Synthesis of new acylsulfamoyl benzoxaboroles as potent inhibitors of HCV NS3 protease

HCV NS3/4A serine protease is essential for the replication of the HCV virus and has been a clinically validated target. A series of HCV NS3/4A protease inhibitors containing a novel acylsulfamoyl benzoxaborole moiety at the P1' region was synthesized and evaluated. The resulting P1-P3 and P2-P4 macrocyclic inhibitors exhibited sub-nanomolar potency in the enzymatic assay and low nanomolar activity in the cell-based replicon assay. The in vivo PK evaluations of selected compounds are also described.     

Novel,potent phenethylamide inhibitors of the hepatitis C virus (HCV) NS3 protease: probing the role of P2 aryloxyprolines with hybrid structures

Synthesis of hybrid HCV NS3 protease/NS4A inhibitors having the 4,4-difluoroaminobutyric acid (difluoroAbu) phenethylamides as P1-P1' and quinolyloxyprolines as P2 fragments led to 7 (IC(50) 54 nM). Molecular modelling suggests that this potent tripeptide inhibitor utilizes interactions in the S1', S1, S2, S3 and S4 sites of the protease.     

Novel,sulfonamide linked inhibitors of the hepatitis C virus NS3 protease

A sulfonamide replacement of the P2–P3 amide bond in the context of macrocyclic HCV NS3 protease inhibitors was investigated. These analogs displayed good inhibitory potency in the absence of any P3 capping group. The synthesis and preliminary SAR are described.     

Depeptidization efforts on P3-P2' alpha-ketoamide inhibitors of HCV NS3-4A serine protease: effect on HCV replicon activity

Depeptidization efforts of the P(3)-P(2) region of P(3) capped alpha-ketoamide inhibitor of HCV NS3 serine protease 1 are reported. We clearly established that N-methylation of the P(2) nitrogen and modification of the P(2)' carboxylic acid terminus were essential for activity in the replicon assay.     

Inhibitors of hepatitis C virus NS3/4A: α-Ketoamide based macrocyclic inhibitors

A novel series of hepatitis C virus (HCV) NS3/4A protease inhibitors bearing a P2-P4 macrocycle and a P1-P1′ α-ketoamide serine trap is reported. The NS3 protease, which is essential for viral replication, is considered one of the most attractive targets for developing novel anti-HCV therapies. The optimization of both the macrocycle and the warhead portions led to the discovery of compounds 8b and 8g with a good activity both in the enzyme as well as in the cell based (replicon) assays with favorable PK profile in a preclinical species.     

Synthesis and biological evaluations of P4-benzoxaborole-substituted macrocyclic inhibitors of HCV NS3 protease

We disclose here a series of P4-benzoxaborole-substituted macrocyclic HCV protease inhibitors. These inhibitors are potent against HCV NS3 protease, their anti-HCV replicon potencies are largely impacted by substitutions on benzoxaborole ring system and P21 groups. P21 2-thiazole-isoquinoline provides best replicon potency. The in vitro SAR studies and in vivo PK evaluations of selected compounds are described herein.     

Phenylglycine as a novel P2 scaffold in hepatitis C virus NS3 protease inhibitors

Molecular modeling and inhibitory potencies of tetrapeptide protease inhibitors of HCV NS3 proposed phenylglycine as a new promising P2 residue. The results suggest that phenylglycine might be capable of interacting with the NS3 (protease-helicase/NTPase) in ways not possible for the common P2 proline-based inhibitors. Thus, a series of tripeptides, both linear and macrocyclic, based on p-hydroxy-phenylglycine in the P2 position were prepared and their inhibitory effect determined. When the p-hydroxy group was replaced by methoxy, isoquinolin-, or quinolinyloxy functions, inhibitors with improved potencies were obtained. The P2 phenylglycine-based inhibitors were further optimized by C-terminal extension to acyl sulfonamides and by P1-P3 cyclization, which gave products with inhibition constants in the nanomolar range ( approximately 75nM).     

Activity of recombinant dengue 2 virus NS3 protease in the presence of a truncated NS2B co-factor, small peptide substrates, and inhibitors

Recombinant forms of the dengue 2 virus NS3 protease linked to a 40-residue co-factor, corresponding to part of NS2B, have been expressed in Escherichia coli and shown to be active against para-nitroanilide substrates comprising the P6-P1 residues of four substrate cleavage sequences. The enzyme is inactive alone or after the addition of a putative 13-residue co-factor peptide but is active when fused to the 40-residue co-factor, by either a cleavable or a noncleavable glycine linker. The NS4B/NS5 cleavage site was processed most readily, with optimal processing conditions being pH 9, I = 10 mm, 1 mm CHAPS, 20% glycerol. A longer 10-residue peptide corresponding to the NS2B/NS3 cleavage site (P6-P4') was a poorer substrate than the hexapeptide (P6-P1) para-nitroanilide substrate under these conditions, suggesting that the prime side substrate residues did not contribute significantly to protease binding. We also report the first inhibitors of a co-factor-complexed, catalytically active flavivirus NS3 protease. Aprotinin was the only standard serine protease inhibitor to be active, whereas a number of peptide substrate analogues were found to be competitive inhibitors at micromolar concentrations.     

Novel potent macrocyclic inhibitors of the hepatitis C virus NS3 protease: use of cyclopentane and cyclopentene P2-motifs

Several highly potent novel HCV NS3 protease inhibitors have been developed from two inhibitor series containing either a P2 trisubstituted macrocyclic cyclopentane- or a P2 cyclopentene dicarboxylic acid moiety as surrogates for the widely used N-acyl-(4R)-hydroxyproline in the P2 position. These inhibitors were optimized for anti HCV activities through examination of different ring sizes in the macrocyclic systems and further by exploring the effect of P4 substituent removal on potency. The target molecules were synthesized from readily available starting materials, furnishing the inhibitor compounds in good overall yields. It was found that the 14-membered ring system was the most potent in these two series and that the corresponding 13-, 15-, and 16-membered macrocyclic rings delivered less potent inhibitors. Moreover, the corresponding P1 acylsulfonamides had superior potencies over the corresponding P1 carboxylic acids. It is noteworthy that it has been possible to develop highly potent HCV protease inhibitors that altogether lack the P4 substituent. Thus the most potent inhibitor described in this work, inhibitor 20, displays a K(i) value of 0.41 nM and an EC(50) value of 9 nM in the subgenomic HCV replicon cell model on genotype 1b. To the best of our knowledge this is the first example described in the literature of a HCV protease inhibitor displaying high potency in the replicon assay and lacking the P4 substituent, a finding which should facilitate the development of orally active small molecule inhibitors against the HCV protease.     

Activity of purified hepatitis C virus protease NS3 on peptide substrates.

The protease domain of the hepatitis C virus (HCV) protein NS3 was expressed in Escherichia coli, purified to homogeneity, and shown to be active on peptides derived from the sequence of the NS4A-NS4B junction. Experiments were carried out to optimize protease activity. Buffer requirements included the presence of detergent, glycerol, and dithiothreitol, pH between 7.5 and 8.5, and low ionic strength. C- and N-terminal deletion experiments defined a peptide spanning from the P6 to the P4' residue as a suitable substrate. Cleavage kinetics were subsequently measured by using decamer P6-P4' peptides corresponding to all intermolecular cleavage sites of the HCV polyprotein. The following order of cleavage efficiency, in terms of kcat/Km, was determined: NS5A-NS5B > NS4A-NS4B >> NS4B-NS5A. A 14-mer peptide containing residues 21 to 34 of the protease cofactor NS4A (Pep4A 21-34), when added in stoichiometric amounts, was shown to increase cleavage rates of all peptides, the largest effect (100-fold) being observed on the hydrolysis of the NS4B-NS5A decamer. From the kinetic analysis of cleavage data, we conclude that (i) primary structure is an important determinant of the efficiency with which each site is cleaved during polyprotein processing, (ii) slow cleavage of the NS4B-NS5A site in the absence of NS4A is due to low binding affinity of the enzyme for this site, and (iii) formation of a 1:1 complex between the protease and Pep4A 21-34 is sufficient and required for maximum activation.     

Synthesis and antiviral activity of novel HCV NS3 protease inhibitors with P4 capping groups

We have synthesized and evaluated a series of novel HCV NS3 protease inhibitors with various P4 capping groups, which include urea, carbamate, methoxy-carboxamide, cyclic carbamate and amide, pyruvic amide, oxamate, oxalamide and cyanoguanidine. Most of these compounds are remarkably potent, exhibiting single-digit to sub-nanomolar activity in the enzyme assay and cell-based replicon assay. Selected compounds were also evaluated in the protease-inhibitor-resistant mutant transient replicon assay, and they were found to show quite different potency profiles against a panel of HCV protease-inhibitor-resistant mutants.     

A collaborative screening program for the discovery of inhibitors of HCV NS2/3 cis-cleaving protease activity

This report describes the development of a cell-based assay for high-throughput screening and detection of small-molecule inhibitors for hepatitis C virus (HCV) NS2/3 protease. The HCV NS2/3 protease is essential for the normal infectious cycle of HCV. Generation of a cell-based assay for this cis-acting viral protease involved reporter constructs in which the NS2/3 protease sequence was inserted between the ,B-lactamase (BLA) reporter and a ubiquitin-based destabilization domain. In stable cell lines, NS2/3 cis cleavage of the NS2/3-BLA fusion protein resulted in differential stability of the cleaved versus uncleaved BLA reporter, providing a robust readout for protease activity. BLA reporter activity was shown to be a function of NS2/3-specific protease activity, by using genetic mutants of the NS2/3 sequence. In addition, the cell-based assay was validated and screened in a 384-well format on a fully automated robotic platform where small-molecule inhibitors of NS2/3 protease activity were identified.     

Synthesis, antiviral activity, and conformational studies of a P3 aza-peptide analog of a potent macrocyclic tripeptide HCV protease inhibitor

BILN 2061 is a macrocyclic tripeptide inhibitor of hepatitis C virus NS3-4A protease that has shown efficacy in the clinic for treating patients infected with HCV. We have synthesized a P3 aza-peptide analog of a potent macrocyclic tripeptide inhibitor closely related to BILN 2061. This aza-derivative was found to be >2 orders of magnitude less active than the parent macrocycle in both isolated enzyme (HCV NS3-4A) and HCV subgenomic replicon assays. NMR studies of P3 aza-peptides revealed these compounds adopt a beta-turn conformation stabilized by an intramolecular H-bonding interaction. Molecular models of these structures indicate a D-like configuration of the P3 aza-residue. Thus, the configurationally undefined nature at P3 in the aza-peptide allows the compound to adopt an H-bond stabilized conformation that is substantially different from that necessary for tight binding to the active site of HCV NS3 protease.     

A loop-mimetic inhibitor of the HCV-NS3 protease derived from a minibody

We have been interested for some time in establishing a strategy for deriving lead compounds from macromolecule ligands such as minibody variants. A minibody is a minimized antibody variable domain whose two loops are amenable to combinatorial mutagenesis. This approach can be especially useful when dealing with 'difficult' targets. One such target is the NS3 protease of hepatitis C virus (HCV), a human pathogen that is believed to infect about 100 million individuals worldwide and for which an effective therapy is not yet available. Based on known inhibitor specificity (residues P6-P1) of NS3 protease, we screened a number of minibodies from our collection and we were able to identify a competitive inhibitor of this enzyme. We thus validated an aspect of recognition by HCV NS3 protease, namely that an acid anchor is necessary for inhibitor activity. In addition, the characterization of the minibody inhibitor led to the synthesis of a constrained hexapeptide mimicking the bioactive loop of the parent macromolecule. The cyclic peptide is a lead compound prone to rapid optimization through solid phase combinatorial chemistry. We therefore confirmed that the potential of turning a protein ligand into a low molecular weight active compound for lead discovery is achievable and can complement more traditional drug discovery approaches.     

Hepatitis C virus NS3 protease inhibitors comprising a novel aromatic P1 moiety

Inhibition of the hepatitis C virus (HCV) NS3 protease has emerged as an attractive approach to defeat the global hepatitis C epidemic. In this work, we present the synthesis and biochemical evaluation of HCV NS3 protease inhibitors comprising a non-natural aromatic P(1) moiety. A series of inhibitors with aminobenzoyl sulfonamides displaying submicromolar potencies in the full-length NS3 protease assay was prepared through a microwave-irradiated, palladium-catalyzed, amidocarbonylation protocol.     

丙型肝炎病毒NS2-3蛋白酶基因在Sf9昆虫细胞中的表达

丙型肝炎病毒(hepatitis C virus,HCV)为单股正链RNA病毒,其基因组长约9.5kb,5'端和3'端各有一个长约345bp和60bp的非编码区,编码区含一个大开放读码框架,编码3 010aa～3 033aa残基的多蛋白前体.     

Cleavage preference distinguishes the two-component NS2B-NS3 serine proteinases of Dengue and West Nile viruses

Regulated proteolysis of the polyprotein precursor by the NS2B-NS3 protease is required for the propagation of infectious virions. Unless the structural and functional parameters of NS2B-NS3 are precisely determined, an understanding of its functional role and the design of flaviviral inhibitors will be exceedingly difficult. Our objectives were to define the substrate recognition pattern of the NS2B-NS3 protease of West Nile and Dengue virises (WNV and DV respectively). To accomplish our goals, we used an efficient, 96-well plate format, method for the synthesis of 9-mer peptide substrates with the general P4-P3-P2-P1-P1'-P2'-P3'-P4'-Gly structure. The N-terminus and the constant C-terminal Gly of the peptides were tagged with a fluorescent tag and with a biotin tag respectively. The synthesis was followed by the proteolytic cleavage of the synthesized, tagged peptides. Because of the strict requirement for the presence of basic amino acid residues at the P1 and the P2 substrate positions, the analysis of approx. 300 peptide sequences was sufficient for an adequate representation of the cleavage preferences of the WNV and DV proteinases. Our results disclosed the strict substrate specificity of the WNV protease for which the (K/R)(K/R)R/GG amino acid motifs was optimal. The DV protease was less selective and it tolerated well the presence of a number of amino acid residue types at either the P1' or the P2' site, as long as the other position was occupied by a glycine residue. We believe that our data represent a valuable biochemical resource and a solid foundation to support the design of selective substrates and synthetic inhibitors of flaviviral proteinases.     

Novel P2–P4 macrocyclic inhibitors of HCV NS3/4A protease by P3 succinamide fragment depeptidization strategy

Hepatitis C represents a serious worldwide health-care problem. Recently, we have disclosed a novel class of P2–P4 macrocyclic inhibitors of NS3/4A protease containing a carbamate functionality as capping group at the P3 N-terminus. Herein we report our work aimed at further depeptidizing the P3 region by replacement of the urethane function with a succinamide motif. This peptidomimetic approach has led to the discovery of novel P2–P4 macrocyclic inhibitors of HCV NS3/4A protease with sub-nanomolar enzyme affinities. In addition to being potent inhibitors of HCV subgenomic replication, optimized analogues within this series have also presented attractive PK properties and showed promising liver levels in rat following oral administration.     

Probing the substrate specificity of the dengue virus type 2 NS3 serine protease by using internally quenched fluorescent peptides

The NS3 (dengue virus non-structural protein 3) serine protease of dengue virus is an essential component for virus maturation, thus representing an attractive target for the development of antiviral drugs directed at the inhibition of polyprotein processing. In the present study, we have investigated determinants of substrate specificity of the dengue virus NS3 protease by using internally quenched fluorogenic peptides containing Abz (o-aminobenzoic acid; synonymous to anthranilic acid) and 3-nitrotyrosine (nY) representing both native and chimaeric polyprotein cleavage site sequences. By using this combinatorial approach, we were able to describe the substrate preferences and determinants of specificity for the dengue virus NS2B(H)-NS3pro protease. Kinetic parameters (kcat/K(m)) for the hydrolysis of peptide substrates with systematic truncations at the prime and non-prime side revealed a length preference for peptides spanning the P4-P3' residues, and the peptide Abz-RRRRSAGnY-amide based on the dengue virus capsid protein processing site was discovered as a novel and efficient substrate of the NS3 protease (kcat/K(m)=11087 M(-1) x s(-1)). Thus, while having confirmed the exclusive preference of the NS3 protease for basic residues at the P1 and P2 positions, we have also shown that the presence of basic amino acids at the P3 and P4 positions is a major specificity-determining feature of the dengue virus NS3 protease. Investigation of the substrate peptide Abz-KKQRAGVLnY-amide based on the NS2B/NS3 polyprotein cleavage site demonstrated an unexpected high degree of cleavage efficiency. Chimaeric peptides with combinations of prime and non-prime sequences spanning the P4-P4' positions of all five native polyprotein cleavage sites revealed a preponderant effect of non-prime side residues on the K(m) values, whereas variations at the prime side sequences had higher impact on kcat.