Rajiv Chopra

Crystal Structure of Human Cdk4 in Complex with a D-Type Cyclin.

The cyclin D1–cyclin-dependent kinase 4 (CDK4) complex is a key regulator of the transition through the G1 phase of the cell cycle. Among the cyclin/CDKs, CDK4 and cyclin D1 are the most frequently activated by somatic genetic alterations in multiple tumor types. Thus, aberrant regulation of the CDK4/cyclin D1 pathway plays an essential role in oncogenesis; hence, CDK4 is a genetically validated therapeutic target. Although X-ray crystallographic structures have been determined for various CDK/cyclin complexes, CDK4/cyclin D1 has remained highly refractory to structure determination. Here, we report the crystal structure of CDK4 in complex with cyclin D1 at a resolution of 2.3 Å. Although CDK4 is bound to cyclin D1 and has a phosphorylated T-loop, CDK4 is in an inactive conformation and the conformation of the heterodimer diverges from the previously known CDK/cyclin binary complexes, which suggests a unique mechanism for the process of CDK4 regulation and activation.

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.

Discovery of benzisoxazoles as potent inhibitors of chaperone heat shock protein 90.

Heat shock protein 90 (Hsp90) is a molecular chaperone that is responsible for activating many signaling proteins and is a promising target in tumor biology. We have identified small-molecule benzisoxazole derivatives as Hsp90 inhibitors. Crystallographic studies show that these compounds bind in the ATP binding pocket interacting with the Asp93. Structure based optimization led to the identification of potent analogues, such as 13, with good biochemical profiles.

Abstract PR02: LEE011: An orally bioavailable, selective small molecule inhibitor of CDK4/6– Reactivating Rb in cancer.

The tumor suppressor Retinoblastoma protein (Rb) is often inactivated in cancer. In many tumors, the Rb protein itself is retained but functionally inactivated by increased CDK4/6 kinase activity. A number of key oncogenic aberrations can result in this increased activity, including inactivation of CDKN2A (p16), translocation or amplification of D-cyclins, amplification of CDK4/6 and mutations/deletions upstream of cyclin D, such as activating mutations of BRAF/PIK3CA and PTEN deletion. Abolishing CDK4/6 kinase activity and subsequent reactivation of Rb in these tumors has been demonstrated to inhibit tumor initiation and growth. Here we will describe LEE011- an orally bioavailable, selective small molecule inhibitor of CDK4/6 kinases. LEE011 inhibits CDK4/6 kinase activity with nM IC50 and is highly selective for these targets. In a number of preclinical tumor models, LEE011 demonstrates a dose dependent anti-tumor activity that tracks well with CDK4/6 inhibition. The primary mechanism for growth inhibitory effect appears to be G1 arrest in vitro , although, in some sensitive in vivo models, regressions are observed. Importantly, given the role of CDK4/6 downstream of other oncogenic driver mutations, LEE011 shows single agent activity in melanomas with activating mutations of BRAF or NRAS, and in breast cancers with intact estrogen receptor and/or activating aberrations of PIK3CA/Her-2. Combining LEE011 with LGX818, a V600E BRAF specific inhibitor, leads to robust anti-tumor activity in melanoma models that are both sensitive and, importantly, those that are resistant to LGX818. Furthermore, combining LEE011 with BYL719, a PIK3CA specific inhibitor, also leads to significant anti-tumor activity in breast cancer models both sensitive and resistant to BYL719. Several clinical studies evaluating LEE011 as single agent and in combinations are underway. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):PR02. Citation Format: Sunkyu Kim, Alice Loo, Rajiv Chopra, Giordano Caponigro, Alan Huang, Sadhna Vora, Sudha Parasuraman, Steve Howard, Nicholas Keen, William Sellers, Christopher Brain. LEE011: An orally bioavailable, selective small molecule inhibitor of CDK4/6– Reactivating Rb in cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr PR02.

3,5-Dioxopyrazolidines, Novel Inhibitors of UDP-N- Acetylenolpyruvylglucosamine Reductase (MurB) with Activity against Gram-Positive Bacteria

ABSTRACT A series of 3,5-dioxopyrazolidines was identified as novel inhibitors of UDP-N-acetylenolpyruvylglucosamine reductase (MurB). Compounds 1 to 3, which are 1,2-bis(4-chlorophenyl)-3,5-dioxopyrazolidine-4-carboxamides, inhibited Escherichia coli MurB, Staphyloccocus aureus MurB, and E. coli MurA with 50% inhibitory concentrations (IC50s) in the range of 4.1 to 6.8 μM, 4.3 to 10.3 μM, and 6.8 to 29.4 μM, respectively. Compound 4, a C-4-unsubstituted 1,2-bis(3,4-dichlorophenyl)-3,5-dioxopyrazolidine, showed moderate inhibitory activity against E. coli MurB, S. aureus MurB, and E. coli MurC (IC50s, 24.5 to 35 μM). A fluorescence-binding assay indicated tight binding of compound 3 with E. coli MurB, giving a dissociation constant of 260 nM. Structural characterization of E. coli MurB was undertaken, and the crystal structure of a complex with compound 4 was obtained at 2.4 Å resolution. The crystal structure indicated the binding of a compound at the active site of MurB and specific interactions with active-site residues and the bound flavin adenine dinucleotide cofactor. Peptidoglycan biosynthesis studies using a strain of Staphylococcus epidermidis revealed reduced peptidoglycan biosynthesis upon incubation with 3,5-dioxopyrazolidines, with IC50s of 0.39 to 11.1 μM. Antibacterial activity was observed for compounds 1 to 3 (MICs, 0.25 to 16 μg/ml) and 4 (MICs, 4 to 8 μg/ml) against gram-positive bacteria including methicillin-resistant S. aureus, vancomycin-resistant Enterococcus faecalis, and penicillin-resistant Streptococcus pneumoniae.

4-(Pyrazol-4-yl)-pyrimidines as selective inhibitors of cyclin-dependent kinase 4/6.

Identification and structure-guided optimization of a series of 4-(pyrazol-4-yl)-pyrimidines as selective CDK4/6 inhibitors is reported herein. Several potency and selectivity determinants were established based on the X-ray crystallographic analysis of representative compounds bound to monomeric CDK6. Significant selectivity for CDK4/6 over CDK1 and CDK2 was demonstrated with several compounds in both enzymatic and cellular assays.

Triad of polar residues implicated in pH specificity of acidic mammalian chitinase

Acidic mammalian chitinase (AMCase) is a mammalian chitinase that has been implicated in allergic asthma. One of only two active mammalian chinases, AMCase, is distinguished from other chitinases by several unique features. Here, we present the novel structure of the AMCase catalytic domain, both in the apo form and in complex with the inhibitor methylallosamidin, determined to high resolution by X‐ray crystallography. These results provide a structural basis for understanding some of the unique characteristics of this enzyme, including the low pH optimum and the preference for the β‐anomer of the substrate. A triad of polar residues in the second‐shell is found to modulate the highly conserved chitinase active site. As a novel target for asthma therapy, structural details of AMCase activity will help guide the future design of specific and potent AMCase inhibitors.

Novel pyrrolyl 2-aminopyridines as potent and selective human beta-secretase (BACE1) inhibitors.

The proteolytic enzyme beta-secretase (BACE1) plays a central role in the synthesis of the pathogenic beta-amyloid in Alzheimers disease. Recently, we reported small molecule acylguanidines as potent BACE1 inhibitors. However, many of these acylguanidines have a high polar surface area (e.g. as measured by the topological polar surface area or TPSA), which is unfavorable for crossing the blood-brain barrier. Herein, we describe the identification of the 2-aminopyridine moiety as a bioisosteric replacement of the acylguanidine moiety, which resulted in inhibitors with lower TPSA values and superior brain penetration. X-ray crystallographic studies indicated that the 2-aminopyridine moiety interacts directly with the catalytic aspartic acids Asp32 and Asp228 via a hydrogen-bonding network.

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)

Fragment-Based Discovery of 7-Azabenzimidazoles as Potent, Highly Selective, and Orally Active CDK4/6 Inhibitors

Herein, we describe the discovery of potent and highly selective inhibitors of both CDK4 and CDK6 via structure-guided optimization of a fragment-based screening hit. CDK6 X-ray crystallography and pharmacokinetic data steered efforts in identifying compound 6, which showed >1000-fold selectivity for CDK4 over CDKs 1 and 2 in an enzymatic assay. Furthermore, 6 demonstrated in vivo inhibition of pRb-phosphorylation and oral efficacy in a Jeko-1 mouse xenograft model.