Background of the Invention The five muscarinic receptors subtypes are designated M1- M5 and all are activated by the binding of acetylcholine (ACh). These receptor subtypes are widely distributed in the central nervous system and in the periphery where they mediate a number of important physiological functions. As a consequence these receptors are a therapeutic target for the treatment of a variety of conditions and potential therapeutic agents are both agonists and antagonists. In the treatment of many conditions it has been thought to be important that the therapeutic agents have a selective action on one or a limited number of subtypes. However, there remains a problem in the art that the muscarinic receptor subtypes are structurally very similar as a consequence of the identity of amino acids in the regions of sequence that are considered to constitute the ACh binding site, i. e. the site of binding of agonists and competitive antagonists. Therefore, it has not been possible to synthesize highly selective muscarinic antagonists and no directly acting muscarinic agonists of any substantial selectivity exist (Caulfield and Birdsall, 1998). A further problem is that synthetic exogenously applied agonists chronically stimulate receptors and this can

result in desensitization and downregulation of the receptor function as well as losing any information content of the pulsatile endogenous ACh signalling mechanism.

However, in addition to the primary site on these receptors at which agonists and competitive antagonists bind, muscarinic receptors are known to also contain an allosteric site. Compounds binding at the allosteric site mediate the binding of the ligands to the primary binding site (GB 2 292 685 A and WO 96/03377). Thus, it is possible that compounds binding at the allosteric site may overcome some of these problems involved in selective modulation of muscarinic receptor subtypes. By way of example, it is known that when brucine and some of its N- substituted analogues bind at the allosteric site, they modulate the response of muscarinic receptors to the primary ligand acetylcholine (ACh) or N-methylscopolamine (NMS), a competitive antagonist of ACh. The modulation caused by compounds binding at the allosteric site can be positive, negative or neutral. A compound which has neutral cooperativity with ACh at one muscarinic receptor subtype binds to the receptor but has no action at any concentration. In contrast, if the same ligand has positive or negative cooperativity at another subtype it has an action at that subtype which is totally selective.

This form of selectivity based on cooperativity can be termed absolute subtype selectivity'... Thus, the allosteric agents can modulate the interaction between the muscarinic receptor and the primary ligand.

Summary of the Invention Broadly, the present invention relates to the finding that muscarinic receptors have a further allosteric site which is characterised herein using compound la, and a

series of related indolocarbazoles represented by formula 1, and compounds 2a and 2b, and a series of related compounds represented by formula 2. These compounds are capable of binding to the allosteric site to modulate the binding of a primary ligand to the receptors, showing positive, negative and neutral cooperativity and selectivity for muscarinic receptor subtypes.

Compounds represented by formula 1: wherein: Ri is hydrogen, lower alkyl, aralkyl, iminoalkyl or an imino protecting group such as an acyl group R19CO, where Rl9 is alkyl or aralkyl ; R2 and R3 are independently an oxygen or two hydrogen atoms; R4-Rll are independently selected from hydrogen or general aromatic substituents e. g. halo, nitro, cyano, lower alkyl, haloalkyl, alkoxy, hydroxy, aralkoxy; R12 is H or lower alkyl ; X is CH2, CH (OR16), where R16 is hydrogen, lower-alkyl or an O-protecting group,

CH (OR16)-CH (OR16) , CH (OR16)-CH (CH2)0-N(R17)2, where n is an integer between 0-5 and Rl, is one or two of the following; alkyl, aralkyl ; R14 is hydrogen, OR16, NHR17, where R17 is alkyl, haloalkyl, aralkyl or an acyl group ; m is an integer between 0-8 ; and, Ris is hydrogen, CO2R18, CONHR18 or an isostere for an ester or substituted ester, where R18 is hydrogen, alkyl or substituted alkyl, aralkyl or substituted aralkyl.

Compounds represented by formula 2: wherein: X is N (H) or C (H); R1, R2 are independently hydrogen, general aromatic substituents e. g. halo, nitro, cyano, lower alkyl, haloalkyl, alkoxy, hydroxy, aralkoxy; R3 is hydrogen, alkyl, iminoalkyl, or aralkyl ; Ri and R2 together and/or R2 and R3 together are a fused aromatic or heterocyclic system optionally with ring substituents; R4, R5, R6 are normal aromatic substituents; or R5, R6 together are a fused alicyclic system with 1-5 rings including steroid ring systems, preferably with substituents at either or both 17a and 17j3 positions, e. g. hydroxy, alkoxy, aralkoxy, alkyl, alkenyl, or alkynyl, and R4 is a substituent compatible with the

synthetic method; or R5, R6 together are a fused alicyclic system with 1-5 rings and R4 is hydrogen or an alkyl group; or R4, Rst R6 are part of a fused alicyclic system ; and a dotted line indicated either the presence or the absence of a bond.

Preferably, the above generally defined substituents are Ci-Ci or in the case of lower alkyl substituents, C1-C6 ; and in either case, optionally including branching and/or halogen substitution.

The formulae of compounds la, 2a and 2b are shown on pages 54 to 56.

Accordingly, in a first aspect, the present invention provides a compound represented by formula 1 or 2 for use in a method of medical treatment.

In a further aspect, the present invention provides the use of a compound for the preparation of a medicament for the treatment of a condition mediated by the binding of a primary ligand to a muscarinic receptor, wherein the compound binds to an allosteric site of the muscarinic receptor which is capable of binding to compound la and/or 2a and thereby modulates the binding of the primary ligand to the muscarinic receptor. Preferred compounds include those represented by formula 1 or 2.

In a further aspect, the present invention provides a method of modulating the response of a muscarinic receptor to a primary ligand, the method comprising contacting the muscarinic receptor with a compound which binds to an allosteric site of the muscarinic receptor which is capable of binding to compound la and/or 2a and

which thereby modulates the binding of the primary ligand to the muscarinic receptor. This method may be carried out in vitro, e. g. as part of a screening method or to otherwise activate or modulate the response of the receptor, or in vivo, e. g. in the treatment of a patient suffering from one of the conditions described herein.

The primary ligand binding to the muscarinic receptor may be an agonist or an antagonist of the receptor's biological activity. Examples of primary ligands include acetylcholine (ACh) or N-methylscopolamine (NMS). Other primary ligands are well known to those skilled in the art.

The use of a compound which binds to this allosteric site, and in particular an allosterically acting compound which has a positive or negative cooperative effect on the binding of the primary ligand, can have the advantage of selectively modulating the natural function of a limited group of the muscarinic receptor subtypes, and more preferably only a single muscarinic receptor subtype. Thus, the invention helps to solve the problem of selectively activating the function of specific muscarinic receptor subtypes in a way which is difficult to achieve using a primary ligand which binds to multiple receptor subtypes, and opens up the possibility of therapeutic treatment based on this selectivity.

Examples of these conditions are discussed below. While it is generally preferred that the binding of the allosteric compound to the muscarinic receptor enhances the binding of the primary ligand (i. e. shows positive cooperativity), compounds which decrease the binding of the primary ligand (i. e. act antagonistically or show negative cooperativity) can also have therapeutic potential. Such compounds have the property of not

blocking 100% of the receptor response when they bind to the allosteric site of the receptor (as competitive antagonists do). These compounds can be used in the treatment of conditions including Alzheimer's disease, motion sickness, depression, bronchitis, gastric and duodenal ulcers, non ulcer dyspepsia, urinary bladder incontinence and retention, sinus bradycardia, Parkinson's disease, incontinence, asthma, chronic obstructive pulmonary disease, irritable bowel syndrome, excessive vagal drive, as a preanaesthetic, for cardiac pacemaker regulation, or for the regulation of sleep.

The allosteric site defined herein is distinct from the common allosteric site'disclosed in the prior art (e. g.

GB 2 292 685 A) which binds to gallamine, strychnine, brucine and N-substituted brucine analogues. In contrast, the present inventors have found a new allosteric site which binds compound la and a series of related indolocarbazoles having formula 1 and compound 2a and a series of related compounds having formula 2.

Preferably, the muscarinic receptor is selected from the Ml, M2, M3, M4 or Ms receptors known in the art. The receptors may be human or an appropriate animal homologue (rat, mouse etc). The generation of transfected cell lines stably or transiently expressing one or more of the Mi-Mg receptor genes from any given species is well in the art and relevant references are cited, for example, in the reviews: Hulme et al, 1990 and Caulfield and Birdsall, 1998.

Exemplary compounds which are. capable of modulating the binding of a primary ligand to a muscarinic receptor by interaction with the allosteric site described for the first time herein include compounds la, 2a and 2b.

In a further aspect, the present invention provides a method for aiding in the identification of compounds capable of modulating the binding of a primary ligand to a muscarinic receptor by binding to an allosteric site of the muscarinic receptor which is capable of binding to compound la and/or compound 2a, the method comprising: (a) contacting the muscarinic receptor and the primary ligand with one or more concentrations of a candidate compound; and, (b) determining whether the candidate compound modulates the binding of a primary ligand to the muscarinic receptor by binding to the allosteric site of the receptor which is capable of binding compound la and/or compound 2a.

The method may comprise the further step of selecting a candidate compound which modulates the binding of the primary ligand to the muscarinic receptor.

In a further aspect, the present invention provides the use of an allosteric site of a muscarinic receptor which is capable of binding to compound la and/or 2a in screening for compounds which are capable of modulating the binding of a primary ligand to a muscarinic receptor by binding to the allosteric site.

The modulation of the binding of the primary ligand may be achieved by a number of mechanisms. The allosteric compound may have a positive or negative cooperative effect at one or more of the muscarinic receptor subtypes, and preferably no effect (neutral cooperativity) at other receptor subtypes. Alternatively or additionally, the binding of the allosteric compound may affect the binding of an agent acting at a different allosteric site such as the common allosteric site which

binds brucine.

Preferably, candidate allosteric compounds are selected if they have a positive or negative cooperative effect on the binding of the primary ligand. Thus, in preferred embodiments, step (b) involves determining whether the candidate compounds bind to the allosteric site and additionally determining how the binding modulates the action of the primary ligand at its binding site. This can be carried out using the assays described herein including equilibrium and/or kinetic binding assays and/or functional assays.

General assays which are suitable or can be adapted for use in the present invention are described in Lazareno and Birdsall (1995), Detection, quantitation and verification of allosteric interactions of agents with labelled and unlabelled ligands at G-protein-coupled receptors: Interactions of strychnine and acetylcholine at muscarinic receptors. Mol. Pharmacol. 48: 362-378; Lazareno et al (1998), Subtype selective positive cooperative interactions between brucine analogues and acetylcholine at muscarinic receptors: radioligand binding studies. Mol. Pharmol. 53: 573-589; Birdsall et al (1999), Subtype selective. positive cooperative interactions between brucine analogues and acetylcholine at muscarinic receptors: functional studies. Mol.

Pharmacol. 55: 778-786.

Examples of specific assays which can be employed are described in the Materials and Methods'section below.

Firstly, the candidate compounds are selected as being allosteric using the criteria defined in the above general and specific assays. The compounds are further

selected as binding to the novel site, e. g. the site which binds compound 2a, using specific kinetic and equilibrium assays. For example, any allosteric compound which is not competitive with the binding of brucine, gallamine or strychnine and is competitive with the binding of 2a to the novel allosteric site is a candidate compound for further investigation or development as a therapeutic. These methods are useful for finding compounds which are positively, negatively or neutrally cooperative with the binding of the primary ligand.

In preferred embodiments, the screening is carried out using a receptor, one or more concentrations of a candidate allosteric ligand (possibly including an assay carried out in the absence of the candidate ligand by way of control) and one or more primary ligands, in the presence or absence of another allosteric ligand.

Preferably, the primary ligand employed in this screening method is acetylcholine (ACh) and/or N-methylscopolamine (NMS), although other suitable primary ligands are well known to those skilled in the art. In one embodiment, the method involves as assay in which the binding of a candidate compound to the allosteric site is determined in using labelled NMS in the absence or presence of one or more concentrations of ACh. Alternatively or additionally, the binding of a candidate compound to the allosteric site is determined using labelled NMS in assays which determine the NMS dissociation rate constant in the presence and absence of one or more concentrations of the candidate compound. Another preferred assay format is an assay of the effects of one or more concentrations of an allosteric ligand on the acceleration of the dissociation rate of NMS from muscarinic receptors produced by 2a (described in Part II below). A further assay is to quantitate the effects of

a test compound, which has been demonstrated in the general assays to be allosteric, on the equilibrium allosteric effects of ligands which are known to bind one or other of the two allosteric sites described in Part Ii Figure 5).

In these methods, the candidate compound may be selected if it enhances the binding of the primary ligand to the muscarinic receptor (otherwise referred to as positive cooperativity).. However, other compounds may be selected if they reduce the binding of the primary ligand to the muscarinic receptor (otherwise referred to as negative cooperativity). Candidate compounds having neutral cooperativity are selected if they bind to one or more of the muscarinic receptor subtypes but have no action on the equilibrium binding of a primary ligand at any concentration.

The allosteric site employed in the work described herein is capable of binding compounds la and/or 2a. In contrast to the first allosteric site disclosed in the prior art, the site described herein does not bind to brucine, gallamine and/or strychnine to any substantial extent at concentrations up to 10-'M.

In a further aspect, the present invention provides a method which comprises, having identified a candidate compound by the above method, the further step of manufacturing the compound in bulk and/or formulating the compound as a pharmaceutical composition.

In a further aspect, the present invention provides the use of a compound as obtainable by the above method for the preparation of a medicament for the treatment of a conditions mediated by the binding of the primary ligand

to the muscarinic receptor. Examples of these conditions discussed below.

Embodiments of the invention will now be described by way of example and not limitation with reference to the accompanying drawings.

Brief Description of the Figures Figure 1: Effect of staurosporine (lf) on the binding of 3H-NMS (210 pM) at M1 receptors in the absence and presence of 2.2 mM ACh, all in the presence of 0.2 mM GTP. The points are individual observations. The lines show the fit to Equation 2 (see Methods), which yielded a log affinity of 5.95 0.06, a slope factor of + 0.05, cooperativity with 3H-NMS of 1.51 0.06, and cooperativity with ACh of 0.27 0.03. The affinity ratio plots of these data are shown in Figure 2.

Figure 2: Affinity ratio plots of five indolocarbazoles (la, lb, le, lf and li) at Ml-M4 receptors. The points were derived from duplicate observations of 3H-NMS binding in the absence and presence of ACh, as described in Methods. The parameter estimates pK (log affinity of the test agent for the free receptor), aNMS (cooperativity with 3H-NMS), and BACH (cooperativity with ACh) were derived from nonlinear regression analysis with Equations 1 or 2 as appropriate (see Methods). The parameter estimates from a number of similar assays are summarised in Table 1.

Figure 3: Effect of la, lb, le, lf and li on the dissociation rate constant (koff) of 3H-NMS at Ml-M4 receptors, expressed as a percent inhibition of the control koff. The points are the mean and range/2 of duplicate observations. The lines show the fit to a

logistic function, as described in Methods. The parameter estimates from a number of similar assays are summarised in Table 2.

Figure 4: Effect of various concentrations of KT5720 (la) on the inhibition of 3H-NMS (50 pM) binding at M1 receptors by ACh in a volume of 3 ml. The points are the mean and range/2 of duplicate observations. The lines show the fit to Equation 1 with the slope factor for KT5720 binding set to 1. The parameter estimates were: log affinity of KT5720 6.6 0. 1, cooperativity with 3H-NMS 1.9 0.1, cooperativity with ACh 1.6 + 0.2. The inset shows affinity ratio plots derived from these parameters (see Methods). The-log ICso values of ACh in the presence of increasing concentrations of KT5720, from independent logistic fits of the curves, were 5.28,5.33, 5.40 and 5.42.

Figure 5: Inhibition by gallamine of 3H-NMS binding at Mi receptors in the presence of various concentrations of (A) KT5720 (la) and (B) staurosporine (1f). The points are individual observations. The lines show the fit of the data to Equation 3, where the cooperativity estimates of gallamine with KT5720 and staurosporine were not significantly different from 1 and 0 respectively and were set to those values. The slope factors for gallamine, KT5720 and staurosporine were not different from 1 and were set at that value. From three such assays, similarly constrained, KT5720 had a log affinity of 6.22 0.17 and cooperativity with 3H-NMS of 0.08. From three such assays, similarly constrained, staurosporine had a log affinity of 5.75 0.11 and cooperativity with 3H-NMS of 1.62 0.13. From these six assays gallamine had a log affinity of 5.05 0.05 and cooperativity with 3H-NMS of 0.11 0.01. The insets show

the effect of the test agent on the-log IC50 of gallamine, obtained from nonlinear regression analysis of the individual curves.

Figure 6: Effect of KT5720 (la) on 3H-NMS dissociation from Mi receptors, alone and in the presence of other allosteric agents, measured at a single time point as described in Methods. The points show the mean and s. e. m of quadruplicate observations obtained in two assays, except for 10-4M gallamine, and 3.10-5M and 3.10-4M brucine , which show the mean and range/2 of duplicate observations. The lines in the top panel show the fits of the individual curves to a hyperbolic function, except for those in the presence of staurosporine. The estimates of the log EC50 of KT5720 derive from those fits. The top panel shows the data as % inhibition of the control koff of 3H-NMS. The lower panel shows shows Ef, the 3H-NMS koff values in the presence of KT5720 and a certain concentration of test agent (gallamine, brucine or staurosporine) as a fraction of the koff values in the presence of that concentration of test agent alone.

Figure 7 : Concentration-dependent effects of the compounds shown on pages 55 and 56 on the 3H-NMS-occupied receptor. The percent inhibition of the dissociation rate constant of 3H-NMS from Ml-M4 receptors was measured at a single time point. The legends indicate the ICso (or EC50) values obtained using nonlinear regression analysis of these curves.

Figures 8 and 9: Concentration-dependent effects of active compounds on the equilibrium binding of 3H-NMS and ACh. The effects are expressed as affinity ratios', i. e. the apparent affinity of the primary'ligand (3H-NMS or ACh) in the presence of a particular concentration of

test agent divided by its apparent affinity in the absence of test agent. Affinity ratios were calculated as described in Methods'. Affinity ratios > 1 indicate positive cooperativity, affinity ratios < 1 indicate negative cooperativity, and affinity ratios of 1 with one primary ligand at concentrations of test agent which modify the binding of the other primary ligand indicate neutral cooperativity. The IC50, or ECso, of a test agent on the affinity ratio of either primary ligand corresponds approximately to the Kd of the test agent for the free receptor. High concentrations of compounds which show neutral or positive cooperativity with 3H-NMS and which strongly inhibit 3H-NMS dissociation may inhibit 3H-NMS binding through a kinetic effect, i. e. lack of equilibration of 3H-NMS binding.

Figure 10: Inhibition of 3H-NMS binding to M3 receptors by ACh, alone and in the presence of three concentrations of 2c (WIN 62577). GTP (0.2 mM) was present. The data were fitted to Equation 1 (see Methods) to yield a log affinity of 2c of 5.31, cooperativity with 3H-NMS of 0.47, and cooperativity with ACh of 1.41. The inset shows affinity ratios (1/dose ratio) for ACh and 3H-NMS, calculated from the parameters of the fit.

Figure 11: Dissociation of 3H-NMS over time, alone and in the presence of three concentrations of 2a. For each receptor subtype, the parameter estimates and standard errors were derived from the nonlinear regression fits of the entire dataset to a version of Equations 2 and 3, where koff and kO¢fx are the dissociation rate constants of 3H-NMS from the free and 2a-liganded receptor respectively, and pKocc is the log Kd of 2a for the 3H-NMS- occupied receptor. The insets show the linearising transformation ln (B/Bo) vs. time, where B is the specific

binding remaining after a certain time and Bo is the initial level of specific binding.

Figure 12: Concentration-effect curves for 2a on 3H-NMS dissociation, alone and in the presence of one or three concentrations of test agent, measured at a single time point. The data were converted to dissociation rate constants (see Methods) and expressed as a % of the control dissociation rate constant. The curves show the fits from nonlinear regression analysis to Equation 1.

For strychnine and gallamine, dissociation of 3H-NMS from the dually or triply liganded receptor was not different from 0 and the cooperative interaction with 2a was not different from 1 (i. e. neutral cooperativity), so these values were fixed. For la (KT 5720), lf (staurosporine) and 2b (WIN 51708), the cooperative interaction with 2a was not different from 0 (i. e. competition), so this value was fixed. The panel marked dose-ratio plots' shows the log of the ratio ECgogent/ECsoaicneof 2a vs log [agent]. Each assay was repeated at least once, with similar results.

Detailed Description Assays As indicated above, the screening assays of the invention are useful in the identification of compounds capable of binding to the allosteric site disclosed herein and modulating the binding of a primary ligand to a muscarinic receptor. The precise format of these assays can be readily devised by the skilled person using the common general knowledge in the art. In one embodiment, the assays will employ (a) a muscarinic receptor of one of the Mi to Me, subtypes, (b) one or more primary ligands or ligand analogues, and (c) one or more candidate compounds. Optionally, the assays may additionally

employ (d) a compound known to act at the allosteric site which is capable of competing with the candidate compound being tested. The assays will generally involve contacting the receptor, the primary ligands or ligand analogues and one or more concentrations of the candidate compound in vitro, under conditions in which the candidate compound can bind or compete for binding at the allosteric site. The results of the assays can be determined by labelling one or more of the candidate compound, the competitive allosteric compound or the primary ligand or ligand analogue, and determining which species interact in the assay system.

In an alternative embodiment, and especially in the context of high throughput screening, it may be desirable that the screening assays involve determining whether the candidate compound binds to the allosteric site disclosed herein in the absence of the primary ligand. These assays could be followed with a separate determination of whether or in what sense the compounds binding to the allosteric site modulate the binding of a primary ligand or ligand analogue to the receptor. These assays could be carried out by contacting (a) a muscarinic receptor and (b) one or more candidate compounds, and optionally, (c) one or more compounds known to act allosterically, under conditions in which compounds (b) and/or (c) can bind or compete for binding to the allosteric site. The binding of the candidate compound or competitive compound to the receptor can be determined by labelling compounds (b) and/or (c).

The above assays may comprise carrying out controls, e. g. carrying out the assay in the absence or presence of the candidate compound (s).

In these assays, the muscarinic receptors can be present in either a free form or alternatively immobilised, e. g. on the surface of a cell expressing the receptor, or a solid support. A preferred format uses cell surface receptors.

The labelling of different types of agents is well known in the art. Broadly, this involves tagging the agent with a label or reporter molecule which can directly or indirectly generate detectable, and preferably measurable, signal. The linkage of reporter molecules may be direct or indirect, e. g. by a covalent bond or a non-covalent interaction. Examples of commonly used labels include fluorochrome, phosphor or laser dyes with spectrally isolated absorption or emission characteristics. Suitable fluorochromes include fluorescein, rhodamine, luciferin, phycoerythrin and Texas Red. Suitable chromogenic dyes include diaminobenzidine. Other detectable labels include radioactive isotopic labels, such as 3H, 14C 32p, 35s, 126j, or 99mTc, and enzyme labels such as alkaline phosphatase, ß-galactosidase or horseradish peroxidase, which catalyze reactions leading to detectable reaction products and can provide amplification of signal.

Other reporters include macromolecular colloidal particles or particulate material such as latex beads that are coloured, magnetic or paramagnetic, and biologically or chemically active agents that can directly or indirectly cause detectable signals to be visually observed, electronically detected or otherwise recorded. These molecules may be enzymes which catalyze reactions that develop or change colour or cause changes in electrical properties. They may be molecularly excitable, such that electronic transitions between

energy states result in characteristic spectral absorptions or emissions.

In the context of high throughput screening, the methods described herein can involve carrying out assays using groups or pools of candidate compounds, rather than individual compounds, to enhance the rate at which candidate compounds can be discarded. Individual groups of compounds having positive results in an assay can then be separated and screened to identify the compound (s) in the group which interact with the allosteric site and modulate the binding of the primary ligand. Appropriate measures should be taken to ensure that any one candidate compound is assayed with different pools of other candidate compounds. This protocol minimises the possible interfering masking effects of competitive antagonists or agonists which may be in one pool but not another.

The candidate compounds used may be natural or synthetic chemical compounds used in drug screening programmes.

Mixtures of naturally occurring materials which contain several characterised or uncharacterised components may also be used.

Other candidate compounds may be based on modelling the 3-dimensional structure of a polypeptide or peptide fragment and using rational drug design to provide candidate compounds with particular molecular shape, size, hydrophobicity, hydrophilicity and charge characteristics.

The amount of candidate compound which may be added to an assay of the invention will normally be determined by trial and error depending upon the type of compound used.

Typically, from about 0.01 to 100,000nM concentrations of candidate compound may be used, for example 0.1 to 100uM.

In a further step, the method of the present invention may involve quantifying the amount of a candidate compound required to modulate the binding of the primary ligand to the muscarinic receptor by more detailed equilibrium or kinetic assays.

The screening methods of the invention may be followed by isolation and/or manufacture and/or use of a candidate compound selected in an assay, and/or further testing to determine whether a candidate compound having a positive, neutral or negative cooperative effect on the binding of the primary ligand to the muscarinic receptor has a biological property which makes it suitable for further development as a lead compound. These include tests to determine its activity in assays of function in membranes, whole cells, whole tissues and/or in vivo, as well as tests of its metabolic stability, bioavailability and duration of action and for the presence of side effects.

In a further aspect, the present invention provides the use of the above compounds in the design or screening for mimetics of the compounds which share the property of binding to the allosteric site of muscarinic receptors which binds to compound la and/or compound 2b.

The designing of mimetics to a known pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a lead compound. This might be desirable where the active compound is difficult or expensive to synthesise or where it is unsuitable for a particular method of administration. Mimetic design,

synthesis and testing may be used to avoid randomly screening large number of molecules for a target property.

There are several steps commonly taken in the design of a mimetic from a compound having a given target property.

Firstly, the particular parts of the compound that are critical and/or important in determining the target property are determined. These parts or residues constituting the active region of the compound are known as its pharmacophore. Once the pharmacophore has been found, its structure is modelled to according its physical properties, e. g. stereochemistry, bonding, size and/or charge, using data from a range of sources, e. g. spectroscopic techniques, X-ray diffraction data and NMR.

Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modelling process.

In a variant of this approach, the three-dimensional structure of the ligand and its binding partner are determined or modelled. This can be especially useful where the ligand and/or binding partner change conformation on binding, allowing the model to take account of this the design of the mimetic.

A template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted. The template molecule and the chemical groups grafted on to it can conveniently be selected so that the mimetic is easy to synthesise, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound. The mimetic or mimetics found by this approach can then be

screened to see whether they have the target property, or to what extent they exhibit it. Further optimisation or modification can then be carried out to arrive at one or more final mimetics for further testing or optimisation, e. g. in vivo or clinical testing.

Pharmaceutical Uses The compounds identified herein as being useful for modulating the binding of a primary ligand to a muscarinic receptor can be formulated and used as pharmaceuticals.

The pharmaceutical compositions may comprise, in addition to one or more of the compounds, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art.

Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.

The precise nature of the carrier or other material may depend on the route of administration, e. g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, or intraperitoneal routes.

Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil.

Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

The pharmaceutical formulations can be prepared by mixing the compounds of the present invention with one or more

adjuvants, such as excipients (e. g. organic excipients including sugar derivatives, such as lactose, sucrose, glucose, mannitol or sorbitol; starch derivatives, such as corn starch, dextrine or carboxymethyl starch; cellulose derivatives, such as crystalline cellulose, low hydroxypropyl-substituted cellulose, carboxymethyl cellulose, carboxymethyl cellulose calcium or internally bridged carboxymethyl cellulose sodium; gum arabic ; dextran; and Pullulan; inorganic excipients including silicates, such as light silicic acid anhydride, synthetic aluminium silicate or magnesium meta-silicic acid aluminate; phosphates, such as calcium phosphate; carbonates, such as calcium carbonate ; and sulphates, such as calcium sulphate); lubricants (e. g. metal stearates, such as stearic acid, calcium stearate or magnesium stearate; talc; colloidal silica; waxes, such as beeswax or spermaceti; boric acid; adipic acid; sulphates, such as sodium sulphate; glycol ; fumaric acid; sodium benzoate; DL-leucine ; sodium salts of aliphatic acids; lauryl sulphates, such as sodium laurylsulphate or magnesium laurylsulphate; silicate. s, such as silicic acid anhydride or silicic acid hydrate; and the foregoing starch derivatives); binders (e. g. polyvinyl pyrrolidone, Macrogol; and similar compounds to the excipients described above); disintegrating agents (e. g. similar compounds to the excipients described above ; and chemically modified starch-celluloses, such as Crosscarmelose sodium, sodium carboxymethyl starch or bridged polyvinyl pyrrolidone) ; stabilisers (e. g. p- hydroxybenzoates, such as methylparaben or propylparaben ; alcohols, such as chlorobutanol, benzyl alcohol or phenylethyl alcohol; benzalkonium chloride; phenols, such as phenol or cresol; thimerosal ; dehydroacetic acid ; and sorbic acid); corrigents (e. g. sweeteners, vinegar or perfumes, such as conventionally used); diluents and the

like.

For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

Preferably, the pharmaceutically useful compound according to the present invention is given to an individual in a prophylactically effective amount'or a therapeutically effective (as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual.

Typically, this will be to cause a therapeutically useful effect in the patient, e. g. using the compounds to regulate the action of the primary ligand at a muscarinic receptor, and preferably one of the muscarinic receptor subtypes. The actual amount of the compounds administered, and rate and time-course of administration, will depend on the nature and severity of the condition being treated. Prescription of treatment, e. g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's

Pharmaceutical Sciences, 16th edition, Oslo, A. (ed), 1980.

In particular, the compounds may be useful in the treatment of conditions mediated by the action of ACh at a muscarinic receptor. By way of example, these include Alzheimer's disease, Parkinson's disease, motion sickness, Huntingdon's chorea, schizophrenia, depression, anxiety, sedation, analgesia, stroke, preanaesthetic, antispasmodic, irritable bowel syndrome, bladder- incontinence or retention, peptic ulcer disease, bronchitis/asthma/chronic obstructive airway disease, sinus bradycardia, pacemaker regulation, glaucoma, achalasia, symptomatic diffuse oesophageal spasm, biliary dyskinesia, scleroderma, diabetes mellitus, lower oesophageal incompetence, intestinal pseudo obstruction, regulation of sleep, control of pupil diameter and non- ulcer dyspepsia.

Depending on the type and severity of condition, the composition can be administered to provide an initial dose of about 0.01 to 20 mg, more preferably 0.02 to 10 mg, of compound/kg of patient weight. As mentioned above, other dosing regimens and the determination of appropriate amount of the compounds for inclusion in the compositions can be readily determined by those skilled in the art.

Materials 3H-NMS (81-86 Ci/mmol) was from Amersham International, UK, and 35S-GTPyS (1000-1400 Ci/mmol) was from NEN, Boston. Brucine sulfate, gallamine triiodide and ACh chloride were from Sigma Chemical Co., Dorset, UK.

Staurosporine was from Sigma and from Alexis Corporation, Nottingham, UK. Go 7874 (li), Go 6976 (lh) and K-252c

(lg) were from Calbiochem, Nottingham, UK. K-252a (lb) and K-252b (lc) were from Alexis and from TCS Biologicals Ltd, Buckingham, UK. KT5823 (le) and KT5720 (la) were from TCS, Calbiochem and Alexis. KT5926 (ld) was from TCS and Calbiochem. WIN 51708 (2b) and WIN 62577 (2c) were from RBI (Semat), St Albans, UK. Analogues of 2 were synthesised using literature methods according to the general scheme: with further chemical modification of the product as necessary. Further assistance for the synthesis of the compounds or related ones is provided in the papers by Bajwa and Sykes (1978-1980) cited in the references section.

Methods Cell culture and membrane preparation: CHO cells stably expressing cDNA encoding human muscarinic Ml-M4 receptors (Buckley et al, 1989) were grown in alpha-MEM medium (GIBCO) containing 10% (v/v) new born calf serum, 50 U/ml penicillin, 50 ug/ml streptomycin and 2 mM glutamine, at 37° under 5% CO2.

Cells were grown to confluence and harvested by scraping in a hypotonic medium (20 mM Hepes + 10 mM EDTA, pH 7.4).

Membranes were prepared at 0°C by homogenization with a

Polytron followed by centrifugation (40,000 x g, 15 min), were washed once in 20 mM Hepes + 0.1 mM EDTA, pH 7. 4, and were stored at-70°C in the same buffer at protein concentrations of 2-5 mg/ml. Protein concentrations were measured with the BioRad reagent using bovine serum albumin as the standard. The yields of receptor varied from batch to batch but were approximately 10,1,2 and 2 pmol/mg of total membrane protein for the M1, M2, M3 and M4 subtypes respectively.

Radioligand binding assays: Unless otherwise stated, frozen membranes were thawed, resuspended in incubation buffer containing 20 mM Hepes + 100 mM NaCl + 10 mM MgCl2 (pH 7.4) and incubated with radioligand and unlabelled drugs for two hours at 30°C in a volume of 1 ml. Membranes were collected by filtration over glass fibre filters (Whatman GF/B) presoaked in 0.1% polyethylenimine, using a Brandel cell harvester (Semat, Herts, UK), extracted overnight in scintillation fluid (ReadySafe, Beckman) and counted for radioactivity in Beckman LS6000 scintillation counters. Membrane protein concentrations (5-50 ug/ml) were adjusted so that not more than about 15% of added radioligand was bound.

Nonspecific binding was measured in the presence of 10-6M QNB (an antagonist with picomolar potency) and accounted for 1-5% of total binding. GTP was present at a concentration of 2x10-4M in assays containing unlabelled ACh. Data points were usually measured in duplicate.

CHO cell membranes do not possess cholinesterase activity (Gnagey and Ellis, 1996 ; Lazareno and Birdsall, 1993) so ACh could be used in the absence of a cholinesterase inhibitor. The compounds were dissolved in dimethyl sulfoxide which, at the highest final concentration of 2%, had no effect on binding.

Experimental designs and data analysis: General data preprocessing, as well as the affinity ratio'calculations and routine plots of the semiquantitative equilibrium assay, were performed using Minitab (Minitab Ltd, Coventry, UK). The other assays were analysed with nonlinear regression analysis using the fitting procedure in SigmaPlot (SPSS Inc., Erkrath, Germany). This procedure is relatively powerful in that it allows the use of two or more independent variables, e. g. concentrations of two drugs.

Equilibrium binding assays for estimation of the affinity of an allosteric agent for the receptor and the magnitude of its cooperativity with 3H-NMS and ACh: The design and analyses have been described in detail (Lazareno and Birdsall, 1995 ; Lazareno et al, 1998).

Briefly, specific binding of a low concentration of 3H-NMS (1-2 times the Kd) was measured in the presence of a number of concentrations of test agent, all in the absence and presence of one or more concentrations of ACh. Specific binding of a high concentration of 3H-NMS (5-10 times Kd) was also measured. Nonlinear regression analysis was used to fit the data to the equation: Equation 1 <BR> <BR> <BR> <BR> <BR> <BR> Bmax. L. KL. (1 +a. (X. K) S)<BR> <BR> <BR> <BR> i+().y.(i+p.(n.(i+a.(.) where BLAX is observed specific bound radioligand, L, A, and X are concentrations of 3H-NMS, ACh and allosteric agent respectively, KL, KA and KX are affinity constants for the corresponding ligands and the receptor, a and B are allosteric constants of X with 3H-NMS and ACh

respectively, n is a logistic slope factor to describe the binding of ACh, and s is a Schild slope'factor to describe the binding of X. According to the allosteric model s should be 1.

Above a certain concentration, some allosteric agents, especially those which exhibit neutral or positive cooperativity with 3H-NMS, may slow the kinetics of 3H-NMS binding so much that the binding does not reach equilibrium. In most cases sufficient incubation time was used to allow 3H-NMS binding in the presence of the agent to reach equilibrium. In a few cases, however, the highest concentration of agent would be predicted to slow 3H-NMS kinetics sufficiently to prevent binding equilibrium from being reached, and in these cases the data were better fitted to the equation: <BR> <BR> <BR> <BR> <BR> <BR> <BR> Equation 2<BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> -t.koff t.koffL.KL<BR> BLAXt=BLAX+(BLo-BLAX).(exp( + ))<BR> 1+α.(X.Kx)s 1+(X.Kx)s+(A.KA)n.(1+ß.(X.Kx)s) where BLAXt is observed specific binding under nonequilibrium conditions, BLAX is the predicted equilibrium binding defined in Equation 1, t is the incubation time, koff is the dissociation rate constant of 3H-NMS, and BLO is the initial amount of bound radioligand, set to zero in this case. This equation assumes that the dissociation of 3H-NMS from the allosteric agent-occupied receptor is negligible, and that the binding kinetics of both ACh and the allosteric agent are fast in comparison with the dissociation rate of 3H-NMS.

If only a single concentration of ACh was used, the data were visualised with affinity ratio'plots, where the affinity ratio is the apparent affinity of the primary' ligand (3H-NMS or ACh) in the presence of a particular concentration of test agent divided by the apparent affinity of the primary ligand in the absence of test agent. Theoretically, the ECso or IC50 of the affinity ratio plot corresponds to the Kd of the test agent at the free receptor, and the asymptotic level corresponds to the cooperativity constant for the test agent and primary ligand (Lazareno and Birdsall, 1995). Affinity ratios were calculated from the specific binding data as follows (Lazareno and Birdsall, 1999): The affinity ratio of 3H-NMS in the presence of a single concentration of test agent is given by the equation: Equation 3 <BR> <BR> <BR> <BR> <BR> <BR> BLX.(BLI-BL)<BR> <BR> <BR> Y-<BR> BLI.BL.(1-q)-BLX.(BL-q.BLI) The affinity ratio of ACh the presence of a single concentration of test agent is given by the equation: Equation 4 <BR> <BR> <BR> <BR> <BR> <BR> BL.BLA.(BLI-BL).(BLX-BLAX)<BR> r A=<BR> <BR> <BR> BLAX.(BL-BLA).[BLI.BL.(1-q)-BLX.(BLX.(BL-q.BLI)] where BL is binding in the presence of the low [3H-NMS] alone; B., is binding in the presence of the high [3H- NMS] ; BLA is binding in the presence of the low [3H-NMS] and ACh; BLX is binding in the presence of the low [3H- NMS] and a particular concentration of test agent ; BLAX is

binding in the presence of the low [3H-NMS], ACh and the same concentration of test agent; L is the low 3H-NMS concentration ; LI is the high 3H-NMS concentration; and q is the ratio of low and high 3H-NMS concentrations, L/L1.

With assays containing a number of ACh concentrations, affinity ratio plots were calculated using the parameter estimates from the fit of the dataset to Equation 1 or 2 as appropriate (Lazareno and Birdsall, 1995).

The affinity ratio of 3H-NMS and ACh, rL and rA respectively, are given by the equations: Equation 5 <BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> 1 +α.X.K<BR> <BR> <BR> x<BR> <BR> rL 1 +X. K x and equation 6 <BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> 1+ß.X.Kx<BR> rA =<BR> <BR> <BR> 1+X.K x where the symbols are as described above.

Off-rate assay to estimate the affinity of an allosteric agent for the 3H-NMS-occupied receptor: A high concentration of membranes (2-4 mg protein/ml) was incubated with a high concentration of 3H-NMS (5 nM) for about 15 minutes. Then 10 pl aliquots were distributed to tubes which were empty or contained 1 ml of 10-6M QNB alone and in the presence of a number of concentrations of allosteric agent (typically n=4). Non-specific binding was measured in separately prepared tubes containing 10 pi membrane and 2 pl of 3H-NMS + QNB. Some time later, about 2.5 dissociation half-lives (see Table 2), the samples were filtered. The data were transformed

to rate constants, koff, using the formula: kOff=ln (Bo/Bt)/t where Bo is initially bound radioligand and Bt is bound radioligand remaining after t minutes dissociation.

These values were finally expressed as % inhibition of the true 3H-NMS dissociation rate constant (koff in the absence of allosteric agent) and fitted to a logistic function using nonlinear regression analysis.

Theoretically the curves should have slopes of 1, and correspond to the occupancy curves of the allosteric agents at the 3H-NMS-occupied receptors, regardless of whether the inhibition of 3H-NMS dissociation is caused by an allosteric change in the shape of the receptor or the trapping of the 3H-NMS in its binding pocket by the bound allosteric agent (Lazareno and Birdsall, 1995).

Initially the curve was fitted without constraints. If the slope factor was not different from 1, and the maximal inhibition (Emax') did not exceed about 100%, then the slope was constrained to 1 and the Emax was fitted. If the fitted Emax exceeded 100% (a physical impossibility, apart from experimental variation or error) then the Emax was constrained to 100 and the slope fitted. With the compounds under study the Emax was often less than 100, and in most such cases the data were well fitted with the slope constrained to 1.

GTPyS binding assay: Membranes expressing Mi receptors (5-20 ug/ml) were incubated with 35S-GTPYS (0.1 nM), GDP (10-'M) and ligands in incubation buffer in a volume of 1 ml for 30-60 minutes at 30°C. Bound label was collected by filtration over glass fibre filters prewetted with water.

Part 1 Results The structures of the compounds examined are shown on page 54. Figure 1 shows effects of compound lf (staurosporine) on equilibrium 3H-NMS binding at Mi receptors in the absence and presence of a fixed concentration of ACh. 3H-NMS binding was increased by staurosporine concentrations up to 10 uM and was reduced at 30 uM. The increase in 3H-NMS binding reflects a decrease in the Kd of 3H-NMS rather than an increase in Bmax (data not shown). The decrease in binding with 30 uM staurosporine is caused by the slowing of 3H-NMS kinetics by high concentrations of staurosporine (see below) and the consequent lack of equilibration of 3H-NMS binding (Lazareno and Birdsall, 1995). The effect of staurosporine on ACh binding is not clear from inspection of Figure 1, but nonlinear regression analysis of the data, which also takes into account the effects of high concentrations of staurosporine on the kinetics of 3H-NMS, provided a good fit to the data (lines in Figure 1) and revealed a 4-fold negative cooperativity between ACh and staurosporine. The independent effects of staurosporine on 3H-NMS and ACh binding across the four receptor subtypes are easier to visualise when the binding data are transformed into affinity ratios (Lazareno and Birdsall, 1995; Lazareno et al, 1998) (Figure 2). In theory, the ECso or ICso of the affinity ratio plot corresponds to the Kd of the test agent for the free receptor, and the asymptotic value corresponds to the cooperativity with the primary ligand. Staurosporine (If) showed positive cooperativity with 3H-NMS at MI and M2 receptors, neutral cooperativity with 3H-NMS at M4 receptors and was inactive or neutrally cooperative at M3 receptors. It had negative cooperativity with ACh at Mi, M2 and M4 subtypes and was neutral with ACh or inactive at

M3 receptors. Staurosporine had Kd values for unoccupied receptors in the uM range (Figure 2, Table 1). In two functional assays with Mi receptors measuring the stimulation by ACh of 35S-GTPyS binding, 10 uM staurosporine reduced basal activity and the Emax by 17% 7% and 25% 4% respectively, and also caused a 0.9 fold decrease in the potency of ACh, which is consistent with the 3.6-fold change predicted from the 3H-NMS binding studies (data not shown).

Staurosporine also inhibited 3H-NMS dissociation (Figure 3). All the curves had slope factors of 1.

Staurosporine was most potent and effective at Mi receptors, causing apparently complete inhibition of 3H-NMS dissociation with an ICso of 1 uM (Table 2). It was 3-4-fold weaker at the other receptor subtypes, and also caused submaximal inhibition of 3H-NMS'dissociation, with the smallest effect, 67% inhibition, seen at M3 receptors. The ICso values for the inhibition of 3H-NMS dissociation correspond in theory to the Kd values of staurosporine for the 3H-NMS-liganded receptors, and the values at Mi and M2 receptors are consistent with the values predicted from the equilibrium binding studies according to the allosteric model (Table 2). There was a 2-fold disparity between predicted and observed values at M4 receptors, probably because of inaccuracies in measuring the small degree of negative cooperativity with 3H-NMS. In equilibrium binding studies at M3 receptors staurosporine had little or no effect on the binding of either 3H-NMS or ACh: the clear inhibition of 3H-NMS dissociation caused by staurosporine over the same concentration range suggests that staurosporine was neutrally cooperative with 3H-NMS and ACh at M3 receptors, rather than inactive.

Go 7874 (li), a ring-opened analogue of staurosporine still bearing a positive charge, showed weak negative cooperativity with 3H-NMS and stronger negative cooperativity with ACh at Mi, M2 and M4 receptors, and the reversed pattern at M3 receptors (Figure 2). It was necessary to introduce a slope factor >1 into the binding equation for Go 7874 in order to fit the data adequately to the allosteric model (Table 1). Go 7874 caused apparently complete inhibition of 3H-NMS dissociation at M1, M2 and M4 receptors, and submaximal inhibition at M3 receptors. The slopes of the curves at Mi, M2 and M4 receptors were also >1 (Figure 3, Table 2). The ternary complex allosteric model does not predict slope factors different from 1, so it cannot provide a complete mechanistic explanation of the data. Nevertheless, the affinity values of Go 7874 for the 3H-NMS-occupied receptor predicted by the model from the equilibrium binding studies are in excellent agreement with the observed values at Mi and M4 receptors (Table 2), and show only a 3-fold discrepancy at M2 and M3 receptors, caused possibly by a combination of inaccuracies in the measurement of the small cooperative effects which occurred in equilibrium studies at the M2 and M3 subtypes (Table 1) and the small. inhibitory effect on 3H-NMS dissociation from M3 receptors.

KT5823 (le), a ring-contracted analogue of staurosporine in which the methylamino group is replaced by a methyl ester, caused a large increase in 3H-NMS binding at Mi and M receptors, and showed neutral or small positive cooperativity with ACh at these receptors. KT5823 was inactive-or. neutrally cooperative with 3H-NMS and ACh at M3 and M4 receptors (Figure 2). The positive cooperativity with NMS at Mi receptors was confirmed in functional studies in which 1mM KT5823 increased the

potency of ACh 1.9 0.9 fold at Mi receptors for stimulating 35S-GTPyS binding, and caused a 3.3 1.7 fold increase in the affinity of unlabelled NMS (n=2, data not shown). KT5823 inhibited 3H-NMS dissociation completely at M1 receptors, 80% at M2 receptors and 30-40% at M3 and M4 receptors (Figure 3). The affinity of KT5823 for the 3H-NMS-occupied receptor estimated from equilibrium studies at Mi and M2 receptors was very similar to the values measured directly. The inhibition of 3H-NMS dissociation seen at M3 and M4 receptors may indicate that KT5823 is neutrally cooperative with 3H-NMS and ACh at these receptors, rather than inactive.

KT5720 (la), a hexyl ester analogue of KT5823, was positively cooperative with both 3H-NMS and ACh at M1 receptors (Figure 1, Table 1). The small (40%) increase in ACh affinity was confirmed in more detailed assays (Figure 5). KT5720 had little or no effect at M3 receptors, and showed neutral cooperativity with 3H-NMS and negative cooperativity with ACh at M4 receptors. The- effects of KT5720 at M2 receptors are unclear: earlier batches had small inhibitory effects with 3H-NMS and ACh (Figure 2), while a later batch had small positive effects with 3H-NMS (data not shown) : no batch-dependent effects were noted at the other subtypes. KT5720 caused incomplete inhibition of 3H-NMS dissociation at M1, M3 and M4 receptors, with little or no effect at M2 receptors (Figure 3). The largest effect was seen with M1 receptors, and, at this subtype alone, low concentrations of KT5720 caused a small but consistent increase in 3H-NMS dissociation. This phenomenon was observed in l0 out of 11 single-time point assays, with the dissociation rate constant (koff) of 3H-NMS increased by 11 1 % (n=10) in the presence of the most effective concentration between 10 and 300 nM KT5720, and in two full time course studies

in which koff in the presence of 0.1 uM KT5720 was increased by 16.3 0.5 % (data not shown). The affinity of KT5720 for the 3H-NMS-occupied receptor estimated from equilibrium studies at M1 and M4 receptors was similar to the values measured directly (Table 2).

K-252a (lb), in which the methoxy group of KT5823 is replaced by a hydroxyl group, showed positive cooperativity with 3H-NMS at M1 receptors and neutral or small negative cooperativity with ACh (Figure 2, Table 1). Little or no effect was seen in equilibrium binding studies with the other subtypes. K-252a inhibited 3H-NMS dissociation at Mi receptors, apparently by 100%. Slope factors > 1 were required to fit the data adequately.

Only small, though consistent, effects on 3H-NMS off rate were seen at the other subtypes (Figure 3, Table 2).

K-252b (1c), K-252c (lg), KT-5926 (1d) and Go 6976 (1h) at concentrations up to 10 uM had little or no effect on equilibrium binding of 3H-NMS and ACh and on 3H-NMS dissociation (data not shown) and were not studied further.

We have attempted to determine whether some of the allosteric effects described above occurred through an interaction at the same site on the receptor at which other known allosteric agents act. Figure 5a shows the interaction between KT5720 (la) and gallamine on equilibrium 3H-NMS binding at Mi receptors. Gallamine had its expected inhibitory effect on 3H-NMS binding, and KT5720 showed the expected positive cooperativity with 3H-NMS. If gallamine and KT5720 were acting at the same site then gallamine should have become less potent in the presence of KT5720 and the nonlinear regression analysis would have indicated strong negative cooperativity

between the two agents. In fact, the analysis revealed neutral cooperativity, i. e. in equilibrium binding studies gallamine and KT5720 interact allosterically at Mi receptors through distinct and apparently non-interacting sites. In similar experiments with staurosporine and gallamine at M1 receptors, however, there was an negatively cooperative or competitive interaction between the compounds (Figure 5b).

In order to study the site (s) on the Mi receptor at which KT5720 acts to affect 3H-NMS dissociation, the concentration-related effect of KT5720 was measured alone and in the presence of two or three concentrations each of gallamine, brucine and staurosporine. Very similar results were obtained in two independent assays, and the combined data are shown in Figure 6. The data in each condition are shown in two forms: as percentage inhibition of the overall control (i. e. true') koff measured in the absence of any test agent, and, for each curve, as a fraction of its own control koff measured in the presence of test agent and the absence of KT5720.

This latter fractional effect'measure has useful properties: if the interaction between KT5720 and the test agent is competitive, then in the presence of test agent the ECso will increase and the asymptotic fractional effect'will also change; if the interaction is noncompetitive and noninteracting (i. e. with neutral cooperativity), and if maximal concentrations of test agent completely inhibit 3H-NMS dissociation, then in the presence of test agent both the ECso and asymptotic levels are unchanged.

The lines in the top panel of Figure 6 (except in the presence of staurosporine) are hyperbolic fits to the data. The effect of low concentrations of KT5720 to

increase 3H-NMS dissociation was apparent in all the curves. When the data are expressed as a fractional effect of own control, the curves for KT5720 in the presence of various concentrations of gallamine or brucine overlap, i. e. they have the same ECso and asymptotic level. There was a small concentration- related increase in potency in the presence of gallamine, but this is probably experimental noise, since a positively cooperative interaction would result in decreases in the asymptotic level of the fraction of own control'plots. These data therefore demonstrate that KT5720 acts at a different site from the site (s) at which gallamine and brucine act to inhibit 3H-NMS dissociation from M1 receptors.

A quite different pattern of results was seen with staurosporine. The stimulating effect of low concentrations of KT5720 became more apparent, and the curves tend to converge at high concentrations of KT5720 more than in the presence of gallamine or brucine. It was not possible to measure ECso values accurately, but inspection of the fractional effect'plot suggests that staurosporine reduced the potency of KT5720. These results indicate that staurosporine and KT5720 compete for the site which mediates inhibition of 3H-NMS dissociation. They strongly suggest that staurosporine can act at different site (s) from gallamine or brucine.

Discussion Five of the nine indolocarbazoles which we have studied act allosterically at muscarinic receptors. Of these, four have similar structures and a number of similarities in their allosteric effects, while the fifth, Go 7874 (li), lacks the tetrahydrofuran/pyran ring system, which may account for its somewhat different effects.

In equilibrium binding studies, the four active staurosporine-like compounds (staurosporine (1f), KT5823 (le), KT5720 (la) and K-252a (lb)) showed only positive or neutral cooperativity with 3H-NMS, or were apparently inactive, while positive, neutral and negative cooperativity was observed with ACh. The four compounds showed their highest affinity, and largest positive effects with 3H-NMS, at the M1 receptor, while they were inactive (or neutrally cooperative with 3H-NMS and ACh) at M3 receptors. These compounds bound with slope factors of 1, except for KT5823 at Mi receptors, and this exception may be partly accounted for by artefacts arising from the strong (7-10 fold) positive cooperativity with 3H-NMS seen with this compound. Go 7874 (li), the other positively charged ligand in addition to staurosporine, also showed selectivity for the M1 receptor but, in contrast to the other four compounds, it showed negative cooperativity with 3H-NMS, and both neutral and negative cooperativity with ACh, and it bound with slope factors greater than 1.

The four staurosporine-like compounds also showed selectivity for the 3H-NMS-occupied M1 receptor, but this was manifest more clearly in the magnitude of inhibition of 3H-NMS dissociation than in the affinity. Again, these compounds bound to the 3H-NMS-occupied receptor with slopes of 1, except for K-252a at Mi receptors. GO 7874 inhibited 3H-NMS dissociation completely from M1, M2 and M4 receptors with slope factors significantly greater than 1.

There seems to be a relationship between the activity of the compounds in equilibrium binding assays and the maximum degree of inhibition of 3H-NMS dissociation: an ad hoc correlation for the current data is that compounds showing less than 50% inhibition of 3H-NMS dissociation at

a particular subtype appear. inactive in equilibrium studies, while those slowing 3H-NMS dissociation by > 50% show activity in equilibrium studies. This rule works in 17/20 cases, the exceptions being staurosporine at M3, Go 7874 at M3 and KT5720 at M4 receptors. The positive relationship between allosteric activity at equilibrium and the degree of inhibition of 3H-NMS dissociation may reflect the degree to which binding of the allosteric agent perturbs the primary ligand recognition site on the receptor. Those cases where the test agent inhibits 3H-NMS dissociation but appears to be inactive at equilibrium may actually reflect a lack of cooperative effect, i. e. neutral cooperativity, rather than a lack of binding of the test agent at equilibrium.

According to the allosteric model, the affinity of a test agent for the 3H-NMS-occupied receptor may be estimated in two independent ways: from direct measurement of effects on 3H-NMS dissociation, and from the product of affinity for the free receptor and cooperativity with 3H-NMS, measured at equilibrium. In this study there are 11 instances where these measures have been determined with sufficient precision to allow comparison. There was good agreement between the measures : three comparisons differed by about 3-fold, one by about 2-fold, and the rest (7) by 60% or less, and there was no obvious bias since in 5 cases the equilibrium estimate was larger than the directly measured value and in 7 cases it was smaller. These results suggest that the data can be accounted for by the allosteric model, even though the steep slopes seen with Go 7874 (li) and K-252a (lb). are not predicted by the model.

The simple model also cannot account for the effects of KT5720 on 3H-NMS dissociation at Mi receptors, with an

initial speeding of dissociation by about 15 % at submicromolar concentrations, followed by submaximal inhibition of dissociation at higher concentrations. In the presence of staurosporine the speeding effect became more prominent, while the potency of KT5720 for slowing 3H-NMS dissociation appeared to be reduced, : that KT5720 may be exerting its effects at two distinct sites, only one of which can also be occupied by staurosporine. In contrast, the presence of gallamine or brucine had no effect on the potency of KT5270 or its fractional asymptotic effect, suggesting that, unlike staurosporine, gallamine and brucine act at a different site from the site (s) by which KT5720 modulates 3H-NMS dissociation, and that there is no interaction (i. e. neutral cooperativity) between the binding of KT5270 and that of brucine or gallamine.

A similar conclusion can be drawn from equilibrium binding studies at M1 receptors, in which KT5720 showed no interaction with gallamine. In contrast, similar equilibrium binding studies at Mi receptors with staurosporine and gallamine revealed a negatively cooperative or competitive interaction. The different interactions with gallamine shown by staurosporine (negative) and KT5720 (neutral) may be related to the fact that staurosporine, like gallamine, is a positively charged molecule, whereas KT5720 is neutral.

These results demonstrate that KT5720, and possibly other indolocarbazoles, bind to an allosteric site on muscarinic receptors which is distinct from the common allosteric site'to which gallamine and most other allosteric agents bind. Previously reported allosteric agents have a positively charged nitrogen which is thought to be important for their action. Staurosporine

and Go 7874 are also positively charged, but the other active indolocarbazoles are neutral, which suggests that there is no necessity for a positively charged nitrogen at this new allosteric site. The observed affinities and cooperativities are sensitive to small changes in the chemical structure of the analogues. For example, increasing the alkyl chain length of the ester function of K-252a or methylation of its hydroxyl group increase affinity 3-15-fold, whereas removal of the methyl group on the ester of K-252a or the alkoxy substitution of the indolocarbazole ring generate apparently inactive compounds.

The agents studied here are known to be potent inhibitors of various protein kinases, and in most cases the agents have much higher affinity for these targets than for muscarinic receptors, but it is worth noting that KT5720 has only about 6-fold higher potency for its preferred target, protein kinase A (PKA), than for the M1 receptor (log affinity of 7.2 at PKA vs. 6.4 at M1 receptors).

One of our aims has been the development of drugs which enhance the affinity of ACh at M1 receptors while having no effect on ACh binding and function at the other subtypes. The detection of the allosteric properties of KT5720 may be a step towards that goal. KT5720 was the most potent compound at Mi receptors with a log affinity for the free receptor of 6.4, and it showed a small (40%) but consistent positive cooperative effect with ACh. In addition it had little or no effect on ACh affinity at the other subtypes, so KT5720 is close to displaying an absolute subtype selectivity'for the M1 receptor, i. e. a positive or negative interaction with ACh at one receptor subtype and neutral cooperativity at the others, so that whatever concentration of agent is administered only the

one receptor subtype is affected functionally (Lazareno and Birdsall, 1995).

The results in this section show a quite potent allosteric interactions of staurosporine and some other indolocarbazole analogues at muscarinic receptors which, at least in the case of KT5720, occur at a site distinct from the common allosteric site'. The active indolocarbazoles cause different maximal effects on 3H-NMS dissociation, and the size of the maximal effect on 3H-NMS dissociation is a good predictor of the activity detected in equilibrium studies, suggesting a common mechanism for the two effects. In general the results from equilibrium and dissociation assays were mutually consistent with the ternary allosteric complex model as the underlying mechanism of the observed effects. Finally, KT5720 is the most potent agent described so far showing positive cooperativity with ACh at M1 receptors.

Part 2 Results We also examined the interaction of a second family of compounds represented by general formula 2 shown on pages 55 and 56. Figure 7 shows. representative data from off rate assays for these structures, and the parameter estimates are summarised in Table 3. Figures 8 and 9 show representative affinity ratio plots from equilibrium assays, and the parameter estimates are summarised in Table 4.

2b (WIN 51708) strongly inhibited 3H-NMS dissociation at M2 and M4 receptors, with about 10-fold M4 selectivity, it caused submaximal inhibition at the M1 receptor and had no effect at the M3 receptor. 2c (WIN 62577), containing a 5-6 double bond, which reduced the affinity for the

3H-NMS-occupied M4 receptor by about 10-fold and led to a smaller maximal effect at M2 receptors. These effects were reflected in the equilibrium assays, where 2b showed a small degree of positive cooperativity with 3H-NMS at M2 receptors and a larger positive effect at M4 receptors, while 2c, which was up to 5-fold less potent, showed only small negative cooperativity with 3H-NMS. With respect to ACh, 2b showed small negative cooperativity at M1 and M3 receptors and larger negative effects at M2 and M4 receptors, while 2c was also negative at M2 and M4 receptors, was almost neutral at M1 receptors, and showed a small (1.5-fold) positive interaction with ACh at M3 receptors. This latter effect was confirmed in more detailed assays (e. g. Figure 10). It is worth noting that the potency and small degree of negative cooperativity with 3H-NMS of both 2b and 2c at M3 receptors should result in some activity at M3 receptors in the off rate assay, whereas no activity was observed.

Removal of the bridgehead nitrogen from 2c gave 2a and led to a 10-fold increase in affinity at M1 and M2- receptors, a 30-fold increase at M3 receptors, but little or no change in affinity at M4 receptors. With a log affinity of 6.5 it was the most potent of these compounds at M1 receptors. It showed 2-5 fold negative cooperativity with both 3H-NMS and ACh across the receptor subtypes. In the off rate assay 2a had the unique effect of speeding 3H-NMS dissociation. It caused a 2-3 fold increase in 3H-NMS off rate at M3 receptors, with smaller effects at the other subtypes, with the order of effectiveness M3>M1>M4>M2. This effect was confirmed in full dissociation assays (Figure 11).

Replacement of the ethynyl and hydroxy groups of 2a with a keto function, in 2f and 2i, resulted in a complete

loss of activity in the off rate assay (Figure 7) and the equilibrium assay (data not shown).

Removal of the ethynyl substituent from 2b and 2c gave rise to the 17-hydroxy analogues, 2d and 2e. The compounds had slightly greater potency than their corresponding analogues in the off rate assay, and larger inhibitory effects at M1 receptors, but still little or no effect at the 3H-NMS-occupied M3 receptor. In equilibrium studies (Figure 8) 2d showed positive cooperativity with 3H-NMS at all subtypes, and its log affinity of 7 at the free M4 receptor was the most potent interaction in this study. 2e showed small negative, neutral and positive effects with 3H-NMS. Both compounds had negative cooperativity with ACh, except 2e which showed a small (30%) positive cooperativity with ACh at M3 receptors.

2g and 2j are analogues of 2b lacking two rings of the steroid moiety of 2b. 2g is reported to be the trans isomer and 2j is reported to be the cis isomer. 2b itself has the trans configuration, so it is very surprising that 2g was virtually inactive in the off rate assay (Figure 7) and equilibrium assays (data not shown), while 2j showed strong activity in the off rate assay (albeit 10-100 fold weaker than 2b), and in equilibrium assays, where it was strongly negative with 3H-NMS and ACh, and only 2-5 fold less potent than 2b.

21 is a truncated form of 2j, and no longer chiral. It was also active, showing similar potency to 2j in the off rate assay (and a bigger effect on M3 receptors), but less potency in equilibrium assays, especially at M2 and M4 receptors (Figure 9), leading to positive cooperativity with 3H-NMS at these subtypes.

2h is the pyrimidoimidazole analogue of 2c, lacking the fused benzene ring. This change caused a reduction in affinity for both free the 3H-NMS-occupied receptor of 2- 20 fold, and stronger negative cooperativity with 3H-NMS and ACh.

2k is an analogue of 2h with the imidazole ring attached to a different portion of the pyrimidine ring. It had a 30-200 fold higher affinity than 2h at 3H-NMS-occupied receptors, or more than 1000-fold at M3 receptors if the small effects at this subtype have been correctly interpreted. It also had 10-50 fold higher affinity for the free receptor than 2h, with Kd values of less than 1 mM at all subtypes. It showed strong positive cooperativity with 3H-NMS at Mi receptors (Figure 9), and was positive with 3H-NMS at M2 and M4 receptors and weakly negative at M3 receptors. It had 2-4 fold negative cooperativity with ACh.

2t, an analogue of 2k but with a pyrimidoimidazole substituent on the 16,17 positions of the steroid backbone, was inactive, reinforcing the importance of substitution in this region for activity.

Most compounds which bind to the primary or allosteric sites of muscarinic receptors have a basic nitrogen, though neutrally charged antagonists have been described.

The two steroid structures (2m and 2n), which do not contain nitrogen, correspond to the steroid portion of 2c, 2h, 2a and 2k. Surprisingly, they show activity in both the off rate and equilibrium assays, and 2m seems to be more potent than 2h in the off rate assay (Figure 7).

The 17-keto analogue, 2s, and the. 2-substituted analogues, 2o-2r appeared to be inactive.

With regard to slope factors, most of the compounds had binding slopes of 1 in equilibrium or kinetic assays. The relatively weak compounds 21,2j and 2h however had steeper slopes indicating a more complex interaction.

The effect of 2a to increase 3H-NMS dissociation provides an opportunity to assess whether 2a binds to the same site on the 3H-NMS-occupied receptor as other allosteric agents. The dissociation rate constant (koff) of 3H-NMS from the M3 receptor was measured at a single time point alone and in the presence of a range of concentrations of 2a, alone and in the presence of one or more concentrations of a second agent. The data were expressed as % of control koff, as described in Methods, and fitted to Equation 1. The results are shown in Figure 12 and Table 6. Strychnine and gallamine reduced the Emax of 2a, indicating that they were acting at a different site from that occupied by 2a, but they did not affect the EC50 of 2a, indicating that there was no cooperative interaction, i. e. they showed neutral cooperativity. In contrast, la (KT 5720), lf (staurosporine) and 2b (WIN 51708) did not appear to alter the Emax of 2a but reduced its potency, and the data were well fitted assuming a competitive interaction, although a strongly negative interaction cannot be ruled out. These results demonstrate that there are two allosteric sites on the M1 and M3 receptors. It is worth noting that 2b (WIN 51708) clearly binds to the 3H-NMS- occupied M3 receptor, even though it does not modify the koff of 3H-NMS.

Discussion This paper describes a new series of compounds which interact allosterically with muscarinic receptors. The initial lead was provided by two commercially available

compounds, 2b (WIN 51708) and 2c (WIN 62577). These compounds are potent antagonists at rat NK1 receptors, but the affinity of these compounds, exemplified by 2b, is reduced 400 fold at the human NK, receptor. In fact our results show that 2b is up to 60 fold more potent on human muscarinic receptors compared to human neurokinin receptors.

These two compounds and the analogues described in this paper exhibit positive, neutral and low negative cooperativities with NMS and especially ACh. This latter characteristic is important in that it suggests that the allosteric enhancers at a specific muscarinic receptor subtype may be synthesised in this series which are neutrally cooperative with ACh (and therefore inactive at any concentrations) at other subtypes. This form of selectivity, based on cooperativity rather than affinity, has been termed absolute subtype selectivity' (Lazareno et al, 1998; Birdsall et al, 1999) and is a direct consequence of the ternary complex allosteric model (Lazareno and Birdsall, 1995) which underpins the analyses of all our binding and functional data.

The ternary complex allosteric model implies that the affinity of a compound for the 3H-NMS-occupied receptor can be estimated in two ways: as the product of affinity for the f-ree receptor and cooperativity with 3H-NMS from equilibrium assays, and as the reciprocal of the IC50 (or EC50) from off rate assays. Table 5 shows a comparison between the pKocc (from equilibrium studies). and the pKoff (from off rate studies) values of those compounds for which there are at least two observations of each type of measure. Of the 32 comparisons, 23 show a discrepancy between the two measures of less than 0.3 log units (2-fold), a value which is readily accounted for by

experimental error. In most other cases the discrepancy can be accounted for by inaccuracies either observed in the data or predicted from the effect of the compound.

The discrepancies of 2b and 2c at Mi receptors may be explained by the small effects in the off rate assay, 2d has small effects at the M3 receptor in the off rate assay, and at the M4 receptor lower concentrations should have been used to define better the parameters. 2j has strong negative cooperativity at M2 and M4 receptors which cannot be measured accurately with the experimental designs used here, and 2k has very small effects in the off rate assay with M3 receptors. That leaves the 3-fold discrepancies for both 2d and 2k at M2 receptors, for which there are no obvious explanations. Two outliers out of 32 observations may be within the expected variability of such data, and overall the data pass this rather stringent test and are therefore consistent with the ternary complex allosteric model as the underlying mechanism which is responsible for effects on both equilibrium binding and on 3H-NMS dissociation.

2b, 2c and and the analogues examined can be considered in simplistic terms as a fusion of a planar aromatic heterocyclic system with an alicyclic ring system, especially a steroid structure. Most surprisingly both the steroid moiety alone, for example 2n, (but not some other analogues) and the heterocyclic ring system (21) are individually capable of interacting allosterically with 3H-NMS and with comparable affinities to each other.

This result implies that these compounds may interact with different but contiguous subdomains of the same pharmacophore.

Binding to the allosteric site is sensitive to the nature of the heterocycle when the steroid ring is kept constant

(compare 2h, 2a and 2k with 2c). Equally the nature of the 17-substituent on the steroid ring seems important in the fused systems. The analogues with a 17-keto function appear inactive, whereas all compounds with a 17ß hydroxyl group are active. The presence of the 17-a ethynyl group has more subtle effects (mainly on the M4 receptor) and these seem to be interrelated with effects of the saturation status of the 5-6 bond.

Another surprising result is that 2j is active whereas 2g appears to be inactive. Both these compounds are racemates and they represent truncated analogues of 2b with the AB rings of the steroid in the cis and trans configurations respectively. Activity was expected to be associated with the trans isomer rather than the cis isomer.

Many of the compounds investigated in this study, in contrast to most muscarinic allosteric agents, do not inhibit the association and dissociation of 3H-NMS completely at high concentrations. They often only produce a 2-fold or less slowing effects on the kinetics and in some instances, especially at M3 receptors, very small effects indeed (Figure 7).

The remarkable finding is that 2a increases the dissociation rate of 3H-NMS with the largest effect being observed at M3 receptors and the smallest effect at M2 receptors. It is noteworthy that the data from equilibrium and off rate studies with 2a are entirely consistent with the allosteric model, i. e. there are no discrepancies between estimates of affinity for the 3H-NMS-occupied receptor from the two types of assay, and the slope factors were 1 or close to 1. The agreement occurs despite the enhancement of 3H-NMS dissociation by

2a being the opposite to the inhibitory effect seen with all the other compounds in this study, and in every other published study of allosteric agents at muscarinic receptors.

This unique effect of 2a allowed us to assess whether other allosteric agents inhibit 3H-NMS dissociation by acting at the same site as 2a. We found that la (KT5720), lf (staurosporine) and 2b bind to the same site as 2a on the 3H-NMS-liganded receptor, but gallamine and strychnine bind to a different site and have no effect on the binding of 2a, i. e. gallamine and strychnine show neutral cooperativity with 2a.

In conclusion, the following general points can be made.

(1) allosteric agents can enhance, inhibit, or have no effect on the dissociation rate of 3H-NMS. (2) There are at least two nonoverlapping allosteric sites, the common'site and the WIN'site ; (3) Both allosteric sites can support a positive cooperative interaction with ACh; (4) The use of 2a provides a test of whether another allosteric agent binds to the WIN'site.

References: The references mentioned herein are all expressly incorporated by reference.

Caulfield and Birdsall, International Union of Pharmacology XVII: classification of muscarinic receptors. Pharmacol. Rev., 50: 279-290,1998.

Hulme et al, Ann. Rev. Pharmacol., 30: 633-73,1990.

Lazareno and Birdsall, Br. J. Pharmacol., 109: 1110-1119, 1993.

Lazareno and Birdsall, Mol. Pharmacol., 48: 362-378,1995.

Lazareno et al, Mol. Pharmacol., 53: 573-589,1998.

Lazareno and Birdsall, Measurement of competitive and allosteric interactions in radioligand binding studies.' In: G protein-coupled receptors. Haga and Berstein, eds, CRC press, Boca Raton, 1-48,1999.

Birdsall et al, Mol. Pharmacol. 55: 778-786,1999.

Buckley et al, Mol. Pharmacol., 35: 469-476,1989.

Gnagey and Ellis, Biochem. Pharmacol., 52: 1767-1775, 1996.

Bajwa and Sykes, J. C. S. Perkin I, 1618-1620,1978.

Bajwa and Sykes, J. C. S. Perkin I, 1816-1820,1979.

Bajwa and Sykes, J. C. S. Perkin I, 3085-3094,1979.

Bajwa and Sykes, J. C. S. Perkin I, 481-486,1980.

Bajwa and Sykes, J. C. S. Perkin I, 1019-1030,1980.

Bajwa and Sykes, J. C. S. Perkin I, 1859-1861,1980. R1 R2 R3 H 0 N 1 a KT5720 OH COO (CH2) 5CH3 t) 1b K-252a OH COOCH3 ruz 1 c K-252b OH COOH H N N J/ ,,, 0 1d KT5926 OH COOCH3 O (CH2) 2CH3 H ts 0 R1 1e KT5823 OCH3 COOCH3 H R2 H 0 N 1f Staurosporine WN N 4) ""Me OMe NHMe H n N R1 p N 1g K-252c H y- 1h Go 6976 CH3 CH2CH2CN R1 RNl2 Ru R2 H 0N 0 Q N \/ 1i Go 7874 HCI N N Me NMe2 Table 1 Equilibrium binding parameters of indolocarbazoles with 3H-NMS and Ach at muscarinic receptors. Assays such as those shown in Figures 2 and 5 were fitted to Equation 1 or 2 as appropriate (see Methods). The results are from at least 3 assays, except * n=2. Empty cells indicate that it was not possible to obtain at least 2 sets of parameter estimates.<BR> <BR> <P>&num Some, but not all, of the values were obtained from analyses in which the affinity for the 3H-NMS occupied receptor was fixed at the mean value obtained from offrate assays and shown in Table 2. M, M2 cooperativity cooperativity name pK slope'H-NMS ACh pK slope'H-NMS ACh la KT 5720 mean 6. 42" 1.00 1.94 1.39 5. 29 1.05 1.56 0.82 sem 0.09 0.00 0. 14 0.09 0.33 0.05 0.60 0.45 1b K-252a mean 5.13'" 1.00 2. 62 0.86 sem 0.14 0.00 0. 68 0.13 le KT 5823 mean 5.70 1.44 8. 11 1.31 5.67' 1.00 3.27 1. 34 sem 0.05 0.14 1. 61 0.44 0.15 0.00 0.19 0.03 1 f staurosporine mean 5.91 1.01 1. 53 0.19 5.13 1. 00 1.49 0.42 sem 0.03 0.01 0. 06 0.04 0.05 0.00 0.06 0.04 # i Go 7874 mean 5.77' 1. 60 0. 54 0.07 5.03' 1.91 0.66 0.24 sem 0.16 0.17 0. 04 0. 00 0.16 0.08 0.00 0.11 M, M4 cooperativity cooperativity name pK slope'H-NMS ACh pK slope'H-NMS ACh la KT 5720 mean 6. 42 1. 00 0.92 0. 55 sem 0.25 0.00 0.01 0. OS I b K-252a mean sem le KT 5823 mean sem I f staurosporine mean 5.31 1. 00 0.88 0.15 sem 0. 09 0.00 0.02 0.03 li Go 7874 mean 5. 12' 1. 50 0.32 0.72 5. 71' 1. 47 0.80 0.10 sem 0.01 0.50 0.09 0.05 0.13 0.21 0. 05 0.00 Table 2 % inhibition of 3H-NMS dissociation from muscarinic receptors by indolocarbazoles.

Curves such as those shown in Figure 4 were fitted to a logistic equation as described in Mothods. The'estd pK'is the product of affinity for the free receptor and cooperativity with 3H-NMS derived from the equilibrium binding assays summarised in Table 1. The'diff is the difference between the observed pK (-log IC50) and'estd pK'. Empty cells indicate that it was not possible to obtain at least 2 sets of parameter estimates. The 3H-NMS dissociation rate constants (minutes~') observed in this study are (mean sem (n)): M, 0.058 0.002 (26); M2 0.34 0.01 (12); M3 0.054 0.002 (10); M4 0.057 ~ 0. 002 (10). M, M2 name pK slope Emax estd pK d ; j' pK slope Emax estd kiff la KT 5720 mean 6.18 1.00 56.79 6.70 -0.52 5.36 sem 0. 15 0.00 3.81 0.09 0.26 n 7 6 5 1b K-252a mean 5.54 1.40 100.00 5.65 1.00 35. 05 sem 0.01 0.07 0.00 0.00 0.00 2.35 n 2 2 Ic KTS823 mean 6. 40 1. 00 103.5 6.58 -0.18 6.21 1.00 77.85 6.18 0.02 sem 0.01 0.00 3.15 0.08 0.05 0.00 1.95 0.18 n 2 4 2 2 If staurosporine mean 6. 01 1. 00 104. 73 6. 10 0. 05 5. 40 1.00 90.77 5.31 0.09 sem 0.08 0.00 5.28 0.04 0.02 0.00 8.34 0.06 n 35 33 li Go 7874 mean 5.70 1.34 100.00 5.50 0.19 5.30 1.31 100.00 4.85 0.45 sem 0.02 0.01 0.00 0.19 0.01 0.01 0.00 0.16 n 2 2 2 2 M, M, name pK slope Emax estd pK diff pK slope Emax estd pK diff I KT5720 mena 6.66 1.00 38.71 6.33 1.00 24.70 6.38 -0.05 sem 0. 15 0.00 1. 52 0.08 0.00 0.21 0.24 n 3 3 2 lb K-252a mean 5.5 1.00 37.10 5.37 1.00 48.95 sem 0.00 0.00 4.20 0.02 0.00 5.75 n 2 2 lc IT 5823 mean 6.45 1.00 29.80 5.88 1.00 41.50 sem 0.08 0.00 5.70 0.04 0.00 4.40 n 2 2 lf staurosporine mean 5.48 1.00 67.10 5.61 1.00 88.00 5.25 0.36 sem 0.07 0.00 2.77 0.08 0.00 6.20 0.08 n 3 3 3 li Go 7874 mean 5.05 1.00 39.50 4.60 0.45 5.71 1.64 100.00 5.60 0.11 sem 0.28 0.00 14. 00 0. 15 0.03 0.35 0.00 0. 10 n 2 2 2 2 Table 3 Parameter estimates from offrate assays. Emax indicates the maximal % inhibition of 3H-NMS dissociation.<BR> <P>E$mpty cells indicate that at least two reliable estimates were not obtained. Estimates could not be obtained for 2f, 2g #<BR> n>=3, exetp *n=2, &num n=1 M1 M2 M3 M4 Name pK slope Emax pK slope Emax pK slope Emax pK slope En 2a 987 mean 6.01 1 -91.90 5.81 1 -34.30 6.08 1 -146.35 . 6.04 1 -66 sem 0.04 0 10.80 0.19 0 12.24 0.06 0 17.99 0.09 0 8 2b WIN 51708 mean 5.55 1 49.71 5.93 1 94.66 6.78 1 95 690 sem 0.12 0 3.51 0.02 0 1.16 0.06 0 4 2c WIN 62577 mean 5 1 64.89 5.31 1.29 65.90 5.63 1 79 691 sem 0.22 0 23.48 0.15 0.29 10.90 0.06 0 3 2d 924 mean 5.85 1.11 92.25 6.08 0.97 102.29 6.10° 1 29.79 6.98 1 101 sem 0.05 0.12 4.36 0.03 0.03 2.26 0.12 0 6.09 0.03 0 3 2e 923 mean 5.32 1 88.52 5.58 0.99 93.12 6.19 1.02 97 sem 0.10 0 1.74 0.01 0.01 3.26 0.04 0.01 1 2h 986 mean 4.68 1.77 101.67 4.34 1.62 100 3.50 1 100 4.63 1.37 99 sem 0.04 0.08 1.67 0.09 0.09 0 0.02 0 0 0.08 0.12 0 2j 926 mean 4.71° 1.44 100 4.51° 1.02 101.10 4.72° 1 48.58 4.73° 1.53 87 cis sem 0.08 0.09 0 0.05 0.02 1.10 0.01 0 3.64 0.12 0.53 15 2k 988 mean 6.86 0.99 101.02 6.21 1.06 94.52 6.71 1 11.21 6.30 1 91 sem 0.04 0.01 1.46 0.05 0.06 1.99 0.16 0 1.47 0.07 0 3 21 925 mean 6.86 0.99 101.02 6.21 1.06 94.52 6.71 1 11.21 6.30 1 91 sem 0.02 0.08 0.22 0.01 0.07 1.88 0.02 0.06 0.77 0.04 0.07 1 2m 983 mean 5.17" 1 31.42 4.26" 1 105.20 4.92" 1 40.17 4.95" 1 90 sem 2n 982 mean 4.33 1 93.76 5.35 1 12.96 4.24 1.39 sem 0.09 0 9.85 0.56 0 1.53 0 0.04 Table 4<BR> Parameter estimates from equilibrium assays.<BR> <P>PK is the estimate of the log affinity of the agent for the free receptor.<BR> <P>'alloN' and 'alloA' are the estimates of cooperativity with 3H-NMS and Ach respectively.<BR> <P>Estimates could not be obtained for 2f, 2g and 2i.<BR> n>=3, except * n=2,&num n=1 Name Mi pK Mi slope M1 alloN M1 M2 M2 slope M2 M2 M3 M3 M3 M3 M4 pK M alloA pK alloN alloA pK slope alloN alloA 2a 987 mean 6.48 1.11 0.28 0.50 6.29 1.01 0.5 0.14 6.60 1.37 0.29 0.53 6.12 sem 0.07 0.13 0.02 0.04 0.10 0.01 0.02 0.05 0.06 0.16 0.01 0.04 0.09 2b WIN 51708 mean 5.77 1.24 0.25 0.44 5.93 1.22 1.70 0.22 5.46 1.33 0.46 0.74 6.18 690 sem 0.07 0.14 0.04 0.07 0.06 0.22 0.21 0.01 0.13 0.33 0.09 0.17 0.03 2c WIN 62577 mean 5.51 1 0.20 0.70 5.25 1 0.62 0.24 5.13 1.40 0.38 1.78 5.90 691 sem 0.08 0 0.06 0.08 0.26 0 0.13 0.13 0.17 0.24 0.14 0.50 0.17 2d 924 mean 5.67 0.73 1.74 0.26 6.5 0.87 1.33 0.36 5.28 1 1.79 0.37 7.02 sem 0.18 0.14 0.26 0.09 0.20 0.13 0.10 0.08 0.29 0 0.30 0.08 0.11 2e 923 mean 5.68 1 0.60 0.35 5.57 1 0.73 0.15 4.87 1 0.37 1.27 5.88 sem 0.03 0 0.04 0.02 0.11 0 0.09 0.09 0.36 0 0.20 0.21 0.14 2h 986 mean 4.81 1.33 0.26 0.15 4.79# 1.04 0.323 0.02 4.52# 1.42 0 0.12 4.99# sem 0.13 0.42 0.26 0.13 0.02 0.04 0.01 0.02 0.06 0.27 0 0.01 0.08 2j 926 mean 5.26# 1.23 0.04 0.18 5.27# 1.35 0.07 0.03 5.12# 1.58 0.02 0.23 5.52# cis sem 0.02 0.29 0.04 0.16 0.06 0.09 0.05 0.02 0.01 0.23 0.02 0.11 0.03 2k 988 mean 6.02 0.79 8.13 0.38 6.57 1.03 1.37 0.46 6.26 1 0.35 0.40 6.13 sem 0.14 0.21 1.53 0.12 0.15 0.26 0.07 0.01 0.05 0 0.09 0.07 0.05 2l 925 mean 4.72# 2 0.79 0.08 4.50# 2.01 1.71 0.02 4.81# 1.66 0.60 0.02 4.62# sem 0.01 0 0.15 0.01 0.09 0.01 0.17 0.02 0.07 0.09 0.07 0.02 0.05 2m 983 mean 4.57" 1 0 0.23 4.96" 1 0.87 0.35 3.90" 1 0 0.20 4.57" sem 2n 982 mean 4.31# 1 0 0.40 4.95# 1 1.02 0.56 4.13# 1 0.28 0.57 4.47# sem 0.09 0 0 0.09 0.36 0 0.03 0.08 0.22 0 0.28 0.05 0.12 Table 5 Comparison of affinity estimates at 3H-NMS-occupied receptors from equilibrium (pKocc) and offrate (pKoff) assays.'diff is the difference between pKocc and pKoff. n>=2 mi M2 Name pKocc sem pKoff sem diff pKocc sem pKoff sem diff 2a 987 5. 92 0.09 6.10 0.04-0.18 6.03 0.12 5.81 0.19 0. 22 2b WIN 51708 5. 14 0.13 5.55 0.12-0.41 6.15 0.06 5.93 0.02 0.22 2c WIN 62577 4. 63 0. 37 5 0. 22-0.37 5.02 0.36 5.31 0.15-0.29 2d 924 5. 90 0. 16 5.85 0.05 0.05 6.67 0.23 6.08 0.03 0.59 2e 923 5. 46 0. 05 5.32 0.10 0.13 5.43 0.17 5.58 0.01-0.16 2h 986 4. 29 0.04 4.34 0.09-0.05 2j ds 926 3. 97 0.32 4.51 0. 05-0. 55 2k 988 6. 91 0. 06 6.86 0.04 0. 05 6.70 0.12 6. 21 0.05 0.49 21 925 4. 60 0. 08 4.77 0.02-0.16 4.73 0.05 4.84 0.01-0.11 2n 982 4. 96 0.35 4.33 0.09 0.63 M3 M4 Name pKocc sem Pkoff sem diff pKocc sem pKoff sem diff 2a 987 6. 06 0.04 6.08 0.06-0.03 5.77 0.09 6.04 0.09-0. 2 2b WIN 51708 6. 67 0.07 6.78 0.06-0. 10 2c WIN 62577 5. 80 0.18 5.63 0.06 0.1 2d 924 5. 52 0.22 6.10 0.12-0.57 7. 35 0.15 6.98 0.03 0. 3 2e 923 6. 06 0. 05 6.19 0. 04-0. 13 2h 986 4. 40 0. 28 4. 63 0.08 -0.22 2j ds 926 3. 85 0.02 4.73 0.12-0. 88 2k 988 5. 77 0.18 6.71 0.16-0.94 6.31 0.06 6.30 0.07 0. 01 21 925 4. 58 0.12 4.60 0.02-0.02 4.69 0.09 4.78 0.04-0.09 2n 982 4. 34 0. 17 4.24 0 0.1 Table 6 Estimates of log affinity values for the 3H-NMS-occupied M3 receptor and maximal % inhibition of H-NMS dissociation rate, derived from competition assays with 2a analysed with the allosteric model (Equation 1 in Appendix 2). The cooperativity values were fixed at 0 (competitive or strong negative cooperative interaction) or 1 (neutral cooperativity, noninteracting). Slope values for the test agent were fixed at 1, except for staurosporine (slope=1.31 ~ 0. 16, n=3).

NamenpK Emax % cooperativity gallamine 2 3. 38 0. 02 1 strychnine 4 4.06 ~ 0. 01 1 KT 5720 3 6.11 ~ 0. 10 50 4 0 staurosporine 3 5.79 0.17 43 1 0 2b WIN 51708 3 5.78 ~ 0. 06 0 20 0 2k 988 2 no effect