Kenneth A. Jacobson - US grants

Work in our laboratory spanning more than two decades has demonstrated that certain drugs may be attached to well-defined carrier molecules and still retain the ability to bind to the receptor site and effect biological activity. This synthetic strategy for the attachment of drugs to carriers is termed the functionalized congener approach. The carrier molecule may be many times larger than the parent drug; indeed there is practically no maximum size limitation for a fully potent analog. Unlike the prodrug approach or the immobilization of drugs for slow release, the functionalized congener approach is designed to produce analogs for which no metabolic cleavage step is necessary for activation. Moreover, the attachment of the drug to a carrier such as a peptide may result in enhanced affinity at an extracellular receptor site and an improvement in the pharmacological profile of the parent drug through energetically favorable interaction with distal sites on a receptor. [unreadable] [unreadable] Purine derivatives containing attached chains to target distal sites of GPCRs have been developed as functionalized congeners that either activate or antagonize adenosine receptors, and a similar strategy has been used for ATP receptors. For example, the 2-position of the purine moiety has been identified for attachment of functionalized chains in ATP derivatives as P2X and P2Y receptor agonists. Reporter groups such as fluorescent dyes have been covalently attached resulting in receptor probes of relatively high affinity. The targeting of distal sites on the calcium sensing receptor has also been studied.[unreadable] [unreadable] Other means of altering pharmacokinetics of a known drug include the prodrug approach. We have prepared prodrugs of adenosine A3 agonists and antagonists, e.g. nuceloside derivatives, that are not themselves biologically active, but are able to be regenerated in biological systems. Studies of the cleavage of the blocked ligands indicates that the prodrugs are suitable as masked forms of the biologically active A3AR agonists and antagonists for future evaluation in vivo.[unreadable] [unreadable] The use of GPCR agonists for therapy has inherent limitations. The distribution of a given receptor in multiple tissues may lead to undesired side effects. Also, the desensitization of a receptor upon repeated agonist exposure may limit agonist utility. We are developing an alternate approach to achieve the beneficial effects of GPCR activation in a more spatially and temporally selective manner than the systemic administration of agonists to the native GPCR. This approach of neoceptors combines small molecule classical medicinal chemistry and gene or cell therapy. By this rational design approach, complementary structural changes are made in the receptor and ligand for selective enhancement of affinity. The spatially-selective activation of a neoceptor would be dependent on cell- or organ-target delivery of the gene. Molecular modeling, of GPCRs has been used widely to arrive at hypotheses for recognition of antagonists and agonists by ligand docking. We have are validated hypotheses for docking of ligands at purine receptors using site-directed mutagenesis. Site-directed mutagenesis and molecular modeling have been used to characterize the ligand binding sites of the P2Y1 and A3 receptors to predict which regions of a given ligand may be amenable to a chain attachment approach. With this knowledge and the ability to tailor-make new analogues of a native agonist, one may design a matched neoceptor and neoligand, i.e. the binding site of a given GPCR may be engineered to recognize synthetic agonist ligands that do not activate the native receptor. Distal sites of interaction on the engineered receptor may be targeted to allow selective enhancement of affinity in a functionalized congener. Based on predictions from molecular modeling, we have designed neoceptors for A2A and A3 adenosine receptors, in which a tailored ligand activates only engineered receptor. The success of the neoceptor strategy for the ARs validates the use of homology modeling, as well as suggests options for future therapeutics.[unreadable] [unreadable] We have explored the application of nanotechnology to the study of GPCRs. For example, dendrimers are tree-like polymers that have multiple functional sites on the periphery for attachment of ligands. We recently reported the first PAMAM dendrimer to which a GPCR ligand had been attached. This was a conjugate to which A2AAR functionalized congeners were covalently attached. The conjugate displayed potent antithrombotic activity in human platelets, while the parent dendrimer was inactive. We are using the multivalency of dendrimer conjugates to test for interactaction with dimeric and higher order multimeric GPCR assemblies.

The extracellular adenosine receptors have a modulatory role in the nervous, circulatory, endocrine and immunological systems. The prospect of harnessing these effects specifically for therapeutic purposes is attractive. We have recently synthesized highly selective A3 adenosine receptor agonists, antagonists, allosteric modulators. A3 agonists are under development for treating cancer, rheumatoid arthritis, and other diseases. Allosteric enhancers promise to be more specific in their action in an affected tissue, than a classical agonist, which can act at all locations of the receptor in the body. Imidazoquinoline and pyridinylisoquinoline derivatives were found to enhance the actions of agonists of the A3 receptor, and thus may prove to be suitable leads for the development of therapeutic agents based on this concept. We have identified two new classes of allosteric modulators of the A3 receptor and are currently exploring the structure activity relationship (SAR). The potential of A3 agonist therapy is of great interest. We are collaborating with Dr. Bruce Liang and Dr. Asher Shainberg on various aspects of the use of adenosine receptor agonists in protection of the heart. We have designed a mixed agonist of A1 and A3 subtypes, both of which are protective in the heart. A mixed A1/A3 agonist is protective in a model of ischemia in skeletal muscle. The adenosine A3 agonist IB-MECA is currently in clinical trials for use in autoimmune inflammatory diseases and cancer conducted by our CRADA partner Can-Fite Biopharma. This compound has already shown clinical efficacy in Phase 2 trials for treatment of rheumatoid arthritis, psoriasis, and dry eye disease. IB-MECA (CF-101) has just entered Phase 3 clinical trials for dry eye disease. The 2-chloro analogue is currently in clinical trials for liver cancer, and it was shown to reduce viral load in several patients that are infected with hepatitis C virus. Other more selective A3 agonists from our lab, such as the conformationally constrained MRS3558, are of interest for their protective properties. One of the issues in the development of adenosine receptor ligands is the species dependence. Some compounds that are very potent at a given human adenosine receptor are weak in rat tissue. We have developed adenosine agonists and antagonists that work generally across species. This is important for testing the same compounds in various animal models before suggesting the feasablity of their use in humans. The key to A3 receptor ligands that are potent and selective across species is the use of the nucleoside structure as the starting point in the design process. Nucleosides tend to bind to that receptor subtype with greater consistency across species than nonpurine heterocycles. Adenosine A3 antagonists may be useful for the treatment of glaucoma. Early efforts to identify antagonists of the A3 receptor in our library involved screening of chemically diverse libraries. One of the limitations of this approach is that the antagonists often bind well only at the human, but not murine A3 receptors. We are currently developing other novel A3 antagonists based on nucleotide structures, that have proven to be generally applicable across species. We are currently studying systematically the SAR of adenosine derivatives that affect efficacy as A3 adenosine receptor agonists. Surprisingly, a commonly used A1-selective agonist, cyclopentyladenosine, was found to act as a pure antagonist at the A3 subtype. Other nucleosides may be chemically modified, especially on the ribose moiety, to have reduced efficacy at the A3 receptor. Some of these analogues derived from highly potent A3 agonists, such as 5'-truncated nucleosides, were found to be A3 antagonists. Several novel nucleoside-based antagonists of the A3 receptor, including a rigid spirolactam derivative MRS1292 and a truncated 4'-thioadenosine derivative (collaboration with Prof. Lak Shin Jeong, EHWA Univ., Seoul Korea) were found to lower intraocular pressure a mouse model of glaucoma (demonstrated by Prof. Mort Civan, Univ. of Pennsylvania). We are using mutagenesis to study the determinants of recognition of adenosine within the binding site of the A2A and A3 receptors, and proposing conformational factors involved in receptor activation. Since the four subtypes of adenosine receptors have been cloned it has been possible to conduct molecular modeling of the receptor protein, based on sequence analyses and homology modeling using the high resolution rhodopsin structure as template. We intend to use such a modeling approach for the design of more selective adenosine receptor agonists and antagonists. Recently this project has also focused on the effects of adenosine agonists and antagonists in the central nervous system and in the heart and on the possibility of therapeutics for treating neurodegenerative and cardiovascular diseases. An A3 agonist, administered chronically, proved to be highly cerebroprotective in an ischemic model in gerbils. A3 agonists cause morphological and biochemical changes in astroglial cells. Adenosine is released in large amounts during myocardial ischemia and is capable of activating both A1 or A3 receptors that occur on cardiac myocytes to exert a potent cardioprotective effect. We have shown that synthetic adenosine agonists,selective for either the A1 or A3 subtype, protect ischemic cardiac myocytes in culture and in the isolated perfused heart and thus might be beneficial to the survival of the ischemic heart. An acutely administered A3 agonist, Cl-IB-MECA, was cardioprotective in cell culture, through the selective activation of A3 receptors without side effects, such as bradycardia, associated with the A1 subtype. The protection was blocked in the presence of a selective A3 receptor antagonist. Inn summary, highly selective adenosine analogues may have therapeutic potential in treatment of cerebral ischemia, stroke and possibly other neurodegenerative disorders as well. It is proposed that modulation of A2B and A3 receptors may be useful in treating asthma and inflammatory diseases. The pharmacolgical properties of novel xanthines developed in our lab that act as selective A2B receptor antagonists are being explored as potential antidiabetic and antiasthamtic agents. The first highly selective A2B receptor antagonist, synthesized in our section, has now been radiolabeled and is used in assaying newly synthesized analogues for affinity at the A2B receptor. We are also screening chemical libraries for novel leads for A2B receptor antagonists. We recently succeeded in identifying new chemotypes for antagonists of the A2A receptor using structure-based drug discovery (collaboration with B. Shoichet, Univ. of California, San Francisco). We also introduced a fluorescence polarization assay for affinity at the same receptor, that avoids the use fo radioactivity in the drug discovery/screening process.

Activators of ATP-gated ion channels (P2X receptors) are being synthesized and investigated for cardioprotection in collaboration with Dr. Bruce Liang (University of Connecticut). MRS2339, synthesized in our lab, is a nucleotide activator of a P2X4R ion channel present in the cardiac muscle cells. We have explored the structure activity relationships of this nucleotide. Certain phosphonate derivatives are more stable to hydrolysis than the phosphate derivative MRS2339 and are being explored in vivo. MRS2339 is currently being licensed by private industry for the treatment of heart failure. The P2X ion channels mediate a number of potent and possibly important biological effects in the cardiovascular, inflammatory, and central nervous systems. Previous studies have shown that extracellular ATP can cause an ionic current in murine, rat and guinea pig cardiac ventricular myocytes. The receptor that mediates this current appears to be a P2X receptor, of which the P2X4 receptor is an important subunit. Activation of P2X receptors leads to the opening of a nonselective cation channel permeable to sodium, potassium, and calcium ions. The current is inward at negative membrane potentials, reverses near 0 mV, and becomes outward at positive potentials. The continuous activation of this receptor channel by endogenous extracellular ATP may assume an important biological function. This constant activation under the resting or negative membrane potentials would produce an inward current, whereas its activation during depolarized portions of the action potential should lead to an outward current. These currents represent a possible ionic mechanism by which the cardiac P2X channel achieves its biological effects. A potential biologically important role of the cardiac P2X receptor was suggested by the finding that cardiac myocyte-specific overexpression of the P2X4 receptor can rescue the hypertrophic and heart failure phenotype of the calsequestrin (CSQ) model of cardiomyopathy. However, little is known regarding regulation of the cardiac P2X receptor in cardiac hypertrophy or failure. Furthermore, it is not clear whether an increased activation of the endogenous P2X receptor channel is beneficial or harmful in the progression of heart failure. The regulation of the P2X receptor-mediated ionic current and its potential role in heart failure was investigated using several novel nucleotide agonists. Chronic administration of a novel nucleotidase-resistant P2 receptor agonist MRS2339, which was capable of inducing this ionic current and was devoid of any vasodilator action, reduced cardiac hypertrophy and increased lifespan. The data suggests that an important biological function of the cardiac P2X current is to favorably modulate the progression of cardiac hypertrophy and failure. Recently we identified uncharged carbocyclic nucleotide analogues (including nonhydrolyzable phosphonates) related structurally to MRS2339, that represent potential candidates for the treatment of heart failure, suggesting this as a viable and structurally broad approach. We also found a beneficial therapeutic effect of 2-cyclohexylthio-adenosine 5-monophosphate in mice with heart failure (HF). Heart failure (HF), despite continuing progress, remains a leading cause of mortality and morbidity. P2X4 receptors (P2X4R) have emerged as potentially important molecules in regulating cardiac function and as potential targets for HF therapy. Transgenic (Tg) P2X4R overexpression can protect against HF. In collaboration with Bruce T. Liang, we have now defined the role of native cardiac P2X4R under basal conditions and during HF induced by myocardial infarction or pressure overload. Mice established with a conditional cardiac-specific P2X4R knockout (KO) were subjected to left coronary ligation-induced post-infarct or transverse aorta constriction-induced pressure overload HF. KO cardiac myocytes did not show P2X4R by immunoblotting or by any response to the P2X4R-specific allosteric enhancer ivermectin. KO hearts showed normal basal cardiac function but depressed contractile performance in post-infarct and pressure overload models of HF by in vivo echocardiography and ex vivo isolated working heart parameters. P2X4R co-immunoprecipitated and co-localized with nitric oxide synthase 3 (eNOS) in wild type cardiac myocytes. Mice with cardiac-specific P2X4R overexpression had increased S-nitrosylation, cGMP, NO formation, and were protected from post infarct and pressure overload HF. Inhibitor of eNOS L-NIO blocked the salutary effect of cardiac P2X4R overexpression in post-infarct and pressure overload HF as did eNOS knockout. This study established a new protective role for endogenous cardiac myocyte P2X4R in HF and demonstrated a physical interaction between the myocyte receptor and eNOS, a mediator of HF protection. We have provided compounds for the study of transient receptor potential cation channel subfamily V member 1 (TRPV1) to our collaborator Dr. Michael Iadarola of NIH. TRPV1 is a high-conductance, nonselective cation channel strongly expressed in nociceptive primary afferent neurons of the peripheral nervous system. In functions as a multimodal nociceptor gated by temperatures greater than 43˚C, protons, and small molecule vanilloid ligands such as capsaicin. The ability to respond to a variety of stimuli (heat, low pH, vanilloids, and endovanilloids) and its altered sensitivity and expression in experimental inflammatory and neuropathic pain models made TRPV1 a major target for the development of novel, nonopioid analgesics. These have been mostly antagonists, which have intolerable side effects in human clinical trials, but recent work shows that potent agonists or enhancers agonists have utility in this context. Here we show that the dihydropyridine derivative 4,5-diethyl-3-(2-methoxyethylthio)-2-methyl-6-phenyl-1,4-dihydropyridine-3,5-dicarboxylate (MRS1477) behaves as a positive allosteric modulator of both proton and vanilloid activation of TRPV1. Under inflammatory mimetic conditions of low pH (6.0) and protein kinase C phosphorylation, addition of MRS1477 further increased sensitivity of already sensitized TPRV1 toward capsaicin. MRS1477 does not affect inhibition by known vanilloid antagonists and remains effective in potentiating activation by pH in the presence of an orthosteric vanilloid antagonist. These results indicate a distinct site on TRPV1 for positive allosteric modulation that may bind endogenous compounds or novel pharmacological agents. Positive modulation of TRPV1 sensitivity suggests that it may be possible to produce a selective analgesia through calcium overload restricted to highly active nociceptive nerve endings at sites of tissue damage and inflammation.

Structure-based drug design strategies have been used to elucidate specific ligand recognition determinants and ultimately lead to the design of new small molecules for a specific target. The structure of the target receptor protein is the first requirement in structure-based drug design approaches. In the absence of a high resolution structure of a given G protein-coupled receptor (GPCR), computational techniques like homology modeling can be used to build a 3D model. Briefly, the best structural template is chosen from the Protein Database (PDB) mainly considering sequence identity and similarity with the target receptor and the quality of the crystallographic structure (e.g. resolution). The sequence of the target receptor is aligned to the template structure using highly conserved residues and the known shared structural features to guide the automated or semi-automated alignment. The sequence alignment and the template structures are the input for the homology modeling. Energy minimization or molecular dynamics can be used to further refine and optimize the resulting 3D models. The ligand-receptor interactions can be identified by means of structure-based approaches, e.g. molecular docking. The information contained in the ligand-receptor complexes from the docking can clarify structural elements for molecular recognition and lead to a further optimization of the compounds and the design of new derivatives. The binding site of a given GPCR can be mapped for each class of small molecule ligands. Also, virtual searching of chemically diverse databases for novel chemotypes to bind to a given GPCR structure has been productive using both structure-based and ligand-based strategies. We have applied mutagenesis and homology modeling to the study of GPCR families for extracellular purines and pyrimidines and used the structural insights gained to assist in the design of novel ligands. These families consist of the adenosine receptors (ARs) and the P2Y (nucleotide) receptors. The structures of the human A2AAR were recently reported in the antagonist-bound state and in the agonist-bound state. These structures can reliably serve as modeling templates, with some adjustment, for other ARs due to the relatively high sequence identity between ARs (average 47% between human subtypes). We achieved the structure-function analysis of P2YRs by indirect means, using mutagenesis and homology modeling based on a template of the high-resolution structure of similar GPCRs, such as the CXCR4 chemokine receptor. We have collaborated with one of the premier centers for X-ray crystallography of membrane-bound proteins, i.e. the lab of Prof. Ray Stevens (Scripps Research Inst.), to report the first X-ray structure of an agonist-bound A2AAR. Automatic docking of known potent nucleosides to the agonist-bound A2AAR crystallographic structure, and to homology models of other subtypes, was performed, resulting in new predictions of stabilizing interactions and a structural basis for previous empirical structure activity relationships. We predicted binding of novel C2 terminal and 5' derivatives of adenosine and used the models to interpret effects on measured binding affinity and efficacy of newly-synthesized agonists. Structures of G protein-coupled receptors (GPCRs) have a proven utility in the discovery of new antagonists and inverse agonists, which may modulate signaling of this important family of clinical targets. However, applicability of active-state GPCR structures to virtual screening and rational optimization of agonists, remains to be assessed. We have studied adenosine 5′ derivatives and evaluated the performance of an agonist-bound A2A adenosine receptor (AR) structure in retrieval of known agonists, and then employed the structure to screenfor new fragments optimally fitting the corresponding subpocket. Biochemical and functional assays demonstrate high affinity of new derivatives that include polar heterocycles. The binding models also explain a modest selectivity gain for some substituents toward the closely related A1AR subtype and the modified agonist efficacy of some of these ligands. The study suggested further applicability of in silico fragment screening to rational lead optimization for GPCRs in general. In order to investigate the usability of homology models and the inherent selectivity of a particular model in relation to close homologs, we constructed multiple homology models for the A1 adenosine receptor (A1AR) and docked, 2.2 M lead-like compounds. High-ranking molecules were tested on the A1AR as well as the close homologs A2AAR and A3AR. While the screen yielded numerous potent and novel ligands (hit rate 21% and highest affinity of 400 nM), it delivered few selective compounds. Moreover, most compounds appeared in the top ranks of only one model. The structure− activity relationship (SAR) for a novel class of 1,2,4- triazole antagonists of the human A2A adenosine receptor (hA2A AR) was explored. Thirty-three analogs of a ligand that was discovered in a structure-based virtual screen against the hA2A AR were tested in AR radioligand binding assays and in functional assays for the A2B AR subtype. As a series of closely related analogs of the initial lead did not display improved binding affinity or selectivity, molecular docking was used to guide the selection of more distantly related molecules. This resulted in the discovery of novel AR antagonists (Ki ≥200 nM) with high ligand efficiency. In light of the SAR for the 1,2,4-triazole scaffold, we also investigated the binding mode of these compounds based on docking to several A2AAR crystal structures The P2Y1 receptor (P2Y1R) is a G protein-coupled receptor naturally activated by extracellular ADP. Its stimulation is an essential requirement of ADP-induced platelet aggregation, thus making antagonists highly sought compounds for the development of antithrombotic agents. Here, through a virtual screening campaign based on a pharmacophoric representation of the common characteristics of known P2Y1R ligands and the putative shape and size of the receptor binding pocket, we have identified novel antagonist hits of microM affinity derived from a N,N-bis-arylurea chemotype. Unlike the vast majority of known P2Y1R antagonists, these drug-like compounds do not have a nucleotidic scaffold or highly negatively charged phosphate groups. Hence, our compounds may provide a direction for the development of receptor probes with altered physicochemical properties. The P2Y12R is an ADP-activated GPCR that is well validated clinically as an important target for antithrombotic drugs. Three homology models of P2Y12R were compared based on different GPCR structural templates: bovine rhodopsin, human A2AAR and CXCR4R. The CXCR4-based model appeared to be the most consistent with known characteristics of P2Y12R. This conclusion was based on various criteria: sequence analysis, deviation from helicity in the second transmembrane helix (TM2), poses of docked ligands highlighting the role of key residues, the accessibility of a conserved disulfide bridge that is reactive toward irreversibly-binding antagonists, and the presence of a shared disulfide bridge between the third extracellular loop (EL3) and the N-terminus. Thus, ligand docking to the CXCR4-based model of the P2Y12R predicted poses of both reversibly and irreversibly-binding small molecules, consistent with observed pharmacology and mutagenesis studies. Farnesyl pyrophosphate was recently identified as an insurmountable antagonist of ADP-induced platelet aggregation mediated by the P2Y12R. Docking of farnesyl pyrophosphate in a P2Y12R model revealed molecular similarities with ADP and a good fit into the binding pocket for ADP.

The cloning of at least fifteen subtypes of P2 nucleotide receptors has presented a unique challenge to medicinal chemists: the design of selective agonists and antagonists for this multiplicity of receptors with few existing leads. These receptors regulate function of the central nervous system, epithelial cells, the immune system, the cardiovascular system, and smooth muscle. Our laboratory is developing selective agonists and antagonists for these receptors, for use both as pharmacological tools for probing receptor function and as potential therapeutic agents. P2X receptors are ligand-gated ion channels. P2Y receptors are G protein coupled receptors linked to the phosphatidyl inositol pathway as second messenger. The human P2Y1 receptor as representative of the P2Y family of metabotropic purine and pyrimidine nucleotide receptors may be modeled based on a rhodopsin template, and the resulting model is highly consistent with pharmacological and mutagenesis results. The entire P2Y receptor family has been modeled, and the clustering into two subfamilies with functionally conserved residues is evident. Charged residues in both the transmembrane and extracellular domains and two disulfide bridges essential for receptor activation have been identified. [unreadable] [unreadable] Selective P2Y1 receptor antagonists related to the adenine nucleotide MRS 2179 (N-methyl-2-deoxyadenosine-3,5-bisphosphate) developed in our lab and it carbocyclic analogues are under development. We have also synthesized nucleotides containing conformationally constrained ribose-like rings, in order to freeze a conformation that provides favorable affinity and/or selectivity at P2 receptors. As a result, we have identified the conformation preference of the P2Y1 receptor for the Northern ring conformation of the ribose. This conclusion applies to both agonists and antagonists. By freezing the ribose substitute in the receptor-preferred conformation, we have enhanced the potency of known agonists at the P2Y1 subtype by 200-300 fold. One ATP derivative containing a methylene carbon joining the second and third phosphate groups was qualitatively altered in its effects on the P2Y1 receptor: The ribose analogue is inactive, and the conformationally constrained analogue (Northern methanocarba) was a moderately potent agonist. The (N)-methanocarba nucleotide MRS2365 is a selective agonist for the P2Y1 receptor in comparison to all other P2Y subtypes, including the P2Y12 and P2Y13 receptors which are otherwise activated by the ribose equivalent. Thus, a constrained ring in the synthetic analogues designed and synthesized in our lab has greatly enhanced potency and selectivity for a P2Y subtype. In platelets, MRS2365 induces the characteristic shape change without progressing to aggregation. This compound promises to be an important tool in the study of platelet function.[unreadable] [unreadable] MRS 2500, a 2-iodoadenine-methanocarba bisphosphate nucelotide, in which the ribose-like ring is locked in the North conformation is the most potent known antaognist of the P2Y1 receptor. This antagonist has been shown to potently inhibit the ADP-induced aggregation of human platelets. This derivative has now been radiolabeled and shown to be useful as a receptor probe. This supports the view that both P2Y1 and P2Y12 receptor activation are needed for ADP-induced aggregation. These pharmacological tools will be useful in validating the possible use of P2Y1 antagonists as antithrombotic agents. Larger quantities of MRS2500 have now been synthesized, making it possible to study the effects in larger animal models. We are continuing the explore the structure activity relationships in this series of potent and selective P2Y1 receptor agonists and antagonists. [unreadable] [unreadable] The use of conformationally constrained nucleotides has also been extended to P2Y2, P2Y4, P2Y6, and P2Y11 subtypes. The Northern methanocarba ring system results in retention of high potency inagonists at all of the above subtypes, except P2Y6. The conformational requirements of the P2Y6 receptor are currently being explored in our section. Indications from molecular modeling and from novel analogues of UDP that the Southern (S) conformation of the ribose ring is favored at this subtype. This observation will allow the design and synthesis of more potent and selective agonists at this subtype. We recently completed the synthesis of the first enantiomerically pure (S) methanocarba nucleosides and nucleotides.[unreadable] [unreadable] Also, a relationship between this subtype and apoptosis, programmed cell death, has been discovered. Astrocytoma cells that express the P2Y6 receptor, when activated by UDP, are protected from apoptosis induced in control cells upon exposure to TNF, tumor necrosis factor. The protection involves activation of protein kinase C and subsequently the signaling kinase known as ERK. This may have relevance for degenerative and inflammatory conditions that involve TNF. Activation of P2Y6 receptors in mouse skeletal muscle, both in vitro and in vivo, has a protective effect. Also, we recently found that astrocytoma cells that express the P2Y12 receptor, when activated by ADP or more potent synthetic agonists, are protected from apoptosis induced in control cells upon exposure to TNF. This receptor occurs in the brain and the results suggest exploring the possbile use of P2Y12 receptor activation in neuroproteection. [unreadable] [unreadable] We are interested in desigining selective probes for other P2Y subtypes. We recently modeled the P2Y2 receptor (important for treatment of cystic fibrosis) and identified a highly potent and selective nucleotide agonist. MRS2698 is 300-fold selective for the human P2Y2 receptor in comparison to the human P2Y4 receptor. Activation of the P2Y2 subtype by UTP in cardiac myocytes is highly protective against ishcemic damage. In vivo studies to test this approach to cardioprotection are currently underway. The first antagonist of the P2Y13 receptor (MRS2211) has been developed. It is based structurally on pyridoxal-5-phosphate antagonists (such as PPADS), for which the SAR is being examined at all of the P2 receptor subtypes.

Hepatitis C (HCV) is the most comon blood-born infection in the United States, where about 35,000 new cases are estimeted to occur each year. There is currently a need for compounds, compositions, and methods that are useful for treating viral infections such as HCV. This project involved novel compounds that inhibit one or more viral proteases. Accordingly, the compounds may be useful for treating viruses, such as HCV. We have explored the structure activity of nucleoside analogues synthesized in our laboratory in models of HCV, including NS5b polymerase assay and the replicon assay. Ring constrained analogues of purine and pyrimidine nucleotides have been explored as inhibitors.