Joel Bard

Molecular Mechanism of Hepatitis C Virus Replicon Variants with Reduced Susceptibility to a Benzofuran Inhibitor, HCV-796

ABSTRACT HCV-796 selectively inhibits hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase. In hepatoma cells containing a genotype 1b HCV replicon, HCV-796 reduced HCV RNA levels by 3 to 4 log10 HCV copies/μg total RNA (the concentration of the compound that inhibited 50% of the HCV RNA level was 9 nM). Cells bearing replicon variants with reduced susceptibility to HCV-796 were generated in the presence of HCV-796, followed by G418 selection. Sequence analysis of the NS5B gene derived from the replicon variants revealed several amino acid changes within 5 Å of the drug-binding pocket. Specifically, mutations were observed at Leu314, Cys316, Ile363, Ser365, and Met414 of NS5B, which directly interact with HCV-796. The impacts of the amino acid substitutions on viral fitness and drug susceptibility were examined in recombinant replicons and NS5B enzymes with the single-amino-acid mutations. The replicon variants were 10- to 1,000-fold less efficient in forming colonies in cells than the wild-type replicon; the S365L variant failed to establish a stable cell line. Other variants (L314F, I363V, and M414V) had four- to ninefold-lower steady-state HCV RNA levels. Reduced binding affinity with HCV-796 was demonstrated in an enzyme harboring the C316Y mutation. The effects of these resistance mutations were structurally rationalized using X-ray crystallography data. While different levels of resistance to HCV-796 were observed in the replicon and enzyme variants, these variants retained their susceptibilities to pegylated interferon, ribavirin, and other HCV-specific inhibitors. The combined virological, biochemical, biophysical, and structural approaches revealed the mechanism of resistance in the variants selected by the potent polymerase inhibitor HCV-796.

ATP-Competitive Inhibitors of the Mammalian Target of Rapamycin: Design and Synthesis of Highly Potent and Selective Pyrazolopyrimidines.

The mammalian target of rapamycin (mTOR), a central regulator of growth, survival, and metabolism, is a validated target for cancer therapy. Rapamycin and its analogues, allosteric inhibitors of mTOR, only partially inhibit one mTOR protein complex. ATP-competitive, global inhibitors of mTOR that have the potential for enhanced anticancer efficacy are described. Structural features leading to potency and selectivity were identified and refined leading to compounds with in vivo efficacy in tumor xenograft models.

A Selective Matrix Metalloprotease 12 Inhibitor for Potential Treatment of Chronic Obstructive Pulmonary Disease (COPD): Discovery of (S)-2-(8-(Methoxycarbonylamino)dibenzo[b,d]furan-3-sulfonamido)-3-methylbutanoic acid (MMP408)

Matrix metalloprotease 12 plays a significant role in airway inflammation and remodeling. Increased expression and production of MMP-12 have been found in the lung of human COPD patients. MMP408 (14), a potent and selective MMP-12 inhibitor, was derived from a potent matrix metalloprotease 2 and 13 inhibitor via lead optimization and has good physical properties and bioavailability. The compound blocks rhMMP-12-induced lung inflammation in a mouse model and was advanced for further development for the treatment of COPD.

Efficacious 11β-Hydroxysteroid Dehydrogenase Type I Inhibitors in the Diet-Induced Obesity Mouse Model

Cortisol and the glucocorticoid receptor signaling pathway have been implicated in the development of diabetes and obesity. The reduction of cortisone to cortisol is catalyzed by 11beta-hydroxysteroid dehydrogenase type I (11beta-HSD1). 2,4-Disubsituted benzenesulfonamides were identified as potent inhibitors of both the human and mouse enzymes. The lead compounds displayed good pharmacokinetics and ex vivo inhibition of the target in mice. Cocrystal structures of compounds 1 and 20 bound to human 11beta-HSD1 were obtained. Compound 20 was found to achieve high concentrations in target tissues, resulting in 95% inhibition in the ex vivo assay when dosed with a food mix (0.5 mg of drug per g of food) after 4 days. Compound 20 was efficacious in a mouse diet-induced obesity model and significantly reduced fed glucose and fasted insulin levels. Our findings suggest that 11beta-HSD1 inhibition may be a valid target for the treatment of diabetes.

An ultra-specific avian antibody to phosphorylated tau protein reveals a unique mechanism for phosphoepitope recognition.

Background: Truly phosphospecific antibodies are difficult to generate and are poorly understood. Results: Avian single chain Fv library selections yielded fully phosphospecific anti-phospho-tau antibodies, enabling the generation of a 1.9 Å co-crystal structure. Conclusion: Phosphospecific antibodies were readily generated and can exhibit unique epitope recognition mechanisms. Significance: High-affinity antibody phosphoepitope recognition has been defined, at high resolution, for the first time. Highly specific antibodies to phosphoepitopes are valuable tools to study phosphorylation in disease states, but their discovery is largely empirical, and the molecular mechanisms mediating phosphospecific binding are poorly understood. Here, we report the generation and characterization of extremely specific recombinant chicken antibodies to three phosphoepitopes on the Alzheimer disease-associated protein tau. Each antibody shows full specificity for a single phosphopeptide. The chimeric IgG pT231/pS235_1 exhibits a KD of 0.35 nm in 1:1 binding to its cognate phosphopeptide. This IgG is murine ortholog-cross-reactive, specifically recognizing the pathological form of tau in brain samples from Alzheimer patients and a mouse model of tauopathy. To better understand the underlying binding mechanisms allowing such remarkable specificity, we determined the structure of pT231/pS235_1 Fab in complex with its cognate phosphopeptide at 1.9 Å resolution. The Fab fragment exhibits novel complementarity determining region (CDR) structures with a “bowl-like” conformation in CDR-H2 that tightly and specifically interacts with the phospho-Thr-231 phosphate group, as well as a long, disulfide-constrained CDR-H3 that mediates peptide recognition. This binding mechanism differs distinctly from either peptide- or hapten-specific antibodies described to date. Surface plasmon resonance analyses showed that pT231/pS235_1 binds a truly compound epitope, as neither phosphorylated Ser-235 nor free peptide shows any measurable binding affinity.

Naphthyl tetronic acids as multi-target inhibitors of bacterial peptidoglycan biosynthesis.

Since the discovery of penicillin in 1929, many important antibiotic agents have made significant contributions to the prevention and treatment of infections caused by bacteria. Despite these remarkable achievements, infections are still the second-leading cause of death worldwide and remain a major public health problem. Clearly, there is great need for novel antibacterial agents to address resistance problems associated with current antibiotics. Toward this end, three broad strategies have been recently employed in the search for new leads: high-throughput screening of large compound libraries, genomics, and combinatorial biosynthesis. Although some limitations of the former approach to targets in bacterial peptidoglycan biosynthesis have been reported, the peptidoglycan biosynthetic pathway remains an attractive target, validated in the clinic with fosfomycin and vancomycin. Peptidoglycan biosynthesis is a complex process, which involves three main stages: a) cytoplasmic soluble enzymes that include MurA–F, b) membrane-bound enzymes that include MraY and MurG, and finally c) transglycosylases and transpeptidases, which act external to the cytoplasmic membrane. The Mur enzymes are unique to bacteria and are involved in essential functions of both Gram-positive and Gram-negative organisms. Another attractive aspect of Mur enzyme inhibitors is the potential to be bactericidal, leading to cell lysis and bacterial death. Inhibitors of peptidoglycan biosynthesis initiate a complex process of gene expression resulting in the induction of MurA and Mur I in Gram-positive bacteria to compensate for the slower rate of peptidoglycan biosynthesis. Several classes of natural products or their semisynthetic derivatives represented by liposidomycins, amphomycins, and muraymycins are inhibitors of MraY, whereas nisin, ramoplanin, and mersacidin are lipid II inhibitors. In the last decade a few small-molecule inhibitors of the Mur enzymes have been reported, including sesquiterpene lactones, 5-sulfonoxyanthranilic acids T6361 and T6362, UDP-MurNAc (MurA), imidazolinones, 4-thiazolidinones, thienopyrazoles, phosphinates (MurB), peptidosulfonamides, 3-cyanothiophenes (MurF), and d-glutamic acid analogues (Mur I). Despite the discovery of small-molecule inhibitors of various Mur enzymes, many limitations have been noted, including poor antibacterial activities in cells. In parallel, a number of new assay formats for the identification of Mur enzyme inhibitors have been described based on different platforms such as ultra-efficient affinity HTS, LC– MS, TLC, HPLC, and solid-support TLC. Our efforts in identifying Mur enzyme inhibitors were based on an initial pathway screen searching for inhibitors of multiple enzymes, MurA–F. Hits in this assay were evaluated against the individual Mur enzymes for lead optimization. Using this strategy, we identified two classes of inhibitors : 3,5-dioxopyrazolidines and pulvinones, with activities against several of the Mur enzymes. Inhibitors of multiple Mur enzymes are attractive given the essential role of each Mur enzyme in peptidoglycan biosynthesis. This strategy may prevent the development of drug resistance through the multi-target hypothesis. Herein we report on the SAR of the naphthyl tetronic acids and highlight their binding to the E. coli enzyme MurB. The target naphthylfuran-2-ones 5a–k were prepared by a three-step process starting from 3-bromo-4-methoxy-5H-furan2-one (1) and the appropriately substituted aldehydes 2 (Scheme 1). Bromofuranone 1 was acquired by bromination of the commercially available 4-methoxy-5H-furan-2-one with Nbromosuccinimide in carbon tetrachloride at reflux. Deprotonation of 2 at C5 with lithium isopropylcyclohexylamide (LICA) followed by an aldol reaction with substituted aldehydes 2 mediated by anhydrous ZnCl2 afforded diastereomeric alcohols, which were converted into their mesylate or chloride derivatives in situ followed by elimination to generate the exocyclic double bond of 3 in the thermodynamically more stable Z configuration. The key step involved a Suzuki cross-coupling of 3 with aryl boronic acids catalyzed by either [PdACHTUNGTRENNUNG(PPh3)4] or [PdCl2ACHTUNGTRENNUNG(dppf)2] to afford the methoxyfuranones 4a–k. Demethylation of methoxyfuranones 4a–k with lithium bromide in the final step afforded the desired naphthylfuran-2ones 5a–k. Purification by silica gel column chromatography was followed by an acid wash of the collected fractions to restore the acidic functionality. A panel of nine enzymes was used to assess the abilities of the naphthylfuranones to inhibit the Mur enzymes, and thus to define SAR trends for multiple enzyme inhibition (Table 1). The four isomeric bis-naphthyl compounds (Entries 1–4) were evaluated to determine whether there is a preference for aor blinked naphthyl groups at either C3 or C5. The trend seems to favor C3 b and C5 a substitution. Replacement of naphthyl with p-chlorophenyl (Entries 5–8) gave compounds 5e–h with good broad-spectrum activity against the Mur enzymes, thus confirming the desired SAR trend. Further optimization of the C5 a methylidene naphthyl derivative 5h by changing the p[a] Dr. T. S. Mansour, Dr. B. Rasmussen, Dr. G. Krishnamurthy, C. Smeltzer, Dr. Y. Yang, Dr. A. Severin, P. J. Petersen, Dr. G. Singh Medicinal Chemistry, Wyeth Research 401 North Middletown Road, Pearl River, NY 10965 (USA) Fax: (+1)845-602-5580 E-mail : mansout@wyeth.com [b] Dr. C. E. Caufield, K. M. Morris, S. Naughton, S. Antane, Dr. D. Quagliato Wyeth Research, CN 8000, Princeton, NJ 08543 (USA) [c] Dr. R. Chopra, K. Svenson, Dr. J. Bard Wyeth Research, Cambridge, MA 02140 (USA)

Discovery of potent and selective matrix metalloprotease 12 inhibitors for the potential treatment of chronic obstructive pulmonary disease (COPD).

Chronic obstructive pulmonary disease (COPD) is an inflammatory lung disease associated with irreversible progressive airflow limitation. Matrix metalloproteinase-12 (MMP-12) has been characterized to be one of the major proteolytic enzymes to induce airway remodeling, destruction of elastin and the aberrant remodeling of damaged alveoli in COPD and asthma. The goal of this project is to develop and identify an orally potent and selective small molecule inhibitor of MMP-12 for treatment of COPD and asthma. Syntheses and structure-activity relationship (SAR) studies of a series of dibenzofuran (DBF) sulfonamides as MMP-12 inhibitors are described. Potent inhibitors of MMP-12 with excellent selectivity against other MMPs were identified. Compound 26 (MMP118), which exhibits excellent oral efficacy in the MMP-12 induced ear-swelling inflammation and lung inflammation mouse models, had been successfully advanced into Development Track status.

Discovery and optimization of 2-(4-substituted-pyrrolo[2,3-b]pyridin-3-yl)methylene-4-hydroxybenzofuran-3(2H)-ones as potent and selective ATP-competitive inhibitors of the mammalian target of rapamycin (mTOR).

We discovered 2-(4-substituted-pyrrolo[2,3-b]pyridin-3-yl)methylene-4-hydroxybenzofuran-3(2H)-ones as potent and selective ATP-competitive inhibitors of the mammalian target of rapamycin (mTOR). Since phenolic OH groups pose metabolic liability, one of the two hydroxyl groups was selectively removed. The SAR data showed the structural features necessary for subnanomolar inhibitory activity against mTOR kinase as well as selectivity over PI3Kalpha. An X-ray co-crystal structure of one inhibitor with the mTOR-related PI3Kgamma revealed the key hydrogen bonding interactions.

Discovery and characterization of novel potent PARP-1 inhibitors endowed with neuroprotective properties: From TIQ-A to HYDAMTIQ

Activation of poly(ADP-ribose) polymerase (PARP) is an important factor in controlling cell survival or death. As a consequence, therapeutic interventions with PARP-1 inhibitors are sought in different pathological conditions such as cancer, cardiovascular and inflammatory diseases, as well as brain ischemia. In the first part of this work, as a continuation of our efforts in the field, we report the design, synthesis and biological appraisal of novel potent PARP-1 inhibitors. A crystallization experiment is carried out to ascertain the mode of binding to PARP-1 of the most potent compound, namely 2-((dimethylamino)methyl)-9-hydroxythieno[2,3-c]isoquinolin-5(4H)-one (HYDAMTIQ), whilst molecular modeling studies are performed to infer the role of water molecules in ligand binding. In the second part of the work, we discuss the results of HYDAMTIQ in models of brain ischemia as well as its preliminary physicochemical and pharmacokinetic characterization. Collectively, the data obtained qualify HYDAMTIQ as a novel lead candidate for advancement to clinical settings of brain ischemia.

Identification of pyrimidine derivatives as hSMG-1 inhibitors.

hSMG-1 kinase plays a dual role in a highly conserved RNA surveillance pathway termed nonsense-mediated RNA decay (NMD) and in cellular genotoxic stress response. Since deregulation of cellular responses to stress contributes to tumor growth and resistance to chemotherapy, hSMG-1 is a potential target for cancer treatment. From our screening efforts, we have identified pyrimidine derivatives as hSMG-1 kinase inhibitors. We report structure-based optimization of this pan-kinase scaffold to improve its biochemical profile and overall kinome selectivity, including mTOR and CDK, to generate the first reported selective hSMG-1 tool compound.