Zhuyan Guo

Characterization of resistance mutations against HCV ketoamide protease inhibitors

An issue of clinical importance in the development of new antivirals for HCV is emergence of resistance. Several resistance loci to ketoamide inhibitors of the NS3/4A protease have been identified (residues V36, T54, R155, A156, and V170) by replicon and clinical studies. Using SCH 567312, a more potent protease inhibitor derived from SCH 503034 (boceprevir) series, we identified two new positions (Q41 and F43) that confer resistance to the ketoamide class. The catalytic efficiency of protease enzymes was not affected by most resistance mutations, whereas replicon fitness varied with specific mutations. SCH 503034 and another ketoamide inhibitor, VX-950 (telaprevir), showed moderate losses of activity against most resistance mutations (< or =10-fold); the highest resistance level was conferred by mutations at A156 locus. Although SCH 503034 and VX-950 bind similarly to the active site, differences in resistance level were observed with specific mutations. Changes at V36 and R155 had more severe impact on VX-950, whereas mutations at Q41, F43 and V170 conferred higher resistance to SCH 503034. Structural analysis of resistance mutations on inhibitor binding is discussed.

Identification of HCV protease inhibitor resistance mutations by selection pressure-based method

A major challenge to successful antiviral therapy is the emergence of drug-resistant viruses. Recent studies have developed several automated analyses of HIV sequence polymorphism based on calculations of selection pressure (Ka/Ks) to predict drug resistance mutations. Similar resistance analysis programs for HCV inhibitors are not currently available. Taking advantage of the recently available sequence data of patient HCV samples from a Phase II clinical study of protease inhibitor boceprevir, we calculated the selection pressure for all codons in the HCV protease region (amino acid 1–181) to identify potential resistance mutations. The correlation between mutations was also calculated to evaluate linkage between any two mutations. Using this approach, we identified previously known major resistant mutations, including a recently reported mutation V55A. In addition, a novel mutation V158I was identified, and we further confirmed its resistance to boceprevir in protease enzyme and replicon assay. We also extended the approach to analyze potential interactions between individual mutations and identified three pairs of correlated changes. Our data suggests that selection pressure-based analysis and correlation mapping could provide useful tools to analyze large amount of sequencing data from clinical samples and to identify new drug resistance mutations as well as their linkage and correlations.

The introduction of P4 substituted 1-methylcyclohexyl groups into Boceprevir®: A change in direction in the search for a second generation HCV NS3 protease inhibitor

In the search for a second generation HCV protease inhibitor, molecular modeling studies of the X-ray crystal structure of Boceprevir1 bound to the NS3 protein suggest that expansion into the S4 pocket could provide additional hydrophobic Van der Waals interactions. Effective replacement of the P4 tert-butyl with a cyclohexylmethyl ligand led to inhibitor 2 with improved enzyme and replicon activities. Subsequent modeling and SAR studies led to the pyridine 38 and sulfone analogues 52 and 53 with vastly improved PK parameters in monkeys, forming a new foundation for further exploration.

The discovery of novel tartrate-based TNF-[alpha] converting enzyme (TACE) inhibitors

A novel series of TNF-alpha convertase (TACE) inhibitors which are non-hydroxamate have been discovered. These compounds are bis-amides of L-tartaric acid (tartrate) and coordinate to the active site zinc in a tridentate manner. They are selective for TACE over other MMPs. We report the first X-ray crystal structure for a tartrate-based TACE inhibitor.

Discovery of MK-8831, A Novel Spiro-Proline Macrocycle as a Pan-Genotypic HCV-NS3/4a Protease Inhibitor.

We have been focused on identifying a structurally different next generation inhibitor to MK-5172 (our Ns3/4a protease inhibitor currently under regulatory review), which would achieve superior pangenotypic activity with acceptable safety and pharmacokinetic profile. These efforts have led to the discovery of a novel class of HCV NS3/4a protease inhibitors containing a unique spirocyclic-proline structural motif. The design strategy involved a molecular-modeling based approach, and the optimization efforts on the series to obtain pan-genotypic coverage with good exposures on oral dosing. One of the key elements in this effort was the spirocyclization of the P2 quinoline group, which rigidified and constrained the binding conformation to provide a novel core. A second focus of the team was also to improve the activity against genotype 3a and the key mutant variants of genotype 1b. The rational application of structural chemistry with molecular modeling guided the design and optimization of the structure-activity relationships have resulted in the identification of the clinical candidate MK-8831 with excellent pan-genotypic activity and safety profile.

Discovery of novel spirocyclopropyl hydroxamate and carboxylate compounds as TACE inhibitors.

We have discovered nanomolar inhibitors of TNF-alpha convertase (TACE) comprised of a novel spirocyclic scaffold and either a carboxylate or hydroxamate zinc binding moiety. X-ray crystal structures and computer models of selected compounds binding to TACE explain the observed SAR. We report the first TACE X-ray crystal structure for an inhibitor with a carboxylate zinc ligand.

Novel Quinoline-Based P2-P4 Macrocyclic Derivatives As Pan-Genotypic HCV NS3/4a Protease Inhibitors.

We have previously reported the discovery of our P2-P4 macrocyclic HCV NS3/4a protease inhibitor MK-5172, which in combination with the NS5a inhibitor MK-8742 recently received a breakthrough therapy designation from the US FDA for treatment of chronic HCV infection. Our goal for the next generation NS3/4a inhibitor was to achieve pan-genotypic activity while retaining the pharmacokinetic profile of MK-5172. One of the areas for follow-up investigation involved replacement of the quinoxaline moiety in MK-5172 with a quinoline and studying the effect of substitution at 4-position of the quinoline. The rationale for this effort was based on molecular modeling, which indicated that such modifications would improve interactions with the S2 subsite, in particular with D79. We wish to report herein the discovery of highly potent inhibitors with pan-genotypic activity and an improved profile over MK-5172, especially against gt-3a and A156 mutants.

Pivotal Role of an Aliphatic Side Chain in the Development of an HDM2 Inhibitor

Introduction of an aliphatic side chain to a key position of a novel piperidine-based HDM2 inhibitor scaffold resulted in significant potency gains, enabling further series progression.

Himbacine-derived thrombin receptor antagonists: c7-spirocyclic analogues of vorapaxar.

We have synthesized several C7-spirocyclic analogues of vorapaxar and evaluated their in vitro activities against PAR-1 receptor. Some of these analogues showed activities and rat plasma levels comparable to vorapaxar. Compound 5c from this series showed excellent PAR-1 activity (K i = 5.1 nM). We also present a model of these spirocyclic compounds docked to the PAR-1 receptor based on the X-ray crystal structure of vorapaxar bound to PAR-1 receptor. This model explains some of the structure-activity relationships in this series.

Generation of a chimeric hepatitis C replicon encoding a genotype-6a NS3 protease and assessment of boceprevir (SCH503034) sensitivity and drug-associated mutations

BACKGROUND Genotype (gt)6 HCV is common amongst HCV-positive populations of the Asia-Pacific region but cell culture models for this gt have only recently been developed. Boceprevir (SCH503034) is a clinically available inhibitor of the HCV NS3 protein. We investigated the efficacy of boceprevir for inhibiting replication of a chimeric gt1b replicon encoding a gt6a NS3 protease and defined the development of mutations in the protease when boceprevir treatment was applied. METHODS We constructed a chimeric gt1b subgenomic replicon encoding a gt6 NS3 protease (NS3p) sequence (gt6NS3p_gt1b). The boceprevir EC50 value against replication of this replicon was determined using quantitative reverse transcriptase PCR. Next-generation sequencing was used to identify nucleotide changes associated with boceprevir resistance. The replication capacities of chimeric replicons containing mutations associated with boceprevir resistance were determined by colony formation efficiency assays. RESULTS The boceprevir EC50 value for the gt6NS3p_gt1b replicon was 535 ±79 nM. Boceprevir-resistant gt6NS3p_gt1b replicon cell lines could be selected and they demonstrated drug-associated amino acid changes that have previously been reported in other HCV gts. Interestingly, no mutations were observed at A156, a position defined for boceprevir resistance in gt1 NS3p, while mutation at N122, which is rarely reported in boceprevir-resistant gt1 proteases, was frequently observed. Re-introduction of these mutations into the chimeric replicon altered their replication capacity, ranging from complete abolishment of replication (A156T) to increasing replication capacity (V36A, N122S). This report provides the first characterization of gt6 HCV resistance to boceprevir. CONCLUSIONS A chimeric HCV replicon encoding gt6 NS3 protease is sensitive to boceprevir and develops drug-resistant mutations at amino acid sites previously reported for other gts. Mutation at N122 also appears to be associated with boceprevir resistance in the gt6 NS3 protease.