Synthesis and SAR of acyclic HCV NS3 protease inhibitors with novel P4-benzoxaborole moieties

We have synthesized and evaluated a new series of acyclic P4-benzoxaborole-based HCV NS3 protease inhibitors. Structure-activity relationships were investigated, leading to the identification of compounds 5g and 17 with low nanomolar potency in the enzymatic and cell-based replicon assay. The linker-truncated compound 5j was found to exhibit improved absorption and oral bioavailability in rats, suggesting that further reduction of molecular weight and polar surface area could result in improved drug-like properties of this novel series.     

Structure-based design of a novel series of azetidine inhibitors of the hepatitis C virus NS3/4A serine protease

Structural homology between thrombin inhibitors and the early tetrapeptide HCV protease inhibitor led to the bioisosteric replacement of the P2 proline by a 2,4-disubstituted azetidine within the macrocyclic β-strand mimic. Molecular modeling guided the design of the series. This approach was validated by the excellent activity and selectivity in biochemical and cell based assays of this novel series and confirmed by the co-crystal structure of the inhibitor with the NS3/4A protein (PDB code: 4TYD).     

The discovery and optimization of naphthalene-linked P2-P4 Macrocycles as inhibitors of HCV NS3 protease

Naphthalene-linked P2-P4 macrocycles within a tri-peptide-based acyl sulfonamide chemotype have been synthesized and found to inhibit HCV NS3 proteases representing genotypes 1a and 1b with single digit nanomolar potency. The pharmacokinetic profile of compounds in this series was optimized through structural modifications along the macrocycle tether as well as the P1 subsite. Ultimately a compound with oral bioavailability of 100% in rat, and a long half-life in plasma was obtained. However, compounds in this macrocyclic series exhibited cardiac effects in an isolated rabbit heart model and for this reason further optimization efforts were discontinued.     

Discovery of novel P2 substituted 4-biaryl proline inhibitors of hepatitis C virus NS3 serine protease

Inhibitors of hepatitis C virus NS3 serine protease often incorporate a large P2 moiety to interact with the surface of the enzyme while shielding part of the catalytic triad. This feature is important in many inhibitors in order to have the necessary potency needed for efficacy. In this Letter we explore some new P2 motifs to further exploit this region of the enzyme. In a continuing effort to replace the often found 4-hydroxyproline P2 core found in the majority of inhibitors for this target, various directly attached aryl derivatives were evaluated. Of these, the 2,4-disubstituted thiazole core proved to be the most interesting. SAR around this motif has lead to compounds with K_i’s in the high picomolar range and provided cellular potencies in the single digit nM range.     

Variability and resistance mutations in the hepatitis C virus NS3 protease in patients not treated with protease inhibitors

The goal of treatment of chronic hepatitis C is to achieve a sustained virological response, which is defined as exhibiting undetectable hepatitis C virus (HCV) RNA levels in serum following therapy for at least six months. However, the current treatment is only effective in 50% of patients infected with HCV genotype 1, the most prevalent genotype in Brazil. Inhibitors of the serine protease non-structural protein 3 (NS3) have therefore been developed to improve the responses of HCV-infected patients. However, the emergence of drug-resistant variants has been the major obstacle to therapeutic success. The goal of this study was to evaluate the presence of resistance mutations and genetic polymorphisms in the NS3 genomic region of HCV from 37 patients infected with HCV genotype 1 had not been treated with protease inhibitors. Plasma viral RNA was used to amplify and sequence the HCV NS3 gene. The results indicate that the catalytic triad is conserved. A large number of substitutions were observed in codons 153, 40 and 91; the resistant variants T54A, T54S, V55A, R155K and A156T were also detected. This study shows that resistance mutations and genetic polymorphisms are present in the NS3 region of HCV in patients who have not been treated with protease inhibitors, data that are important in determining the efficiency of this new class of drugs in Brazil.     

Antiviral compounds from traditional Chinese medicines Galla Chinese as inhibitors of HCV NS3 protease

Under the guidance of bioassay, the EtOAc extract fraction of the Traditional Chinese Medicine (TCM) Galla Chinese was found to be efficient in inhibiting the NS3 protease of HCV and purified the fraction to get three polyphenol compounds 1,2,6-tri-O-galloyl-β-D-glucose (1), 1,2,3,6-tetra-O-galloyl-β-D-glucose (2), and 1,2,3,4,6-penta-O-galloyl-β-D-glucose (3), which were identified as inhibitors of Hepatitis C Virus (HCV) NS3 protease. Compounds 1, 2, and 3 inhibited HCV NS3 protease with IC₅₀ of 1.89, 0.75, and 1.60 μM, respectively.     

Mechanistic role of NS4A and substrate in the activation of HCV NS3 protease

Hepatitis C virus (HCV) NS3 protease is the key enzyme for its maturation. Three hypotheses have been advanced in the literature to demonstrate the mechanism of the activation of the HCV NS3 protease. A virus-encoded protein NS4A and substrate are proposed to be involved in the activation of the HCV NS3 protease. However, the three hypotheses are not completely consistent with one another. Multiple molecular dynamics simulations were performed on various NS3 protease systems: free NS3 protease, NS3/4A, NS3/inhibitor, and NS3/4A/inhibitor complexes, to further unravel the mechanism of the activation of the NS3 protease. Simulation results suggest that the binding of NS4A induces a classic serine protease conformation of the catalytic triad of the NS3 protease. NS4A rearranges the secondary structure of both the N-terminus and catalytic site of the NS3 protease, reduces the mobility of the global structure of the NS3 protease, especially the catalytic site, and provides a rigid and tight structure, except for the S1 pocket, for the binding and hydrolysis of substrates. The binding of substrate also contributes to the activation of the NS3 protease by an induced-fit of the classic serine protease catalytic triad. However, the global structure of the NS3 protease is still loose and highly flexible without stable secondary structural elements, such as helix α0 at the N-terminus and helix α1 and β-sheet E1-F1 at the catalytic site. The structure of the NS3 protease without NS4A is not suitable for the binding and hydrolysis of substrates.     

Peptide backbone replacement of hepatitis C virus NS3 serine protease C-terminal cleavage product analogs: Discovery of potent succinamide inhibitors

A number of potent peptidic inhibitors of the NS3 protease have been described in the literature based on a substrate-based approach. In an on-going effort to reduce the peptidic character of this class of inhibitors, two novel series of analogs have been prepared in which the usual P3 amino acid residue is replaced by a succinamide fragment. This new backbone modification not only reduces the peptidic nature of traditional inhibitors but also provides new SAR opportunities for the capping group. Optimization of each of these two series resulted in inhibitors with sub-nanomolar potencies.     

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.     

HCV NS3/4A蛋白酶胞内荧光检测方法的建立

目的:建立丙型肝炎病毒NS3/4A丝氨酸蛋白酶胞内荧光检测方法。方法:利用EGFP分子内合适位点可以插入一定长度外源片段而不影响荧光性能的特性,构建EGFP分子内插入NS3/4A蛋白酶识别序列NS5AB的EGFP-5AB重组分子。将EGFP-5AB与NS3/4A蛋白酶共表达,若短肽链被切断,则EGFP的两个部分解离,荧光消失,从而可以监测HCV NS3/4A蛋白酶的存在。通过将NS5AB插入三种不同位点,寻找最合适的插入位点;将EGFP-5AB转染进入不同宿主细胞,验证其在不同细胞的表达情况并选择最佳宿主细胞。结果:确定EGFP 173-174氨基酸位点是合适的插入位点;确定CHO-K1为理想的荧光检测系统宿主细胞;在构建的细胞模型中,能够检测到EGFP被切割后的条带,但检测不到荧光信号,说明EGFP-5AB蛋白被有效切割,该方法可以检测到NS3/4A丝氨酸蛋白酶的存在。结论:成功构建了一种在哺乳动物细胞中检测NS3/4A蛋白酶切割活性的荧光检测方法。     

Vinylated linear P2 pyrimidinyloxyphenylglycine based inhibitors of the HCV NS3/4A protease and corresponding macrocycles

With three recent market approvals and several inhibitors in advanced stages of development, the hepatitis C virus (HCV) NS3/4A protease represents a successful target for antiviral therapy against hepatitis C. As a consequence of dealing with viral diseases in general, there are concerns related to the emergence of drug resistant strains which calls for development of inhibitors with an alternative binding-mode than the existing highly optimized ones. We have previously reported on the use of phenylglycine as an alternative P2 residue in HCV NS3/4A protease inhibitors. Herein, we present the synthesis, structure–activity relationships and in vitro pharmacokinetic characterization of a diverse series of linear and macrocyclic P2 pyrimidinyloxyphenylglycine based inhibitors. With access to vinyl substituents in P3, P2 and P1′ positions an initial probing of macrocyclization between different positions, using ring-closing metathesis (RCM) could be performed, after addressing some synthetic challenges. Biochemical results from the wild type enzyme and drug resistant variants (e.g., R155 K) indicate that P3–P1′ macrocyclization, leaving the P2 substituent in a flexible mode, is a promising approach. Additionally, the study demonstrates that phenylglycine based inhibitors benefit from p-phenylpyrimidinyloxy and m-vinyl groups as well as from the combination with an aromatic P1 motif with alkenylic P1′ elongations. In fact, linear P2–P1′ spanning intermediate compounds based on these fragments were found to display promising inhibitory potencies and drug like properties.     

Structure-activity relationships of 4-hydroxy-4-biaryl-proline acylsulfonamide tripeptides: A series of potent NS3 protease inhibitors for the treatment of hepatitis C virus

The design and synthesis of a series of tripeptide acylsulfonamides as potent inhibitors of the HCV NS3/4A serine protease is described. These analogues house a C4 aryl, C4 hydroxy-proline at the S2 position of the tripeptide scaffold. Information relating to structure-activity relationships as well as the pharmacokinetic and cardiovascular profiles of these analogues is provided.     

Mechanistic studies of electrophilic protease inhibitors of full length hepatic C virus (HCV) NS3

The inhibition mechanism of electrophilic peptide-based protease inhibitors of full-length hepatitis C virus (HCV) NS3 has been investigated by determining the K_i-values for a series of compounds differing in the electrophilicity and acidity of the C-terminal residue at pH-values above and below the pK_a of the catalytic histidine (6.85) and at two different ionic strengths. Electrophilic compounds with a pentafluoroethyl ketone group showed stronger inhibition at pH 8 than pH 6, as expected for a mechanism requiring an unprotonated catalytic histidine. However, the difference was only significant at high ionic strength. In contrast, electrophilic compounds with an acidic C-terminal group or a cyclic P1 residue showed a lower inhibitory effect at pH 8 than at pH 6, inconsistent with a mechanism-based inhibition. Moreover, all electrophilic compounds had an unexpectedly strong inhibition at pH 6, when mechanism-based inhibition is unlikely. The results suggest that for some of the electrophilic compounds the reactive group may not be properly positioned in the active site and that binding of these inhibitors is a result of non-covalent interactions. The nature of these interactions is discussed.     

HCV NS3 serine protease-neutralizing single-chain antibodies isolated by a novel genetic screen

Hepatitis C virus (HCV) infection is a major world-wide health problem causing chronic hepatitis, liver cirrhosis and primary liver cancer. The high frequency of treatment failure points to the need for more specific, less toxic and more active antiviral therapies for HCV. The HCV NS3 is currently regarded as a prime target for anti-viral drugs, thus specific inhibitors of its activity are of utmost importance. Here, we report the development of a novel bacterial genetic screen for inhibitors of NS3 catalysis and its application for the isolation of single-chain antibody-inhibitors. Our screen is based on the concerted co-expression of a reporter gene, of recombinant NS3 protease and of fusion-stabilized single-chain antibodies (scFvs) in Escherichia coli. The reporter system had been constructed by inserting a short peptide corresponding to the NS5A/B cleavage site of NS3 into a permissive site of the enzyme beta-galactosidase. The resulting engineered lacZ gene, coding for an NS3-cleavable beta-galactosidase, is carried on a low copy plasmid that also carried the NS3 protease-coding sequence. The resultant beta-galactosidase enzyme is active, conferring a Lac+ phenotype (blue colonies on indicator 5-bromo-4-chloro-3-indolyl beta-D-galactoside (X-gal) plates), while induction of NS3 expression results in loss of beta-galactosidase activity (transparent colonies on X-gal plates). The identification of inhibitors, as shown here by isolating NS3-inhibiting single-chain antibodies, expressed from a compatible high copy number plasmid, is based on the appearance of blue colonies (NS3 inhibited) on the background of colorless colonies (NS3 active). Our source of inhibitory scFvs was an scFv library that we prepared from spleens of NS3-immunized mice and subjected to limited affinity selection. Once isolated, the inhibitors were validated as genuine and specific NS3 binders by an enzyme-linked immunosorbent assay and as bone fide NS3 serine protease inhibitors by an in vitro catalysis assay. We further show that upon expression as cytoplasmic intracellular antibodies (intrabodies) in NS3-expressing mammalian cells, three of the scFvs inhibit NS3-mediated cell proliferation. Although applied here for the isolation of antibody-based inhibitors, our genetic screen should be applicable for the identification of candidate inhibitors from other sources.     

Discovery of novel HCV inhibitors: Synthesis and biological activity of 6-(indol-2-yl)pyridine-3-sulfonamides targeting hepatitis C virus NS4B

A novel series of 6-(indol-2-yl)pyridine-3-sulfonamides was prepared and evaluated for their ability to inhibit HCV RNA replication in the HCV replicon cell culture assay. Preliminary optimization of this series furnished compounds with low nanomolar potency against the HCV genotype 1b replicon. Among these, compound 8c has identified as a potent HCV replicon inhibitor (EC₅₀ = 4 nM) with a selectivity index with respect to cellular GAPDH of more than 2500. Further, compound 8c had a good pharmacokinetic profile in rats with an IV half-life of 6 h and oral bioavailability (F) of 62%. Selection of HCV replicon resistance identified an amino acid substitution in HCV NS4B that confers resistance to these compounds. These compounds hold promise as a new chemotype with anti-HCV activity mediated through an underexploited viral target.     

HCV NS3/4A蛋白酶抑制剂细胞筛选模型

丙型肝炎病毒(hepatitis C virus,HCV)持续感染可导致慢性丙型肝炎,并可发展为肝硬化和肝细胞癌。HCV NS3/4A蛋白酶在HCV复制和致病机制中起着非常关键的作用,已成为HCV研究热点。由于候选药物分子必须具有易进入细胞、稳定性好等特征,建立一种细胞水平上酶活性的测定系统对于筛选抗NS3/4A药物无疑有着重要意义。目前已有多种NS3/4A蛋白酶筛选系统开发出来,本文将对此作一综述。     

Molecular dynamics study on drug resistance mechanism of HCV NS3/4A protease inhibitor: BI201335

Hepatitis C virus (HCV) infection is a serious threat to global health. NS3 serine protease is one of the most advanced HCV drug targets. However, the high mutation rate makes many protease inhibitors ineffective and allows viral replication to continue. To investigate the structural basis of the molecular mechanism of HCV resistance to inhibitors, molecular dynamics and molecular mechanics Poisson–Boltzmann/surface area calculations were carried out on HCV NS3 serine protease–BI201335 complexes. The drug resistance to BI201335 is explained by the fact that seven single mutations weaken the biological activity by lessening the sum of electrostatic interactions in the gas phase and polar solvation. The computational results demonstrate that the mutations affect the BI201335 binding through direct and indirect mechanisms. Seven single mutations lead to significant changes in the conformation, such as the shifts of the side chain of His57 and Lys136 and the movement of the P2 group of BI201335 towards the solvent. Furthermore, the contributions of Lys136 significantly decrease, which is the most major binding attraction. The shifts of the side chain of His57 induce the lack of hydrogen bond between His57 with Asp81 expert for D168G mutation. Detailing the molecular mechanisms of BI201335 drug resistance provides some helpful insights into the nature of mutational effect and aid the rational design of potent inhibitors combating HCV.     

HCV NS3/4A蛋白酶与Faldaprevir类似物的分子识别机制及其复合物运动模式

Faldaprevir类似物(Faldaprevir analogue molecule,FAM)能有效抑制HCV NS3/4A蛋白酶的催化活性,是一种潜在抗HCV先导化合物。通过生物信息学统计分析了已报道的HCV NS3/4A蛋白酶晶体结构,得到了FAM-HCV NS3/4A蛋白酶晶体结构。对FAM-HCV NS3/4A蛋白酶复合物进行了20.4 ns的分子动力学模拟,重点从氢键和结合自由能两个角度分析了二者分子识别中的关键残基及结合驱动力。氢键和范德华力是促使FAM特异性结合到蛋白V132?S139、F154?D168、D79?D81和V55的活性口袋中的主要驱动力,这与实验数据较为吻合。耐药性突变实验分析了R155K、D168E/V和V170T定点突变对FAM分子识别的影响,为可能存在的FAM耐药性提供了分子依据。最后,用自由能曲面和构象聚类两个方法探讨了体系的构象变化,给出体系的4种优势构象,为后续的基于HCV NS3/4A蛋白酶结构的Faldaprevir类似物抑制剂分子设计提供一定的理论帮助。     

P3-P4 ureas and reverse carbamates as potent HCV NS3 protease inhibitors: Effective transposition of the P4 hydrogen bond donor

A series of tripeptidic acylsulfonamide inhibitors of HCV NS3 protease were prepared that explored structure-activity relationships (SARs) at the P4 position, and their in vitro and in vivo properties were evaluated. Enhanced potency was observed in a series of P4 ureas; however, the PK profiles of these analogues were less than optimal. In an effort to overcome the PK shortcomings, modifications to the P3-P4 junction were made. This included a strategy in which one of the two urea N–H groups was either N-methylated or replaced with an oxygen atom. The former approach provided a series of regioisomeric N-methylated ureas while the latter gave rise to P4 reverse carbamates, both of which retained potent NS3 inhibitory properties while relying upon an alternative H-bond donor topology. Details of the SARs and PK profiles of these analogues are provided.     

Phosphonate inhibitors of West Nile virus NS2B/NS3 protease

West Nile virus (WNV) is a member of the flavivirus genus belonging to the Flaviviridae family. The viral serine protease NS2B/NS3 has been considered an attractive target for the development of anti-WNV agents. Although several NS2B/NS3 protease inhibitors have been described so far, most of them are reversible inhibitors. Herein, we present a series of α-aminoalkylphosphonate diphenyl esters and their peptidyl derivatives as potent inhibitors of the NS2B/NS3 protease. The most potent inhibitor identified was Cbz-Lys-Arg-(4-GuPhe)^P(OPh)₂ displaying K_i and k₂/K_i values of 0.4 µM and 28 265 M^?1s^?1, respectively, with no significant inhibition of trypsin, cathepsin G, and HAT protease.