Sylvie Goulet

Peptide-based inhibitors of the hepatitis C virus serine protease

Hexapeptide DDIVPC-OH is a competitive inhibitor of the hepatitis C virus (HCV) NS3 protease complexed with NS4A cofactor peptide. This hexapeptide corresponds to the N-terminal cleavage product of an HCV dodecapeptide substrate derived from the NS5A/5B cleavage site. Structure-activity studies on Ac-DDIVPC-OH revealed that side chains of the P4, P3 and P1 residues contribute the most to binding and that the introduction of a D-amino acid at the P5 position improves potency considerably. Furthermore, there is a strong preference for cysteine at the P1 position and conservative replacements, such as serine, are not well tolerated.

Binding Site Characterization and Resistance to a Class of Non-nucleoside Inhibitors of the Hepatitis C Virus NS5B Polymerase

The virally encoded NS5B RNA-dependent RNA polymerase has emerged as a prime target in the search for specific HCV antivirals. A series of benzimidazole 5-carboxamide compounds inhibit the cellular RNA replication of a HCV subgenomic replicon and we have advanced our understanding of this class of inhibitors through a combination of complementary approaches that include biochemical cross-linking experiments with a photoreactive analogue followed by mass spectrometry analysis of the enzyme. A novel binding site has been localized for these inhibitors at the junction of the thumb domain and the N-terminal finger loop. Furthermore, the isolation and characterization of resistant replicon mutants that co-localize to this region distinguished this class of compounds from other non-nucleoside NS5B inhibitors that bind to distinct allosteric sites. Resistant mutations that emerged with the benzimidazole 5-carboxamide and related compounds were found at three amino acid positions in the thumb domain: Pro495 with substitutions to Ser, Leu, Ala, or Thr; Pro496 substitutions to Ser or Ala; and a V499A substitution. Mutations at each of these positions conferred different levels of resistance to this drug class: the Pro495 changes provided the greatest shifts in compound potency, followed by moderate changes in potency with the Pro496 substitutions, and finally only minor shifts in potency with V499A. Combinations that include the benzimidazole 5-carboxamide polymerase inhibitors and compounds that bind other sites or other HCV targets, including HCV protease inhibitors, are complementary in cell culture models of HCV RNA replication at suppressing the emergence of resistant variants. This novel class of compounds and unique binding site expand the diversity of HCV antivirals currently under development and offer the potential to improve the treatment of chronic HCV infection.

Studies on the C-terminal of hexapeptide inhibitors of the hepatitis C virus serine protease

Replacement of the C-terminal carboxylic acid functionality of peptide inhibitors of hepatitis C virus (HCV) NS3 protease (complexed with NS4A peptide cofactor) by activated carbonyl groups does not produce any substantial increase in potency. These latter inhibitors also inhibit a variety of other serine and cysteine proteases whereas the carboxylic acids are specific. Norvaline was identified as a chemically stable replacement for the P1 residue of Ac-DDIVPC-OH which was also compatible with activated carbonyl functionalities.

Highly potent and selective peptide-based inhibitors of the hepatitis C virus serine protease : Towards smaller inhibitors

Structure-activity studies on a hexapeptide N-terminal cleavage product of a dodecamer substrate led to the identification of very potent and highly specific inhibitors of the HCV NS3 protease/NS4A cofactor peptide complex. The largest increase in potency was accomplished by the introduction of a (4R)-naphthalen-1-yl-4-methoxy substituent to the P2 proline. N-Terminal truncation resulted in tetrapeptides containing a C-terminal carboxylic acid, which exhibited low micromolar activity against the HCV serine protease.

Discovery of a potent and selective noncovalent linear inhibitor of the hepatitis C virus NS3 protease (BI 201335).

C-Terminal carboxylic acid containing inhibitors of the NS3 protease are reported. A novel series of linear tripeptide inhibitors that are very potent and selective against the NS3 protease are described. A substantial contribution to the potency of these linear inhibitors arises from the introduction of a C8 substituent on the B-ring of the quinoline moiety found on the P2 of these inhibitors. The introduction of a C8 methyl group results not only in a modest increase in the cell-based potency of these inhibitors but more importantly in a much better pharmacokinetic profile in rats as well. Exploration of C8-substitutions led to the identification of the bromo derivative as the best group at this position, resulting in a significant increase in the cell-based potency of this class of inhibitors. Structure-activity studies on the C8-bromo derivatives ultimately led to the discovery of clinical candidate 29 (BI 201335), a very potent and selective inhibitor of genotype1 NS3 protease with a promising PK profile in rats.

Novel inhibitor binding site discovery on HIV-1 capsid N-terminal domain by NMR and X-ray crystallography.

The HIV-1 capsid (CA) protein, a domain of Gag, which participates in formation of both the mature and immature capsid, represents a potential target for anti-viral drug development. Characterization of hits obtained via high-throughput screening of an in vitro capsid assembly assay led to multiple compounds having this potential. We previously presented the characterization of two inhibitor series that bind the N-terminal domain of the capsid (CA(NTD)), at a site located at the bottom of its helical bundle, often referred to as the CAP-1 binding site. In this work we characterize a novel series of benzimidazole hits. Initial optimization of this series led to compounds with improved in vitro assembly and anti-viral activity. Using NMR spectroscopy we found that this series binds to a unique site on CA(NTD), located at the apex of the helical bundle, well removed from previously characterized binding sites for CA inhibitors. 2D (1)H-(15)N HSQC and (19)F NMR showed that binding of the benzimidazoles to this distinct site does not affect the binding of either cyclophilin A (CypA) to the CypA-binding loop or a benzodiazepine-based CA assembly inhibitor to the CAP-1 site. Unfortunately, while compounds of this series achieved promising in vitro assembly and anti-viral effects, they also were found to be quite sensitive to a number of naturally occurring CA(NTD) polymorphisms observed among clinical isolates. Despite the negative impact of this finding for drug development, the discovery of multiple inhibitor binding sites on CA(NTD) shows that capsid assembly is much more complex than previously realized.

Discovery of benzimidazole-diamide finger loop (Thumb Pocket I) allosteric inhibitors of HCV NS5B polymerase: Implementing parallel synthesis for rapid linker optimization

Previously described SAR of benzimidazole-based non-nucleoside finger loop (Thumb Pocket I) inhibitors of HCV NS5B polymerase was expanded. Prospecting studies using parallel synthesis techniques allowed the rapid identification of novel cinnamic acid right-hand sides that provide renewed opportunities for further optimization of these inhibitors. Novel diamide derivatives such as 44 exhibited comparable potency (enzymatic and cell-based HCV replicon) as previously described tryptophan-based inhibitors but physicochemical properties (e.g., aqueous solubility and lipophilicity) have been improved, resulting in molecules with reduced off-target liabilities (CYP inhibition) and increased metabolic stability.

Discovery of a novel series of non-nucleoside thumb pocket 2 HCV NS5B polymerase inhibitors.

A novel series of non-nucleoside thumb pocket 2 HCV NS5B polymerase inhibitors were derived from a fragment-based approach using information from X-ray crystallographic analysis of NS5B-inhibitor complexes and iterative rounds of parallel synthesis. Structure-based drug design strategies led to the discovery of potent sub-micromolar inhibitors 11a-c and 12a-c from a weak-binding fragment-like structure 1 as a starting point.

Optimization of a 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione series of HIV capsid assembly inhibitors 2: Structure–activity relationships (SAR) of the C3-phenyl moiety

Detailed structure-activity relationships of the C3-phenyl moiety that allow for the optimization of antiviral potency of a series of 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione inhibitors of HIV capsid (CA) assembly are described. Combination of favorable substitutions gave additive SAR and allowed for the identification of the most potent compound in the series, analog 27. Productive SAR also transferred to the benzotriazepine and spirobenzodiazepine scaffolds, providing a solution to the labile stereocenter at the C3 position. The molecular basis of how compound 27 inhibits mature CA assembly is rationalized using high-resolution structural information. Our understanding of how compound 27 may inhibit immature Gag assembly is also discussed.

Herpes simplex virus ribonucleotide reductase subunit association inhibitors: the effect and conformation of β-alkylated aspartic acid derivatives

Incorporating beta-alkylated aspartic acid derivatives into herpes simplex virus ribonucleotide reductase subunit association inhibitors can improve inhibitor potency up to 50 times over the corresponding inhibitors containing an unsubstituted aspartic acid. A combination of NMR studies, conformational analysis, and molecular mechanics calculations suggests that the beta-alkyl group improves inhibitor potency by favoring the bioactive conformation of the critical aspartic acid carboxyl group. Further support for this hypothesis is provided by a potent conformationally restricted aspartic acid derivative in which the carboxyl group is locked in the putative bioactive conformation.