A, wherein the ligation is catalyzed by a sortase enzyme.

[004Q7] Clause 11 A The method of clause 10 A, wherein the sortase enzyme is obtained from a prokaryote.

[00408] Clause 12 A. A method for selecting a modulator of a G protein-coupled receptor (GPCR), which method comprises (i) contacting the complex of any one of clauses l A- TA with one or more compounds under conditions to allow for measurement of an activity of the one or more compounds at the chimeric GPCR, (ii) measuring the presence or absence of activity of the one or more compounds, and (iii) selecting at least one compound that displays the activity at the chimeric GPCR.

[00409] Clause 13A. The method of clause 12A, further comprising

measuring a reference activity at (a) an equivalent chimeric GPCR without C-terminal phosphorylation, (b) an equivalent chimeric GPCR in the absence of b-arrestin, (c) an equivalent chimeric GPCR in the presence of G protein, and/or (d) an equivalent GPCRwith a native C- terminus, for the at least one compound that displays activity at the chimeric GPCR; and

selecting a compound that exhibits a difference m the activity' at the chimeric GPCR compared to the reference activity.

[00410] Clause 14 A. The method of clause 13 A, wherein the difference in the activity is an enhancement in the activity at the chimeric GPCR compared to the reference activity.

[00411] Clause 15 A. The method of clause 14 A, wherein the enhancement m the activity at the chimeric GPCR is greater than the enhancement in the activity' measured for a reference ligand.

[00412] Clause 16A. The method of clause 14 A, wherein the enhancement in the activity' at the chimeric GPCR is less than the enhancement in the activity measured for the reference ligand.

[00413] Clause 17A The method of clause 13 A, wherein the difference in the activity is a decrease in the activity' at the chimeric GPCR compared to the reference activity'.

[00414] Clause ISA. The method of clause 17 A, wherein the decrease in the activity at the chimeric GPCR is greater than the decrease in the activity measured for a reference ligand.

[00415] Clause 19A. The method of clause 17A, wherein the decrease in the activity at the chimeric GPCR is less than the decrease in the activity measured for a reference ligand.

[00416] Clause 20A. The method of clause 12 A, further composing

measuring a reference activity at (a) an equivalent chimeric GPCR without C-terminal phosphorylation, (b) an equivalent chimeric GPCR in the absence of b-arrestin, (c) an equivalent chimeric GPCR in the presence of G protein, and/or fd) an equivalent GPCRwith a native C- termmus, for the at least one compound that displays activity at the chimeric GPCR; and selecting a compound that exhibits substantially no difference in the activity at the chimeric GPCR compared to its reference activity.

[00417] Clause 21 A. The method of any one of clauses 12A-20A, wherein the activity is a binding activity.

[00418] Clause 22A. The method of any one of clauses 12A-20A, wherein the activity is a functional activity.

[00419] Clause 23A. The method of clause 22A, wherein the functional activity is agonism of the chimeric GPCR.

[0042Q] Clause 24A The method of clause 22 A, wherein the functional activity is positive allosteric modulation of the chimeric GPCR.

[00421] Clause 25 A. The method of clause 22A, wherein the functional activity is neutral antagonism of the chimeric GPCR.

[00422] Clause 26A. The method of clause 22 A, wherein the functional activity is inverse agonism of the chimeric GPCR.

[00423] Clause 27A. The method of clause 22A, wherein the functional activity is negative allosteric modulation of the chimeric GPCR.

[00424] Clause 28 A. The method of any one of clauses 12A-20A, wherein the activity measurement is signaling activity.

[00425] Clause 29 A. The method of clause 28A, which comprises selecting at least one compound that binds to the chimeric GPCR and activates at least one signaling pathway over one or more other signaling pathways mediated by the chimeric GPCR. [00426] Clause 30 A. The method of clause 29 A, wherein the compound preferentially activates a Parr-dependent signaling pathway over a G protein-dependent signaling pathway or the compound preferentially activates a G protein-dependent signaling pathway over a Parr- dependent signaling pathway.

[00427] Clause 31 A. The method of clause 30A, wherein the Parr-dependent signaling pathway is Mitogen- Activated Protein Kinase (MAPK) signaling, receptor transactivation, receptor trafficking, protein ubiquitination, transcriptional regulation, GPCR desensitization, or GPCR internalization.

[00428] Clause 32A. The method of any one of clauses 29A-31 A, wherein the compound activates a signaling pathway that is different than the signaling pathway activated by a reference ligand.

[00429] Clause 33 A. The method of any one of clauses 29A-31 A, wherein the compound activates one of a plurality of signaling pathways activated by a reference ligand.

[00430] Clause 34A. A method of identifying a biased ligand for a G protein-coupled receptor (GPCR) comprising:

(i) contacting the complex of any one of clauses 1 A-7A with one or more compounds under conditions to allow for binding of the one or more compounds to the GPCR, and

(ii) selecting a compound that binds to the GPCR and exhibits a change in an activity measurement compared to a reference activity measurement for the compound at (a) an equivalent GPCR without C-terminal phosphorylation; (b) an equivalent GPCR m the absence of b-arrestin, (c) an equivalent GPCR in the presence of G protein, and/or (d) an equivalent GPCRwith a native C-terminus. [00431] Clause 35 A. The method of clause 34, wherein the change in the activity measurement is an enhancement in the activity measurement.

[00432] Clause 36A. The method of clause 34A or clause 35A, wherein the

enhancement in the activity measurement for the compound is greater than the enhancement m the activity measurement for a reference ligand for the GPCR.

[00433] Clause 37A. The method of clause 34 A or clause 35A, wherein the

enhancement in the activity' measurement for the compound is less than the enhancement in the activity measurement for a reference ligand for the GPCR.

[00434] Clause 38A. The method of any one of clauses 35A-37A, wherein the enhancement in the activity measurement is an allosteric enhancement in the activity

measurement.

[00435] Clause 39A The method of clause 34A, wherein the change in the activity measurement is a decrease in the activity measurement.

[00436] Clause 40A. A method of identifying a biased ligand for a G protein-coupled receptor (GPCR) comprising:

(i) contacting the complex of any one of clauses 1 A-7A with one or more compounds under conditions to allow for binding of the one or more compounds to the GPCR, and

(ii) selecting a compound that binds to the GPCR and exhibits substantially no change m an activity measurement compared to a reference activity measurement for the compound at (a) an equivalent GPCR without C-temunal phosphorylation; (b) an equivalent GPCR in the absence of b-arrestin, (c) an equivalent GPCR in the presence of G protein, and/or (d) an equivalent GPCRwith a native C-termmus. [00437] Clause 41 A. The method of any one of clauses 34A-40A, wherein the activity measurement is binding affinity.

[00438] Clause 42A. The method of any one of clauses 34A-40A, wherein the activity measurement is functional potency or efficacy.

[00439] Clause 43A. The method of any one of clauses 34A-42A, wherein the compound blocks G protein-mediated signal transduction.

[00440] Clause 44A. The method of any one of clauses 12A-43 A, wherein the compound is a small molecule, a protein, a peptide, a nucleic acid molecule, or a DNA-encoded compound.

[00441] Clause 45 A The method of clause 44A, wherein the compound is a small molecule

[00442] Clause 46A. The method of clause 45A, wherein the small molecule is a therapeutic agent.

[00443] Clause 47A The method of any one of clauses 12A-46A, wherein the complex is immobilized on a solid support

[00444] Clause 48 A. The method of any one of clauses 12A-47A, wherein the one or more compounds are DNA-encoded compounds.

[00445] Clause IB. A compound of formula (I), or a pharmaceutically acceptable salt thereof,

wherein

R ¹ is an aiyl group optionally substituted with 1-5 substituents independently selected from the group consisting of Ci-ealkyl, Ci-ehaloaikyl, Cb-ieycloalkyi, halogen, cyano, -OH, -OCi- ealkyl, -OCi-ehaloaikyl, -ML·, -MiCi-ealkyl, -N(Ci-6alkyl)2, -OCs-ecycloalkyl, -MICs- 6cycloalkyl, -N(Cj-6alkyl)(C3-6cycloalkyl), and -N(C3-6cycloalkyi)2, and optionally the aryl is phenyl wherein two substituents join to form a 5- to 7-membered non-aromatic fused ring containing 1-2 heteroatom groups selected from NR ^la and O;

X ¹ is O, N(H), N(Cr.4alkyl), S, S(O), S(0) ₂ , C(O), or CR ^ib R ^lc ;

R ^la is H or Craalkyl;

R ^lb and R ^lc are each independently hydrogen or Ci-ralkyl, or R ^ib and R ^lc together with the

carbon to which they are attached form a Cs-ecycloalkyl ring;

R ² is hydrogen, Ci-ealkyl, or C-3-7cycloalkyl;

R is hydrogen, Ci-ealkyl, Cs-rcycloalkyi, or aryl, the aryl being optionally substituted with 1 -5 substituents independently selected from the group consisting of Ci-6alkyi, Ci-ehaloalkyl, C3- rcycloalkyl, halogen, cyano, -OH, -OCi-ealkyl, -OCi-ehaloalkyl, -NIL·, -NHCi-6alkyl, - N(Ci-6alkyi)2, -OCs-ecycloalkyl, -NIKN-ecycloalkyL -N(C _l -6alkyi)(C3-6cycloalkyl), and - N(C ₃ -6cy cl oalky 1 ) ₂ ;

alternatively, R ² and R ³ together with the nitrogen to which they are attached form a 4- to 8- membered heterocyclic ring optionally containing one additional heteroatom selected from N, O, and S, and being optionally substituted with 1 -4 substituents independently selected from the group consisting of Ci-ealkyl, Ci-ehaloalkyl, halogen, cyano, -OH, oxo, -OCi-ealkyl, -NKb, -NHCi-ealkyl, and -N(Ci-6alkyl) ₂ ;

R ⁴ is hydrogen, Ci-ealkyl, or Cbycycloalkyi:

R ⁵ is CRR-R· ·';

alternatively, R ⁴ and R ³ together with the nitrogen to which they are attached form a 4- to 8- membered heterocyclic ring optionally containing one additional heteroatom selected from N, O, and S, and being optionally substituted with 1-4 substituents independently selected from the group consisting of Ci-ealkyl, Ci-ehaloalkyl, halogen, cyano, -OH, oxo, -OCi-ealkyl, -NHz, -NHCi-ealkyl, and -N(Ci-ealkyl)2;

R ^5a is aryl or -Ci walkylene-aryl, wherein each aryl in R ^3a is optionally substituted with 1 -5

substituents independently selected from the group consisting of Ci-ealkyl, Ci-ehaloalkyl, Cs- 7cycloalkyl, halogen, cyano, -OH, -OCi-ealkyl, -OCi-ehaloalkyl, -NH2, -NHCi-ealkyl, - NfCi-ealkyl)?, -OCri-ecyeloalkyl, -NHCi-ecycloalkyl, -N(Ci- ₆ alkyl)(C _3-6 cycloalkyl), and - N(C ₃ -6cy cloalky 1)2 ;

R ^5b is X ² or -Ci-salkylene-X ² ; and

SO2NH2, -S O2NHC 1 -ral ky 1 , or -S0 ₂ N(C alkyl) ₂ .

[00446] Clause 2B. The compound of clause IB, or a pharmaceutically acceptable salt thereof, wherein

R ¹ is phenyl optionally substituted with 1-3 substituents independently selected from the group consisting of Ci-6alkyl, Ci-ehaloalkyl, Cs-ycydoalkyl, halogen, cyano, -OH, -OCi-ealkyl, - OCi-6haloalkyl, -NHz, -NHCi-ealkyl, -N(Ci-6alkyl)2, -OC3-6cycloalkyl, -NHCs-ecycloalkyl, -N(Ci-6alkyl)(C3-6cycloalkyl), and -N(C3-6cycloalkyl)2, wherein optionally two substituents join to form a 5- to 7-membered non-aromatic fused ring containing 1-2 heteroatom groups selected from NR ^la and O. [00447] Clause 3B. The compound of clause 2B, or a pharmaceutically acceptable salt thereof, wherein

R ¹ is phenyl optionally substituted with 1-3 substituents independently selected from the group consisting of Ci-ealkyl, C -shaloalkyl, Cs-/cycloalkyl, halogen, -OCi-ealkyl, -OCurJiaioalkyl, or -OC3-6cycloalkyl, wherein optionally two substituents join to form a 5- to 7-membered non-aromatic fused ring containing 1-2 oxygen atoms.

[00448] Clause 4B. The compound of clause 3B, or a pharmaceutically acceptable salt thereof, wherein R ^! is

[00449] Clause 5B. The compound of any of clauses 1B-4B, or a pharmaceutically acceptable salt thereof, wherein X ^! is S.

[00450] Clause 6B. The compound of any of clauses 1B-5B, or a pharmaceutically acceptable salt thereof, wherein is hydrogen or Ci-ealkyl.

[00451] Clause 7B. The compound of any of clauses 1B-6B, or a pharmaceutically acceptable salt thereof, wherein R ³ is Ci-ealkyl, C3-7cycloalkyl, or aryl, the ary! being optionally substituted with 1-5 substituents independently selected from the group consisting of Ci-6alkyl, Ci- ehaioalkyl, C ₃ -7cycloalkyl, halogen, cyano, -OH, -OCi-ea!kyl, -OCi-ehaloalkyl, -NH2, -NHCi- ealkyl, -N(Ci-6alkyl) ₂ , -OCs-ecycloalkyl, -NHC3-6cycloalkyl, -N(Ci-6alkyl)(C3-6cycloalkyl), and -N(C3-6cycloalkyl)2. [00452] Clause 8B. The compound of clause 7B, or a pharmaceutically acceptable salt thereof, wherein the aryl is optionally substituted with 1-3 substituents independently selected from the group consisting of Ci-ealkyl, halogen, and Ci-ehaloalkyl.

[00453] Clause 9B. The compound of clause 8B, or a pharmaceutically acceptable salt thereof, wherein the aryl is selected from

halogen

[00454] Clause 10B. The compound of any of clauses 1B-9B, or a pharmaceutically acceptable salt thereof, wherein R ⁴ is hydrogen.

[00455] Clause 1 IB. The compound of any of clauses 1B-10B, or a pharmaceutically acceptable salt thereof, wherein R ⁵ is -CHR ^5a R ^5b .

[00456] Clause 12B. The compound of clause 1 IB, or a pharmaceutically acceptable salt thereof, wherein R ^5a is -Ciaalkylene-aryl, the aryl being optionally substituted with 1-5 substituents independently selected from the group consisting of Ci-ealkyl, Ci-ehaloalkyl, C3- 7cycloalkyl, halogen, cyano, -OH, -OCi-6alkyl, -OCi -bhaloalkyl, -NH2, -NHCi-ealkyl, -N(Ci 6alkyl)2, -OCs-ecycloalkyl, -NHCs-ecycloalkyl, -N(Ci-6alk\d)(C3-6cycloalkyl), and -N(C3- 6cycloaikyl)2.

[00457] Clause 13B. The compound of clause 12B, or a pharmaceutically acceptable salt thereof, wherein R ^5a is -Ctb-phenyl, the phenyl being optionally substituted with 1-3 substituents independently selected from the group consisting of Ci-ealkyl, Ci-6haloalkyi, C ₃ - 7cydoaikyl, halogen, cyano, -OH, -OCi-ealkyi, -OCi-ehaloalkyl, -NH2, -NHCi-ealkyl, -N(Ci- ealkyl):?, -OCsycycloalkyl, -MTCr-eeycloalkyi, -N(Ci-6alkyl)(C3-6cycloalkyl), and -N(C3- 6cycioalkyl)2.

[00458] Clause 14B. The compound of clause 13B, or a pharmaceutically acceptable salt thereof, wherein the phenyl in R ^5a is optionally substituted with 1-3 substituents

independently selected from the group consisting of Ci-ealkyl, -OH, and C -shaloalkyl.

[00459] Clause 15B. The compound of clause 14B, or a pharmaceutically acceptable salt thereof, wherein R ^5a is

[00460] Clause 16B. The compound of any of clauses 1B-15B, or a pharmaceutically acceptable salt thereof, wherein R ^5b is X ² or -CH2---X ² .

[00461] Clause 17B. The compound of any of clauses 1B-16B, or a pharmaceutically acceptable salt thereof, wherein X ² is -C(0)OH or -C(Q)NH2.

[00462] Clause 18B. The compound of clause G7B, or a pharmaceutically acceptable salt thereof, wherein X ² is -C(0)NH ₂ .

Clause 19B. The compound of any of clauses 1B-18B, or a pharmaceutically acceptable salt thereof, wherein R ⁵ is

[00464] Clause 20B. The compound of clause 1 B, selected from the group consisting of (R)-N-(4-amino-l-(4-(tert-butyl)phenyl)-4-oxobutan-2-yl)-5-( N-isopropylsulfamoyl)-2-((4- methoxyphenyl)thio)benzamide;

(R)-N-(4-amino-l-(4-(tert-butyl)phenyl)-4-oxobutan-2-yl)-5-( N-cyclopentylsulfamoyl)-2-((4- methoxyphenyl)thio)benzamide;

(R)-N-(4-amino-l-(4-(tert-butyl)phenyl)-4-oxobutan-2-yl)-5-( N-isopropyl-N-methylsulfamoyl)-

2-((4-methoxyphenyl)thio)benzamide;

(R)-N-(4-amino-l-(4-(tert-butyl)phenyl)-4-oxobutan-2-yl)-2-( (4-methoxyphenyl)thio)-5-(N-(m- toiyl)sulfamoyl)benzamide;

(R)-N-(4-amino-l -(4-(tert-butyl)phenyl)-4-oxobutan-2-yl)-5-(N-(3-bromophenyl )sulfamoyl)-2-

((4-methoxyphenyl)thio)benzamide;

(R)-N-(4-amino-l -(4-(tert-butyl)phenyl)-4-oxobutan-2-yl)-5-(N-(4-fluoropheny l)sulfamoyl)-2-

((4-methoxyphenyl)thio)benzamide;

(R)-N-(4-amino-l -(4-(tert-butyl)phenyl)-4-oxobutan-2-yl)-5-(N-(3-fluoropheny l)sulfamoyl)-2-

((4-methoxyphenyl)thio)benzamide;

N-isopropyl-4-((4-methoxyphenyl)thio)-N-methyl-3-(piperidine -l -carbonyl)benzenesulfonamide (R)-N-(4-amino-l-(4-(tert-butyl)phenyl)-4-oxobutan-2-yl)-5-( N-isopropyl-N-methylsulfamoyl)-

(R)-N-(4-amino-l-(4-(tert-butyl)phenyl)-4-oxobutan-2-yl)-5-( N-isopropyl-N-methylsulfamoyl)-

2-((3-methoxyphenyl)thio)benzamide;

(5-(N-isopropyl-N-methylsulfamoyl)-2-((4-methox> phenyl)thio)benzoyl)-D-tyrosine; and (R)-N-(l-amino-l -oxo-3-phenylpropan-2-yl)-5-(N-isopropyl-N-methylsulfamoyl)- 2-((4- methoxy pheny l)thio)benzamide; or

a pharmaceutically acceptable salt thereof.

[00465] Clause 21 B. A pharmaceutical composition comprising the compound of any of clauses 1B-20B, or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. [00466] Clause 22B. A method of treating a disease or condition ameliorated by b2 receptor activation comprising administering to a subject m need thereof a therapeutically effective amount of the compound of any of clauses 1B-20B, a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of clause 21B.

[00467] Clause 23B. The method of clause 22B, wherein the disease or condition is an obstructive airway disease, bronchospasm, or pre-term labor.

[00468] Clause 24B. The method of clause 23B, wherein the disease or condition is chronic obstructive pulmonary disease or asthma.

[00469] Clause 25B. The method of any of clauses 22B-24B, wherein the administration is inhalation administration.

[00470] Clause 26B. The method of any of clauses 22B-25B, wherein the compound, or a salt thereof, binds to an allosteric site of the [32 receptor in the subject.

[00471] Clause 27B. The method of clause 26B, wherein the binding of the compound or its salt to the allosteric site stabilizes an active conformation of the b2 receptor.

[00472] Clause 28B. The method of any of clauses 22B-27B, further comprising administering a therapeutically effective amount of an additional pharmaceutical agent selected from the group consisting of PDE3 and/or PDE4 inhibitors, 5-lipoxygenase (5-LO) inhibitors; 5- lipoxygenase activating protein (FLAP) antagonists; dual inhibitors of 5-lipoxygenase (5-LO) and antagonists of platelet activating factor (PAF); leukotnene antagonists (LTRAs) including antagonists of LTB4, LTC4, LTD4, and LTE4; P2-adrenoceptor agonists; cromolyn sodium, theophylline and aminophylhne; inhaled glucocorticoids, interleukin- 5 inhibiting monoclonal antibodies, and monoclonal antibodies that inhibit IgE binding to the high-affinity IgE receptor, or a pharmaceutically acceptable salt thereof. [00473] Clause 29B. The method of clause 28B, wherein the additional pharmaceutical agent is a b2 receptor agonist, or a pharmaceutically acceptable salt thereof.

[00474] Clause 30B. The method of clause 29B, wherein the b2 receptor agonist is selected from the group consisting of albuterol, levalbuterol, arformoterol, salbutamol, formoterol, indacaterol, olodaterol, terbutaline, ritodrine, hexoprenaline, metaproterenol, nylidrin, orciprenaline, and sa!meterol.

[00475] Clause 3 IB. The method of clause 29B or 30B, wherein the therapeutically effective amount of the compound of any of clauses 1B-20B, the pharmaceutically acceptable salt thereof, or the pharmaceutical composition of clause 21B is an amount that reduces tolerance to the effects of the b2 receptor agonist compared to the tolerance m a reference subject receiving treatment with the b2 receptor agonist alone.

[00476] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

The use of the terms“a” and“an” and“the” and“at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term“at least one” followed by a list of one or more items (for example,“at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms“comprising,” “having,”“including,” and“containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[00478] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.