CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of priority based on U.S. Provisional Application Nos. 61/087,519, 61/138,250 and 61/151,095 filed August 8, 2008, February 4, 2009, and February 9, 2009, respectively. The disclosure of each priority application is incorporated herein by reference in its entirety.

GOVERNMENT INTEREST

[0002] This invention was made with U.S. government support under grant nos. HL64035 and HL22231, awarded by the National Institutes of Health. The U.S. government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] The p ²¹ activated kinase- 1 (Pakl) belongs to a family of serine/threonine protein kinases directly activated by small GTPases, Cdc42 and Racl. Pakl is abundant in the heart and localizes to cell and nuclear membranes, intercalated discs and to Z-discs in ventricular myocytes. The active form of Pakl in cardiomyocytes increases Ca2+ sensitivity of myofilament force development through activation of PP2A (Ke 2004, p 194-200) and in SA nodal cells, Pakl inhibits isoproterenol-stimulated activation of L-type Ca2+ channel and delayed rectifier potassium channels (Ke 2007, p 1317-1327). Other studies have shown that in endothelial cells, Pakl activation induces dephosphorylation of myosin regulatory light chain and inhibition of thrombin-induced barrier dysfunction (Ke 2007, p 281-288) and in HeLa cells, expression of constitutively active Pakl induces loss of stress fibers and dissolution of focal adhesion complexes (Manser 1997, p 1129-1143). These studies suggest a role of Pakl in cytoskeletal function and reorganization. In transgenic mice expressing an active Racl in the heart, hypertrophy developed followed by dilated cardiomyopathy with altered intracellular partitioning of Pakl in the ventricle myocytes (Sussman 2000, p 875- 886).

[0004] A prominent post-translational modification of Pakl is autophosphorylation, which is correlated with its activity (Manser 1997, p 1129-1143; Zhao 1998, p 2153-2163). Pakl is autophosphorylated at seven serine/threonine sites most of which occur at the N-terminal half of the kinase. Substitution of threonine 423, the last autophosphorylation site, with glutamic acid renders the kinase constitutively active (Manser 1997, p 1129-1143). Although there is abundant expression of Pakl in cardiomyocytes, smooth muscle and endothelial cells, the function of Pakl in the cardiovascular system remains poorly understood (Sheehan 2007, p 963-973). Moreover, potential modifications in autophosphorylation of native Pakl in failing heart and in other pathological conditions have not been defined.

[0005] Studies in skeletal muscle have shown that Pakl activity was responsive to insulin treatment (Tsakiridis 1996, p 19664-19667) suggesting that Paks are also phosphorylated by tyrosine kinase (Bagheri-Yarmand 2001, p 29403-29409; He 2004, p 96-101; Yang 2004, p 658-667). Tyrosine phosphorylation of Pakl may also play an important role in regulation of cardiac function.

SUMMARY OF THE INVENTION

[0006] In one embodiment, the invention provides a method of treating a cardiac disorder in a mammalian subject comprising administering to the subject a Pakl agonist in amount effective to treat the disorder. Also provided is a method of treating a cardiac disorder in a subject in which treatment with a standard of care therapeutic is contraindicated, wherein the method comprises administering to the subject a Pakl agonist in amount effective to treat the disorder. In the context of methods of the invention, "standard of care" refers to a treatment that is generally accepted by clinicians for a certain type of patient diagnosed with a type of illness. In one embodiment, the contraindicated standard of care therapeutic is a β-blocker.

[0007] Exemplary cardiac disorders include, but are not limited to systolic heart failure, ischemia, arrhythmias, atrial fibrillation, atherosclerosis and angina pectoris. In some embodiments, the Pakl agonist is a sphingolipid or analog thereof. In one embodiment, the Pakl agonist is selected from the group consisting of a ceramide (including, but not limited to C2-ceramide and C6-ceramide), safingol, D-sphingosine and sphingosine 1-phosphate and molecules structurally related to C2/C6 ceramide and demonstrate the same functional effects in cardiac cells as the active Pakl does including inhibition of heart rate (pacemaking activity), such as fingolimod (FTY720) and FTY720 derivatives that contains one or more unsaturated carbon bonds in its hydrophobic side chain.

[0008] Some embodiments of the methods described herein further comprise administering a standard of care therapeutic to the subject. For cardiac disorders, for example, an aspect of the invention is to improve standard of care therapy with co-therapy by administering one or more Pakl agonists described herein. Exemplary standard of care therapeutics include, but are not limited to, β-blockers (if not contraindicated), nitrates and calcium-channel blockers. In one embodiment, the Pakl agonist and the standard of care therapeutic are administered concurrently (e.g., in either a single formulation or separate formulations). In another embodiment, the Pakl agonist and the standard of care therapeutic are administered sequentially.

[0009] In some embodiments, the subject is human. In one embodiment, the human is asthmatic.

[0010] Methods of identifying a Pakl agonist are also provided. The method comprises contacting a candidate compound with purified Pakl, wherein a compound that enhances autophosphorylation of Pakl compared to Pakl autophosphorylation in the absence of the compound is identified as a Pakl agonist. Another embodiment of the invention includes the identification of Pakl antagonists. For example, the invention provides a method of identifying a Pakl antagonist comprising contacting a candidate compound with purified Pakl in the presence of a Pakl agonist, wherein a compound that inhibits autophosphorylation of Pakl in the presence of the agonist compared to Pakl autophosphorylation in the absence of the compound is identified as a Pakl antagonist. Exemplary candidate compounds include, but are not limited to, a nucleic acid, a polypeptide or fragment thereof, an antibody or antigen-binding fragment thereof, and a small molecule.

[0011] In some embodiments, the contacting step of the method is performed in a reaction mixture that includes a radioactive phosphorous reagent (including, but not limited to, ( P gamma ATP), and phosphorylation is determined by detecting a radioactive phosphorous attached to the purified Pakl polypeptide.

[0012] In some embodiments, the methods comprise purifying the Pakl polypeptide from a cardiac sample. In one embodiment, the purified Pakl is isolated from a heart muscle homogenate with a decoy Pakl peptide. In some embodiments, the decoy Pakl peptide comprises a portion of a Pakl sequence linked to a heterologous peptide (including, but not limited to an hemagglutinin (HA) epitope).

[0013] In some embodiments, the decoy Pakl peptide comprises amino acids 131-150 of the human Pakl polypeptide sequence set forth in SEQ ID NO: 1. In one embodiment, the decoy Pakl peptide comprises YNSKKTSNSQKYMSFTDKSAYPYDVPDYA (SEQ ID NO: 3). In another embodiment, the decoy Pakl peptide comprises MSNNGLDIQDKPPAPPMRNTSTYPYDVPDYA (SEQ ID NO: 4). [0014] Other aspects of the invention include use of a Pakl agonist described herein in the manufacture of a medicament for the treatment of a cardiac disorder. In some embodiments, the medicament is for the treatment of a cardiac disorder where treatment with a standard of care therapeutic is contraindicated. In one embodiment, the medicament is for the treatment of a cardiac disorder selected from the group consisting of systolic heart failure, ischemia, arrhythmias, atrial fibrillation, atherosclerosis and angina pectoris.

[0015] This document is intended to be related as a unified disclosure, and it should be understood that all combinations of features described herein are contemplated, even if the combination of features are not found together in the same sentence, or paragraph, or section of this document.

BRIEF DESCRIPTION OF THE FIGURES

[0016] Figure IA shows the localization and expression of Pakl in rat ventricle myocytes detected by immuno-fluorescence and confocal microscopy.

[0017] Figure IB is a schematic representation of purification of Pakl from cardiac muscle.

[0018] Figure 1C. The decoy peptide is derived from the autoinhibitory region of Pakl. The autoinhibitory domain of Pakl is downstream of p21 binding domain (PBD) and by dimerization domain (DI). The vertical bars represent the proline rich sequences in Pakl.

[0019] Figure 2A is a picture of a gel demonstrating the purity of Pakl at various stages of the affinity chromatography method described in Example 1. Lane 1 : Cardiac muscle extracts resolved on a SDS page. Lane 2: The cardiac muscle extract after fractionation by 25% and 50% (NH4)2SO4 precipitation. Lane 3 : Pakl purified from the fractionated muscle sample. Lane M: The molecular weight markers.

[0020] Figure 2B shows the detection of purified Pakl by Western blotting analysis. Pakl was detected as a single band with an antibody from Santa CruZ (sc-881).

[0021] Figure 2C shows purified Pakl detected by mass spectrometry. Peaks match the Pakl peptides produced by theoretical trypsin digestion. The positions of amino acids of each peptide from Pakl were denoted in quotation: LSAIFR (amino acids 490-495 of SEQ ID NO: 1), ELLQHQFLK (amino acids 514-522 of SEQ ID NO: 1), SVIEPLPVTPTR (amino acids 204-215 of SEQ ID NO: 1), KELIINEILVMR (amino acids 309-320 of SEQ ID NO: 1 ), ECLQALEFLHSNQVIHR (amino acids 372-388 of SEQ ID NO: 1). [0022] Figure 3A demonstrates that autophosphorylation of Pakl is stimulated by sphingo sine-phosphate in vitro.

[0023] Figure 3B demonstrates that autophosphorylation of Pakl is stimulated by D- erythro-sphingosine in vitro.

[0024] Figures 4A-4C demonstrate that autophosphorylation of Pakl is stimulated by C2- ceramide, C6-ceramide and saphingol, respectively, in vitro.

[0025] Figures 5A and 5B demonstrate that autophosphorylation of Pakl is stimulated by FTY720.

DETAILED DESCRIPTION

[0026] The present invention is based on the discovery that compounds that stimulate or enhance of Pakl autophosphorylation can be used for the treatment of various cardiac disorders.

[0027] I. Screening Assays for Modulators of Pakl Autophosphorylation

[0028] One aspect of the present invention is directed to methods of identifying compounds that modulate Pakl autophosphorylation. Such methods provide lead compounds and therapeutics for modulating Pakl activity and cellular responses that are influenced by Pakl. Generally, such methods involve introducing one or more candidate compounds (alone or simultaneously) into a controlled system containing a purified Pakl polypeptide, to determine whether the candidate compound modulates the autophosphorylation of Pakl.

[0029] To identify a candidate compound as being capable of modulating (stimulating/enhancing or inhibiting/decreasing) Pakl autophosphorylation, one measures Pakl autophosphorylation in the absence of the added candidate compound. A candidate agonist/antagonist compound is then contacted with Pakl and the autophosphorylation (or lack thereof) of Pakl is determined in the presence of the candidate agonist/antagonist compound. A candidate antagonist compound would decrease or inhibit Pakl autophosphorylation, relative to the same parameter in its absence, whereas a candidate agonist will increase or stimulate Pakl autophosphorylation, relative to the parameter in its absence.

[0030] In one aspect, the invention provides methods of identifying a Pakl agonist comprising contacting a candidate compound with purified Pakl, wherein a compound that enhances autophosphorylation of Pakl compared to Pakl autophosphorylation in the absence of the compound is identified as a Pakl agonist. Such screening techniques are useful in the general identification of a compound that will modulate Pakl autophosphorylation, with such compounds being useful as therapeutic agents. For example, those compounds that stimulate or increase phosphorylation of Pakl are useful in the treatment of a cardiac disorder because it is shown herein that enhanced Pakl autophosphorylation results in the deophosphorylation of cardiac troponin I in cardiomyocytes

[0031] In another aspect, the invention provides methods of identifying a Pakl antagonist comprising contacting a candidate compound with purified Pakl, wherein a compound that reduces or inhibits autophosphorylation of Pakl compared to Pakl autophosphorylation in the absence of the compound is identified as a Pakl antagonist. Such screening techniques are useful in the general identification of a compound that will modulate Pakl autophosphorylation, with such compounds being useful as therapeutic agents. For example, those compounds that inhibit or decrease autophosphorylation of Pakl are useful in the treatment of disorders with aberrant Pakl autophosphorylation.

[0032] In some embodiments, the Pakl polypeptide used in the screening methods described here is first purified by affinity chromatography using a decoy Pakl polypeptide comprising an hemagglutinin (HA) epitope.

[0033] In some embodiments, the decoy Pakl polypeptide comprises a portion of the Pakl polypeptide, wherein, the decoy Pakl polypeptide comprises amino acids 131-150 of SEQ ID NO: 1 (YNSKKTSNSQKYMSFTDKSA). In another embodiment, the decoy Pakl polypeptide comprises amino acids 1-22 of SEQ ID NO: 1

(MSNNGLDIQDKPPAPPMRNTST). In some embodiments, the Pakl polypeptide or decoy Pakl polypeptide is fused to other sequences to make fusion proteins, and/or contain additional moieties such as labels and tags. For example, in one embodiment, the Pakl polypeptide or portion thereof is linked to a nine amino acid HA epitope (YPYDVPDYA, SEQ ID NO: 2).

[0034] In some embodiments, the purified Pakl polypeptide used in the screening methods described herein comprises a full-length Pakl polypeptide (amino acids 1-545 of SEQ ID NO: 1; Genbank Accession No. AAC50590. In other embodiments, the purified Pakl polypeptide comprises a continuous portion of the full-length Pakl polypeptide that comprises one or more serine/threonine autophosphorylation sites selected from the group consisting of S21, S57, T84, S144, S149, S199, S204, T212, S220, S223, T225, T230 and T423 of SEQ ID NO: 1.

[0035] In one aspect, phosphotyrosine levels are measured by standard in vitro techniques well known in the art, such as, and without limitation, enzyme-linked immunosorbant assay (ELISA), radio immunoassay (RIA), Western blot, or immunofluoroescence-based assays. In one aspect, Pakl activity ismeasured in biological samples using fluorescent microscopy with fluorescently labeled anti-Pakl and anti-phosphotyrosine antibodies. Detection is correlated, for example, by a brighter staining signal in a fluorescent microscopy assay, the presence of more staining in a fluorescent microscopy assay, or by decreased fluid levels of phosphorylated Pakl as detected by Western blot, ELISA, RIA or other immunofluoroescence-based assays, such as fluorescence resonance electron transfer (FRET). It is also contemplated to measure phosphorylated Pakl using antibodies that specifically recognize Pakl and which differentiate between a phosphorylated versus unphosphorylated form.

[0036] An anti-phosphotyrosine antibody or anti-phosphorylated Pakl antibody suitable for use in the method of the invention optionally comprisea a label, such as a radioisotope, a fluorophore, a fluorescing protein (e.g., natural or synthetic green fluorescent proteins), a dye, an enzyme, a substrate, or the like. Such labels include, without limitation, biotin molecules, alkaline phosphatase, fluorophores (e.g., fluoroisothiocyanate, phycoerythrin, Texas red, Alexa Fluor stains, and other fluorescent dyes well known in the art), radioisotopes (e.g., ³ H, Europium ^3"1" , ³² P), genetically engineered peptide tags such as a histidine (HiS ₆ ) tag linked to the aggregating polypeptide, a myc-tag, a Hemagluttinin (HA) tag, and the like. Biotin, fluorophores, and other contemplated small molecules comprising a label can be linked to the polypeptide by means well-known in the art such as a commercially produced Biotinylation kit (Sigma Chem. Co., St. Louis, MO), or alternative methods commonly used in organic chemistry to attach a small molecule to a peptide or protein (see e.g., Current Protocols in Protein Chemistry, John Wiley & Sons, 2001). Genetically engineered tags, e.g., His ₆ and myc-tags, are operably linked to the polypeptide using standard recombinant DNA methods well known in the art (see e.g., Current Protocols in Molecular Biology, supra), or using conventional peptide synthesis techniques. Such labels facilitate quantitative detection with standard laboratory machinery and techniques.

[0037] High-content screens (HCS) provide for analysis of multiple parameters in a single screening assay. For example, in one aspect, autophosphorylation of a Pakl polypeptide, or a portion thereof, is measured using phosphotyro sine- specific antibodies that fluoresce in a particular excitation channel (e.g., Alexa Fluor 488 excites at 488 nm). Antibodies to additional cell markers which excite at different wavelength ranges (i.e., in different channels) are then added to the same assay.

[0038] In one aspect, the candidate compound is a substance capable of modulating (enhancing/stimulating or decreasing/inhibiting) Pakl autophosphorylation. The candidate compound may be a small molecule, a nucleic acid molecule, a peptide, a polypeptide, an antibody, a synthetic compound, and a naturally-occurring compound. Screening of chemical libraries consisting of both chemically synthesized and natural compounds and combinatorial libraries, are specifically contemplated.

[0039] In certain aspects, chemical libraries contain known compounds, proprietary structural analogs of known compounds, or compounds that are identified from natural product screening.

[0040] Natural product libraries are collections of materials isolated from natural sources, typically, microorganisms, animals, plants, or marine organisms. Natural products are isolated from their sources by fermentation of microorganisms followed by isolation and extraction of the fermentation broths or by direct extraction from the microorganism or tissue (plant or animal) themselves. Natural product libraries include polyketides, non ribosomal peptides, and variants (including non naturally occurring variants) thereof. See Cane et al., Science, 282:63 68 (1998), incorporated herein by reference.

[0041] Combinatorial libraries are composed of large numbers of related compounds, such as peptides, oligonucleotides, or other organic compounds as a mixture. Such compounds are relatively straightforward to design and prepare by traditional automated synthesis protocols, PCR, cloning or proprietary synthetic methods. Of particular interest are peptide and oligonucleotide combinatorial libraries.

[0042] Still other libraries of interest include peptide, protein, peptidomimetic, multiparallel synthetic collection, recombinatorial, and polypeptide libraries. For a review of combinatorial chemistry and libraries created thereby, see Myers, Curr. Opin. Biotechnol., 8:701 707 (1997), incorporated herein by reference.

[0043] In other aspects, modulators of Pakl autophosphorylation identified by assessment of the candidate compounds are formulated into compositions which optionally include pharmaceutically acceptable (i.e., sterile and non-toxic) liquid, semisolid, or solid diluents that serve as pharmaceutical vehicles, excipients, or media. Candidate compounds suitable for administration as therapeutics will exhibit acceptable toxicity levels as would be known in the art or determinable by one of skill in the art using routine experimentation.

[0044] In some embodiments, compositions comprising one or more modulators of Pakl autophosphorylation are packaged in forms convenient for delivery. In some embodiments, the compositions are enclosed within a capsule, caplet, sachet, cachet, gelatin, paper, or other container. The dosage units are packaged, e.g., in tablets, capsules, suppositories or cachets.

[0045] Antibodies are contemplated as inhibitors of Pakl autophosphorylation. The term "antibody" is used in the broadest sense and includes fully- assembled antibodies, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (including bispecific antibodies), chimeric antibodies, human antibodies, humanized antibodies, antibody fragments that can bind an antigen (including, Fab', F'(ab) ₂ , Fv, single chain antibodies, diabodies), and recombinant peptides comprising the foregoing as long as they exhibit the desired biological activity. Multimers or aggregates of intact antibodies and/or fragments, including chemically derivatized antibodies, are contemplated. Antibodies of any isotype class or subclass, including IgG, IgM, IgD, IgA, and IgE, IgGl, IgG2, IgG3, IgG4, IgAl and IgA2, or any allotype, are contemplated. Standard techniques are employed to generate polyclonal or monoclonal antibodies directed against Pakl and to generate useful antigen-binding fragments thereof or variants thereof. Such protocols can be found, for example, in Sambrook et al., Molecular Cloning: a Laboratory Manual. Second Edition, Cold Spring Harbor, N. Y.: Cold Spring Harbor Laboratory (1989); Harlow et al. (Eds), Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y. (1988). Peptibodies are also contemplated. The term "peptibody" refers to a molecule comprising an antibody Fc domain attached to at least one peptide, which has specific binding properties. The production of peptibodies is generally described in PCT publication WO 00/24782, the disclosure of which is incorporated herein by reference.

[0046] Hn one embodiment, the invention provides in vitro screening methods. In such screening methods, the purified Pakl or a fragment thereof is either free in solution, fixed to a support, or expressed in or on the surface of a cell. Either the polypeptide or the candidate compound is optionally labeled, thereby permitting determination of binding.

[0047] II. Therapeutic Methods [0048] In one embodiment, methods of treating a cardiac disorder in a subject are provided comprising administering to the subject a Pakl agonist in amount effective to treat the disorder. Exemplary cardiac disorders include, but are not limited to, systolic heart failure, ischemia, arrhythmias, atrial fibrillation, atherosclerosis and angina pectoris.

[0049] A "Pakl agonist" as described herein means a compound capable of enhancing or stimulating Pakl autophosphorylation. In one embodiment, the Pakl agonist is a sphingolipid. In another embodiment, the Pakl agonist is selected from the group consisting of ceramide (including, but not limited to, C ₂ -ceramide and C _ό -ceramide), safingol, D- sphingosine and sphingosine 1 -phosphate, and FTY720 (fingolimod) as well as its derivatives with one or more unsaturated carbon bonds introduced to its hydrophobic side chain. In another embodiment, the Pakl agonist is a compound identified in the screening methods described herein.

[0050] In another embodiment, Pakl agonists are administered to subject in which treatment with a standard of care therapeutic is contraindicated. For example, methods of treating a cardiac disorder in a subject in which treatment with a β-blocker is contraindicated (i.e., a diabetic subject) are also provided. Such methods comprise administering to the subject a Pakl agonist in amount effective to treat the disorder. The term "β-blocker" or "beta-blocker" as used herein refers to one or more agents that antagonize the effects of beta- stimulating catecholamines by blocking the catecholamines from binding to the beta- adrenergic receptors. Exemplary beta-blockers include, but are not limited to, acebutolol, alprenolol, atenolol, betantolol, betaxolol, bevantolol, bisoprolol, carterolol, celiprolol, chlorthalidone, esmolol, labetalol, metoprolol, nadolol, penbutolol, pindolol, propranolol, oxprenolol, sotalol, teratolo, timolol and combinations, mixtures and/or salts thereof.

[0051] In some embodiments, the method further comprises administering of a standard of care therapeutic (one that is not contraindicated) to the subject. For cardiac disorders, for example, an aspect of the invention is to improve standard of care therapy with co-therapy with one or more of the Pakl agonists described herein. In some embodiments, the standard of care therapeutic comprises a β-blocker, a statin-related agent (including, but not limited to, lovastatin, atorvastatin, pravastatin, simvastatin and fluvastatin), nicotinic acid, a fibrate (including, but not limited to, bezafibrate, ciprofibrate, clofibrate, gemfibrozil and fenofibrate), bile acid resin(s) (including, but not limited to, cholestyramine, colestipol and cholsevelam), salicylic acid, and a phytosterol (including, but not limited to, stigmastanol, sitosterol, sitostanol and policosanol), [0052] In some aspects, the methods are used to treat subjects with a cardiac disorder who do not respond to treatment with standard of care therapeutics. In other aspects, the methods are used to augment treatment with a standard of care therapeutic. Treatment with a Pakl agonist as described herein in conjunction with a second therapeutic is required in some instances because, e.g., the patient may only partially respond to Pakl agonist therapy.

[0053] In one embodiment, the subject is a mammal, and in other aspects the subject is human. Practice of methods of the invention in other mammalian subjects, especially mammals that are conventionally used as models for demonstrating therapeutic efficacy in humans (e.g., primate, porcine, canine, or rabbit animals), is also contemplated.

[0054] In various aspects, the Pakl agonist and standard of care therapeutic are administered concurrently or sequentially. In embodiments where the Pakl agonist and standard of care therapeutic are administered separately, one would generally ensure that a significant period of time did not expire between the times of each delivery, such that the Pakl agonist and standard of care therapeutic(s) would still be able to exert an advantageously combined effect. In such instances, it is contemplated that both modalities would be administered within about 12-24 hours of each other. In some situations, it may be desirable to extend the time period for treatment significantly, from several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8). Repeated treatments with one or both agents is specifically contemplated.

[0055] III. Pharmaceutical Compositions and Routes of Administration

[0056] In some embodiments, the compounds identified in the methods described herein are delivered directly or in pharmaceutical compositions along with suitable carriers or excipients, as is well known in the art. The methods described herein comprise administration of an effective amount of a Pakl agonist for the treatment of a cardiac disorder. Typical pharmaceutical compositions are formulated to include various components as described in U.S. Patent No. 7,220,547, the disclosure of which is incorporated by reference for its disclosure of pharmaceutical composition formulations.

[0057] An effective amount, e.g., dose, of compound or drug can readily be determined by routine experimentation, as can an effective and convenient route of administration and an appropriate formulation. Various formulations and drug delivery systems are available in the art. (See, e.g., Gennaro, Ed. (2000) Remington's Pharmaceutical Sciences, supra; and Hardman, Limbird, and Gilman, Eds. (2001) The Pharmacological Basis of Therapeutics, supra.)

[0058] Suitable routes of administration may, for example, include oral, rectal, topical, nasal, pulmonary, ocular, intestinal, and parenteral administration. Primary routes for parenteral administration include intravenous, intramuscular, and subcutaneous administration. Secondary routes of administration include intraperitoneal, intra-arterial, intra- articular, intracardiac, intracisternal, intradermal, intralesional, intraocular, intrapleural, intrathecal, intrauterine, and intraventricular administration. The indication to be treated, along with the physical, chemical, and biological properties of the drug, dictate the type of formulation and the route of administration to be used, as well as whether local or systemic delivery would be preferred.

[0059] Pharmaceutical dosage forms of a compound of the invention may be provided in an instant release, controlled release, sustained release, or target drug-delivery system. Commonly used dosage forms include, for example, solutions and suspensions, (micro-) emulsions, ointments, gels and patches, liposomes, tablets, dragees, soft or hard shell capsules, suppositories, ovules, implants, amorphous or crystalline powders, aerosols, and lyophilized formulations. Depending on route of administration used, special devices may be required for application or administration of the drug, such as, for example, syringes and needles, inhalers, pumps, injection pens, applicators, or special flasks. Pharmaceutical dosage forms are often composed of the drug, an excipient(s), and a container/closure system. One or multiple excipients, also referred to as inactive ingredients, can be added to a compound of the invention to improve or facilitate manufacturing, stability, administration, and safety of the drug, and can provide a means to achieve a desired drug release profile. Therefore, the type of excipient(s) to be added to the drug can depend on various factors, such as, for example, the physical and chemical properties of the drug, the route of administration, and the manufacturing procedure. Pharmaceutically acceptable excipients are available in the art, and include those listed in various pharmacopoeias. (See, e.g., the U.S. Pharmacopeia (USP), Japanese Pharmacopoeia (JP), European Pharmacopoeia (EP), and British pharmacopeia (BP); the U.S. Food and Drug Administration (www.fda.gov) Center for Drug Evaluation and Research (CEDR) publications, e.g., Inactive Ingredient Guide (1996); Ash and Ash, Eds. (2002) Handbook of Pharmaceutical Additives, Synapse Information Resources, Inc., Endicott N.Y.; etc.) [0060] Pharmaceutical dosage forms of a compound of the present invention may be manufactured by any of the methods well-known in the art, such as, for example, by conventional mixing, sieving, dissolving, melting, granulating, dragee-making, tabletting, suspending, extruding, spray-drying, levigating, emulsifying, (nano/micro-) encapsulating, entrapping, or lyophilization processes. As noted above, the compositions of the present invention can include one or more physiologically acceptable inactive ingredients that facilitate processing of active molecules into preparations for pharmaceutical use.

[0061] Other aspects and advantages of the present invention will be understood upon consideration of the following illustrative examples, which are not intended to be limiting in any way.

EXAMPLES

Example 1: Purification of Pakl from a cardiac sample

[0062] Preparation of Muscle Sample. Frozen bovine ventricle muscle (200 g) was cut into small pieces and homogenized in a blender containing 1 liter homogenization buffer (50 mM Tris base, 5 mM EDTA, 2 mM EGTA, 1 mM DTT, 0.5 mM benzamidine, 0.1 mM PMSF, pH 7.2). The homogenized muscle sample was centrifuged at 450Og for 30 minutes. The supernatant fraction was saved and precipitated with ammonium sulfate (NH4)2SO4 at concentration 25% and 50% (w/v). Precipitates formed by (NH4)2SO4 addition between concentration 25% (w/v) and 50% (w/v) were saved and dialyzed against the homogenization buffer. After dialysis, the sample was filtered and lyophilized. For purification of Pakl from smaller amounts of sample, the tissues or cells were homogenized in mammalian extraction buffer (50 mM Tris, pH 7.5; 150 mM NaCl; 0.1% Nonide P40; Complete Protease inhibitor cocktail tablets [Roche Diagnostics, cat # 11 873 580 001]). The ratio of tissue:buffer was about 1:5 (v/v). The homogenate was centrifuged in a bench-top centrifuge at 4 ⁰ C. The supernatant fraction was saved for direct application onto the affinity column.

[0063] Peptide Synthesis and the Matrix. The peptides were synthesized in the University of Illinois at Chicago protein core facility. The decoy peptide

(YNSKKTSNSQKYMSFTDKSA-YPYDVPDYA) (SEQ ID NO: 3) contained the Pakl sequence from amino acids 131-150 linked to a 9 amino acid of HA epitope . The peptide derived from the N-terminal region of Pakl with the sequence

MSNNGLDIQDKPPAPPMRNTST-YPYDVPDYA (SEQ ID NO: 4) was also used in some purification. The Anti-HA Affinity Matrix (Cat. No. 1 815 016) was purchased from Roche Applied Science (9115 Hague Road, PO Box 50414, Indianapolis, IN 46250-0414). The affinity matrix contains antibody specifically bind to HA sequence in protein or peptide.

[0064] Affinity Chromatography. The column was first washed with 20 ml equilibration buffer (20 mM Tris, pH 7.5; 0.1 M NaCl; 0.1 mM EDTA). Decoy peptide (3 mg) was applied to the column to saturate the matrix with the peptide. The muscle sample (the lyophilized extract) was dissolved in buffer (20 mM Tris, pH 7.5; 0.1 M NaCl; 0.1 mM EDTA) containing protease inhibitor cocktail tablets. The sample was loaded onto the affinity column and then washed with 20 ml washing buffer (20 mM Tris, pH 7.5; 0.1 M NaCl; 0.1 mM EDTA; 0.05% Tween-20). The Pakl bound to the gel was eluted with equilibration buffer containing HA peptide (YPYDVPDYA) (SEQ ID NO: 2) at 1 mg/ml. HA peptide was removed from the sample by dialysis in buffer containing 20 mM HEPES, 5 mM NaCl, pH 7.2. The bound Pakl could also be eluted with 5 ml glycine buffer, pH 2.5 (50 mM glycine-HCl; 0.1 % Triton X-100; 0.15 M NaCl).

[0065] Data presented in Figure 2A demonstrate that Pakl is the major component in the eluted fraction as indicated by the resolution of the samples by SDS-PAGE with Coomassie brilliant blue staining (Figure 2A, lane 3). Western blotting analysis (Figure 2B) using an antibody (sc-881) identified the bands shown in Figure 2A as Pakl. The affinity purification procedure yielded about 200 μg of total protein from the 5 mg muscle protein extract prepared from the frozen ventricle with a yield of 4%. Therefore, the yield of final affinity purification product from the frozen ventricle is about 0.016% (w/w). Pakl bound to the matrix was eluted with 3 mg of HA peptide that displace the decoy peptide from the matrix. The HA peptide in the eluant was separated from Pakl protein by use of dialysis tubing permeable to proteins smaller than 12-14 kD (Figure IB). The bound Pakl can also be eluted with Glycine (0.1 M, pH2.0). However, elution with glycine sometimes produces an extra protein bands with molecular weight of 16 kD. Mass spectrometry analysis indicated that the major MS peaks from the digested protein purified matches with Pakl peptides after trypsin digestion (Figure 2C).

Example 2 - Autophosphorylation of Pakl by various sphingolipids

[0066] The following Example demonstrates that various sphingolipids, including C ₂ - ceramide and C _ό -ceramide) induced autophosphorylation of Pakl.

[0067] Kinase Assay. Pakl kinase activity was assayed in the following reaction buffer: 50 mM HEPES, pH 7.3, 10 mM MgC12, 1 mM DTT, 0.05% Triton X-100) with 10 μCi of [γ32P] ATP (Perkin Elma. [γ-32P] -Adenosine 5 ^V -triphosphate, Specific Activity: 3000Ci (lllTBq)/mmole, 5OmM Tricine (pH 7.6), Concentration: 5mCi/mL, Catal NEG502H250UC) at 37 ⁰ C for 2 h. The kinase reaction was stopped by adding SDS sample buffer followed by boiling for 5 min. In view of reports of sphingosine-1 inducing an increase in Pakl and Pak2 activity in vitro (Bokoch 1998, p 8137-8144; Roig 2001, p 195- 199), the effect of sphingosine and sphingosine-1 phosphate on Pakl autophosphorylation was also tested (Figure. 3A). The tested agonists (obtained from Calbiochem) included the following: C2 ceramide (N-acetyl-D-erythro-sphingosine), C6 ceramide (N-Hexanoyl-D- erythro-sphingosine) and safingol (Dihydro-L-threo-sphingosine). D-sphingosine (S 6879) and sphingosine 1 -phosphate (S 9666) were purchased from Sigma. Sphingosines were dissolved in DMSO. The total reaction volume was 20 μl. DMSO (2 μl) was included in each reaction including the control. Incorporation of 32P from gamma 32P into Pakl was directly detected by autoradiography after resolve the in vitro reaction products on SDS PAGE. Interestingly, sphingosine-1 phosphate had no significant effect on autophosphorylation of Pakl at 25 and 50 μM. When the sphingosine phosphate concentration increased to 100 and 200 μM, there was an increase in Pakl autophosphorylation. In purified Pakl sample, a 48 kD Pakl degradation products can sometimes be detected (Figure IA, lane 3). When Pakl activity increased, phosphorylation of the degradation products increased accordingly (Figure 3).

[0068] Results indicated that autophosphorylation of Pakl purified from cardiac muscle was highly responsive to C2 and C6 ceramide (Figure 4A and B). Autophosphorylation of the purified Pakl increased in the presence of 50 and 100 μM of D-Sphingosine-1 (Figure 3B), but saphingol (Dihydro-L-threo-Sphingosine) only slightly regulated Pakl autophosphorylation (Figure 4C). By gel filtration, the purified Pakl demonstrated a single major peak. The same experiments were repeated three times each.

[0069] Autophosphorylation of Pakl, which correlates with its activity, provided a measure of Pak-1 enzyme function. Initial studies (data not shown) indicated that autophosphorylation of the wt Pakl was much weaker than the constitutively active Pakl as shown by others (Manser 1997, p 1129-1143). In all the in vitro assays, we used 4 μg of Pakl protein. The smaller band (-48 kD) that could be Pakl degradation products was also phosphorylated. The purified Pakl also demonstrated kinase activity towards the decoy peptide (YNSKKTSNSQKYMSFTDKSA) (amino acids 131-150 of SEQ ID NO: 1). The decoy peptide contains Pakl autophosphorylation sites. The two serine residues that are auto-phosphorylated are situated at the end of the auto-inhibitory sequence. The decoy peptide slightly inhibited autophosphorylation of Pakl. However, it was also phosphorylated and there was a slight increase of the total kinase activity in the presence of the decoy peptide (data not shown).

[0070] Discussion:

[0071] The purified Pakl retained low catalytic activity as demonstrated by autophosphorylation in vitro. The constitutively active Pakl (T423E) has a much higher level of autophosphorylation than the endogenous Pakl under the same conditions. We found that autophosphorylation of the purified Pakl is enhanced by D-sphingosine-1, N-acetyle- sphingosine (C2 ceramide) and by hexanoyl-sphingosine (C6 ceramide). This is consistent with an earlier observation that the activities of Pakl and Pak2 are activated by sphingosine (Bokoch 1998, p 8137-8144; Roig 2001, p 195-199).(11, 12)(18, 19). Our finding that active Pakl induces dephosphorylation of cTnl and C-protein through activation of PP2A (Ke, 2004, p 194-200) provides a plausible mechanism for induction of protein dephosphorylation by sphingosine. For example, C2-ceramide is a known activator of PP2A (Dobrowsky 1993, p 15523-15530)(13)(20) and may be involved in Bcl2 dephosphorylation (Ruvolo 1999, p 20296-20300). In different types of cells, L- type Ca2+ channel activity was demonstrated to be depressed by C2 ceramide by an unknown mechanism (Chik 1999, p 175-183; Chik 2004, p 5682-5690). C2 ceramide also inhibited proliferation of T-cells stimulated by growth signals (O'Byrne 2000, p 225-230). Moreover, C2 ceramide has pre-conditioning effects in heart, which has been attributed to generation of reactive oxygen species (Furuya 2001, p 226-232; Lecour 2006, p 1702-1706). (18, 19)(25, 26). Our data indicate that other mechanisms for C2 ceramide involving activation of Pakl and PP2A need to be considered. To our knowledge, this is the first time demonstration that Pakl is directly stimulated by C2 and C6 ceramide. The mechanism whereby Pakl is activated by sphingosine analogs is still not clear. Our studies indicate that C2 and C6 ceramide had stronger effect than sphingosine- 1 activating Pakl. Saphingosine only had a small stimulatory effect on Pakl autophosphorylation. Zenke et al. suggest that sphingosine activates Pak through the P21 binding domain (PBD) (Zenke 1999, p 32565-32573). However, in another study, Cdc42 and sphingosine- 1 appears to have a cooperative effect activating the kinase (Chong 2001, p 17347-17353).

[0072] The peptide used to decoy native Pakl from cardiac muscle is derived from the auto-inhibitory region of Pakl (Manser 1997, p 1129-1143) (Figure 1C). The auto-inhibitory region is both downstream of and partially overlaps with the P21 binding domain (PBD) (Zhao 1998, p 2153-2163). A peptide derived from the N-terminal region (the first 21 amino acids of Pakl) that binds to Nek in vitro (Bokoch 1998, p 8137-8144; Zhao 2000, p 3906- 3917) also efficiently retained Pakl in the column (data not shown). The peptide interacts with the catalytic center of the kinase and may regulate the conformation of the catalytic center as demonstrated by NMR studies (Pirruccello 2006, p 312-326). Expression of the peptide plus the P21 binding region produced many cellular changes including inhibition of cell cycle progression (Thullberg 2007, p 1820-1828). The auto-inhibitory region was also deleted in GST-Pakl which was used for in vitro kinase studies (Polverino 1995, p 26067- 26070).

[0073] Pakl is auto-phosphorylated at multiple serine/threonine sites. There are at least six autophosphorylation sites at the N-terminal half of Pakl (Manser 1997, p 1129-1143). Autophosphorylation at the N-terminus of Pakl may change its intracellular localization or/and activity (Zhao 2005, p 201-214). It is unclear at which site autophosphorylation occurs first in the activation of Pakl. Furthermore, uncertainty still remains as to whether autophosphorylation is through inter-molecular or intra-molecular kinase reactions. Since Pakl dimer has an anti-parallel conformation, autophosphorylation sites at the N-terminal half of one Pakl are therefore placed in proximity to the catalytic domain of the other Pakl (Lei 2000, p 387-397). This suggests that inter-molecular autophosphorylation may occur at the N-terminal autophosphorylation sites.

[0074] The purified Pakl retained low catalytic activity as demonstrated by autophosphorylation in vitro. The constitutively active Pakl (T423E) has a much higher level of autophosphorylation than the endogenous Pakl under the same conditions. We found that autophosphorylation of the purified Pakl is enhanced by D-sphingosine-1, N-acetyle- sphingosine (C2 ceramide) and by hexanoyl-sphingosine (C6 ceramide). This is consistent with an earlier observation that the activities of Pakl and Pak2 are activated by sphingosine (11, 12)(18, 19). Our finding that active Pakl induces dephosphorylation of cTnl and C- protein through activation of PP2A (1), provides a plausible mechanism for induction of protein dephosphorylation by sphingosine. For example, C2-ceramide is a known activator of PP2A (13)(20) and may be involved in Bcl2 dephosphorylation (14)(21). In different types of cells, L-type Ca2+ channel activity was demonstrated to be depressed by C2 ceramide by an unknown mechanism (15, 16)(22, 23). C2 ceramide also inhibited proliferation of T-cells stimulated by growth signals (17)(24). Moreover, C2 ceramide has pre-conditioning effects in heart, which has been attributed to generation of reactive oxygen species (18, 19)(25, 26). Our data indicate that other mechanisms for C2 ceramide involving activation of Pakl and PP2A need to be considered. To our knowledge, this is the first time demonstrating that Pakl is directly stimulated by C2 and C6 ceramide.

[0075] Pakl contains a few proline-rich sequences at its N-terminus. The proline-rich sequences interact with proteins, such as Nek and Pix. A prominent feature of these proline- rich sequences in Pakl is that they are followed by autophosphorylation sites. Peptide decoy was first designed to identify proteins that interact with the proline rich sequences in the heart. It turned out Pakl is the only major protein that binds to the decoy peptides, including the peptide containing 22 amino acids (MSNNGLDIQDKPP APPMRNTST, amino acids 1-22 of SEQ ID NO: 1) at the N-terminal region of the kinase which contains an autophosphorylation site at Serine 21. Substitution of the serine residue for aspartic acid abolished Nek binding to the peptide in vitro (Zhao 2000, p 3906-3917). In vivo, phosphorylation at the autophosphorylationautophosphorylation sites could be a mechanism to release the "auto-substrate" from the catalytic center of the kinase.

[0076] Our understanding of Pakl function in myocardial cells is still in its infancy, and we think the method reported here is an important step in defining these functions and their mechanisms. Signaling molecules such as Pakl are often extensively modified by post- translational mechanisms, and in many cases these modifications are associated with pathophysiological conditions. Our method is suitable for study of post-translational modifications of Pakl in response to different extra-cellular signals and in pathological conditions such as heart failure, arrhythmias and ischemia. The Pakl kinase purified by this method can also be used for in vitro kinase assay to identify novel activators or inhibitors. Moreover, the method can be adapted for use in purification of Pak isoforms from mammalian tissues other than heart.

[0077] Interaction between lipids and a protein kinase may have profound effect on cellular functions (Argraves 2002, p 439-444). The purified Pakl is stimulated by more than one sphingosine species. It is likely that some other sphiongosine related lipids may also regulate Pakl activity. Modification of the side chain and the polar groups of sphingosines may produces agonists or antagonists that have even more potent effect on Pakl autopho sphoryl ation .

Example 3 — Autophosphorylation of Pakl by FTY720 [0078] FTY720 (fingolimod) is a drug candidate undergoing clinical trials for organ transplantation and multiple sclerosis. However, the same compound produces cardiac effect (such as a decrease of heart rate) at the same or even a lower dose than that used in the clinical studies intended for organ transplantation and multiple sclerosis. The intracellular target for FTY720 remains controversial.

[0079] The kinase assay described above in Example 2 was repeated using FTY720. The results of this assay are set forth below in Table 1.

[0080] FTY720 is a structural analogue of sphingosine-1 and C2/C6-ceramide. Example 2 demonstrates C2- and C6- ceramides can activate Pakl activity in vitro and induces dephosphorylation of cardiac troponin cardiomyoctes. We have evidence that FTY720 has the same functional property on Pakl and in ventricle myocytes. Therefore, like C2- and C6- ceramides, FTY720 can serve as a β-blocker competitor for ischemic heart diseases including angina and systolic heart failure. The anti-(β) adrenergic effect of FTY720 is through activation of Pakl in the heart. Our current observation further enforced by our previous work on Pakl fuction related to heart diseases. Our studies also indicate that even better drug candidates can be designed or screened by testing their effects on Pakl activation or inhibition using C2/C6 ceramides and FTY720 as prototypes.

[0081] All of the above U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet, are incorporated herein by reference, in their entirety.

[0082] From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. References cited:

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