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.     

Homology modeling and molecular dynamics simulations of Dengue virus NS2B/NS3 protease: insight into molecular interaction

The pathogenic West Nile virus (WNV) and Dengue virus (DV) are growing global threats for which there are no specific treatments. Both viruses possess a two component NS2B/NS3 protease which cleaves viral precursor proteins. Whereas for the WNV protease two crystal structures in complex with an inhibitor have been solved recently, no such information is available for the DV protease. Here, we report the generation of a homology model of DV NS2B/NS3 protease. Since it is known from the related WNV protease that it adopts a distinct conformation in free and in inhibitor‐complexed form, a special emphasis was given to the analysis of the protease flexibility. Therefore, several models of DV NS2B/NS3 protease complexed with the peptidic inhibitor (Bz‐Nle(P4)‐Lys(P3)‐Arg(P2)‐Arg(P1)‐H) were generated. The first DV protease model (DV‐1) was constructed using the available crystal structure of the apo DV NS2B/NS3 protease. The second model (DV‐2) was built taking the WNV NS3/NS2B protease in the inhibitor‐complexed form as the template structure. Molecular dynamics simulations which were carried out for the WNV crystal structures as well as for the DV models provided an understanding of the role of NS2B for maintaining the protease in the active conformation. It was also demonstrated that NS2B is not only important for maintaining NS3 in the active form, but is also essential for establishing the interaction between residues from the S2 pocket and the peptidic inhibitor. The DV NS2B/NS3 model in the productive conformation can now be used for structure‐based design purposes. Copyright © 2009 John Wiley & Sons, Ltd.     

Identification of novel thiadiazoloacrylamide analogues as inhibitors of dengue-2 virus NS2B/NS3 protease

Dengue virus is endemic throughout tropical and subtropical regions, and cause severe epidemic diseases. The NS2B/NS3 protease is a promising drug target for dengue virus. Herein, we report the discovery and modification of a novel class of thiadiazoloacrylamide derivatives with potent inhibitory activity against the NS2B/NS3 protease. Thiadiazolopyrimidinone 1 was firstly determined as a new chemical structure against NS2B/NS3 from a commercial compound library. Then, we sought to identify similar compounds with the thiadiazoloacrylamide core that would exhibit better activity. A series of analogues were synthesized and fourteen of them were identified with strong inhibitory activities, in which the nitrile group in the linker part was discovered as an essential group for the inhibitory activity. The best of these (8b) demonstrated an IC₅₀ at 2.24 μM based on in vitro DENV2 NS2B-NS3pro assays.     

Exploring small molecules with pan-genotypic inhibitory activities against hepatitis C virus NS3/4A serine protease

Among the many Hepatitis C virus (HCV) genotypes and subtypes, genotypes 1b and 3a are most prevalent in United States and Asia, respectively. A total of 132 commercially available analogs of a previous lead compound were initially investigated against wild-type HCV genotype 1b NS3/4A protease. Ten compounds showed inhibitory activities (IC₅₀ values) below 10 µM with comparable direct binding affinities (K_D values) determined by surface plasmon resonance (SPR). To identify pan-genotypic inhibitors, these ten selected compounds were tested against four additional genotypes (1a, 2a, 3a, and 4) and three drug-resistant mutants (A156S, R155K, and V36M). Four new analogs have been identified with better activities against all five tested genotypes than the prior lead compound. Further, the original lead compound did not show activity against genotype 3a NS3/4A, whereas four newly identified compounds exhibited IC₅₀ values below 33 µM against genotype 3a NS3/4A. Encouragingly, the best new compound F1813-0710 possessed promising activity toward genotype 3a, which is a huge improvement over the previous lead compound that had no effect on genotype 3a. This intriguing observation was further analyzed by molecular docking and molecular dynamics (MD) simulations to understand their different binding interactions, which should benefit future pan-genotypic inhibitor design and drug discovery.     

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。     

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).     

MD simulations reveal alternate conformations of the oxyanion hole in the Zika virus NS2B/NS3 protease

Recent crystallography studies have shown that the binding site oxyanion hole plays an important role in inhibitor binding, but can exist in two conformations (active/inactive). We have undertaken molecular dynamics (MD) calculations to better understand oxyanion hole dynamics and thermodynamics. We find that the Zika virus (ZIKV) NS2B/NS3 protease maintains a stable closed conformation over multiple 100-ns conventional MD simulations in both the presence and absence of inhibitors. The S1, S2, and S3 pockets are stable as well. However, in two of eight simulations, the A132-G133 peptide bond in the binding pocket of S1' spontaneously flips to form a 3₁₀-helix that corresponds to the inactive conformation of the oxyanion hole, and then maintains this conformation until the end of the 100-ns conventional MD simulations without inversion of the flip. This conformational change affects the S1' pocket in ZIKV NS2B/NS3 protease active site, which is important for small molecule binding. The simulation results provide evidence at the atomic level that the inactive conformation of the oxyanion hole is more favored energetically when no specific interactions are formed between substrate/inhibitor and oxyanion hole residues. Interestingly, however, transition between the active and inactive conformation of the oxyanion hole can be observed by boosting the valley potential in accelerated MD simulations. This supports a proposed induced-fit mechanism of ZIKV NS2B/NS3 protease from computational methods and provides useful direction to enhance inhibitor binding predictions in structure-based drug design.     

Characterisation of the role of zinc in the hepatitis C virus NS2/3 auto-cleavage and NS3 protease activities

Cleavage of the hepatitis C virus polyprotein between the non-structural NS2 and NS3 proteins is mediated by a poorly characterised auto-proteolytic activity that maps to the C terminus of NS2 and the N terminus of NS3, but is distinct from the NS3 protease activity responsible for downstream cleavages in the polyprotein. We have exploited the fact that the minimal precursor (residues 904-1206 of the HCV polyprotein) can be expressed as an insoluble protein in Escherichia coli and subsequently refolded into a form active for both auto-cleavage and NS3 protease activity, to further characterise the NS2/3 auto-cleavage activity. We show that both activities are zinc-dependent and show an absolute requirement for cysteine residues 1123, 1125 and 1171 within NS3. In contrast cysteine 922 (within NS2) is only required for NS2/3 auto-cleavage activity and histidine 1175 is only required for NS3 activity. Although the complete NS3 protease domain (including the C-terminal alpha-helix) is required for NS2/3 auto-cleavage, the activity of the NS3 protease is not essential. Lastly we show that the NS2/3 auto-cleavage activity is more sensitive to zinc chelation by 1,10-phenanthroline than the NS3 protease activity. This observation is consistent with different conformations of the precursor competent for either NS2/3 auto-cleavage or NS3 protease activity; these two conformations can be distinguished by their relative strength and geometry of zinc coordination.     

Mechanistic and kinetic characterization of hepatitis C virus NS3 protein interactions with NS4A and protease inhibitors

The mechanism and kinetics of the interactions between ligands and immobilized full‐length hepatitis C virus (HCV) genotype 1a NS3 have been characterized by SPR biosensor technology. The NS3 interactions for a series of NS3 protease inhibitors as well as for the NS4A cofactor, represented by a peptide corresponding to the sequence interacting with the enzyme, were found to be heterogeneous. It may represent interactions with two stable conformations of the protein. The NS3–NS4A interaction consisted of a high‐affinity (K_D = 50 nM) and a low‐affinity (K_D = 2 µM) interaction, contributing equally to the overall binding. By immobilizing NS3 alone or together with NS4A it was shown that all inhibitors had a higher affinity for NS3 in the presence of NS4A. NS4A thus has a direct effect on the binding of inhibitors to NS3 and not only on catalysis. As predicted, the mechanism‐based inhibitor VX 950 exhibited a time‐dependent interaction with a slow formation of a stable complex. BILN 2061 or ITMN‐191 showed no signs of time‐dependent interactions, but ITMN‐191 had the highest affinity of the tested compounds, with both the slowest dissociation (k_off) and fastest association rate, closely followed by BILN 2061. The k_off for the inhibitors correlated strongly with their NS3 protease inhibitory effect as well as with their effect on replication of viral proteins in replicon cell cultures, confirming the relevance of the kinetic data. This approach for obtaining kinetic and mechanistic data for NS3 protease inhibitor and cofactor interactions is expected to be of importance for understanding the characteristics of HCV NS3 functionality as well as for anti‐HCV lead discovery and optimization. Copyright © 2010 John Wiley & Sons, Ltd.     

Reporter substrates for assessing the activity of the hepatitis C virus NS3-4A serine protease in living cells

We describe a versatile system for monitoring the activity of the NS3-4A serine protease of the hepatitis C virus (HCV) in mammalian cells. The system relies on coexpression of the protease and of an artificial substrate containing a reporter domain and an intracellular targeting sequence separated by a NS3-4A-specific cleavage site. We constructed two different substrates suitable for different applications. The first substrate secretory alkaline phosphatase-1 (SEAP-1) harbors the NS3-4A cleavage site inserted between the SEAP and a membrane anchor featuring an endoplasmic reticulum retention sequence. The arrangement of this substrate is such that SEAP is secreted in the extracellular medium depending on the NS3 protease activity. We show that SEAP-1 can be used to evaluate the activity of NS3-4A inhibitors in living cells. In the second substrate (CD8-1), SEAP is replaced by the extracellular domain of the lymphocyte surface antigen CD8 alpha. The arrangement of this substrate is such that the CD8 alpha domain is transported to the cell surface upon NS3-4Ap cleavage and remains associated with the plasma membrane as an integral membrane protein. We show that CD8-1 can be used for selecting cells capable of supporting HCV replication.     

Development of a cell-based assay for monitoring specific hepatitis C virus NS3/4A protease activity in mammalian cells

The hepatitis C virus (HCV) contains a positive-sense RNA genome that encodes a unique polyprotein precursor, which must be processed by proteases to enable viral maturation. Virally encoded NS3/4A protease has thus become an attractive target for the development of antiviral drugs. To establish an assay system for monitoring NS3/4A protease activity in mammalian cells, this study describes a substrate vector, pEG(Delta4AB)SEAP, in which enhanced green fluorescent protein (EGFP) was fused to secreted alkaline phosphatase (SEAP) through the NS3/4A protease decapeptide recognition sequence, Delta4AB, which spans the NS4A and NS4B junction region. Secretion of SEAP into the culture medium was demonstrated to depend on the cleavage of Delta4AB by HCV NS3/4A protease. We demonstrated that the accumulation of SEAP activity in the culture medium depends on time up to 60h with the coexpression of active NS3/4A protease. The amount of SEAP in the culture medium was around 10 times greater than that of cells with coexpression of inactive NS3/4A mutant protease. This strategy has made it possible to monitor NS3/4A activity inside mammalian cells. Moreover, by using cells containing the HCV subgenomic replicon, the EG(Delta4AB)SEAP reporter can be used to detect the anti-HCV activity of interferon-alpha (IFN-alpha). Consequently, this EG(Delta4AB)SEAP reporter can be used to screen for NS3/4A protease inhibitors in the cellular environment and for anti-HCV drugs in replicon cells.     

Complex of NS3 protease and NS4A peptide of BK strain hepatitis C virus: a 2.2 A resolution structure in a hexagonal crystal form.

The crystal structure of the NS3 protease of the hepatitis C virus (BK strain) has been determined in the space group P6(3)22 to a resolution of 2.2 A. This protease is bound with a 14-mer peptide representing the central region of the NS4A protein. There are two molecules of the NS3(1-180)-NS4A(21'-34') complex per asymmetric unit. Each displays a familiar chymotrypsin-like fold that includes two beta-barrel domains and four short alpha-helices. The catalytic triad (Ser-139, His-57, and Asp-81) is located in the crevice between the beta-barrel domains. The NS4A peptide forms an almost completely enclosed peptide surface association with the protease. In contrast to the reported H strain complex of NS3 protease-NS4A peptide in a trigonal crystal form (Kim JL et al., 1996, Cell 87:343-355), the N-terminus of the NS3 protease is well-ordered in both molecules in the asymmetric unit of our hexagonal crystal form. The folding of the N-terminal region of the NS3 protease is due to the formation of a three-helix bundle as a result of crystal packing. When compared with the unbound structure (Love RA et al., 1996, Cell 87:331-342), the binding of the NS4A peptide leads to the ordering of the N-terminal 28 residues of the NS3 protease into a beta-strand and an alpha-helix and also causes local rearrangements important for a catalytically favorable conformation at the active site. Our analysis provides experimental support for the proposal that binding of an NS4A-mimicking peptide, which increases catalytic rates, is necessary but not sufficient for formation of a well-ordered, compact and, hence, highly active protease molecule.     

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。     

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.     

Designing cyclic peptide inhibitor of dengue virus NS3-NS2B protease by using molecular docking approach

Peptides are preferred for designing inhibitors because of their high activity and specificity. Seven cyclopentapeptide inhibitors were designed in this study against dengue virus type 2 (DEN-2) NS3-NS2B protease: CKRRC, CGRRC, CRGRC, CRTRC, CTRRC, CKRKC and CRRKC. Docking analysis was performed to study the enzyme-inhibitor binding interactions. The free energy binding and estimated Ki values for all the inhibitors were found to be small (within micromolar range), indicating that the inhibitors bind considerably well to the binding site. The results showed that the cyclopentapeptide CKRKC was the best peptide inhibitor candidate with estimated free binding energy of -8.39 kcal/mol and Ki of 0.707 μM when compared to the standard inhibitor Bz-Nle-Lys-Arg-Arg-H that has been experimentally tested and shown to exhibit Ki value of 5.8 μM. Several modes of weak interactions were observed between the cyclopentapeptide CKRKC and the active site of DEN-2 NS3-NS2B protease. Thus, the cyclopentapeptide is proposed as a potential inhibitor to the NS3-NS2B protease activities of DEN-2. While these preliminary results are promising, further experimental investigation is necessary to validate the results.     

The interdomain interface in bifunctional enzyme protein 3/4A (NS3/4A) regulates protease and helicase activities

Hepatitis C (HCV) protein 3/4A (NS3/4A) is a bifunctional enzyme comprising two separate domains with protease and helicase activities, which are essential for viral propagation. Both domains are stable and have enzymatic activity separately, and the relevance and implications of having protease and helicase together as a single protein remains to be explored. Altered in vitro activities of isolated domains compared with the full‐length NS3/4A protein suggest the existence of interdomain communication. The molecular mechanism and extent of this communication was investigated by probing the domain–domain interface observed in HCV NS3/4A crystal structures. We found in molecular dynamics simulations that the two domains of NS3/4A are dynamically coupled through the interface. Interestingly, mutations designed to disrupt this interface did not hinder the catalytic activities of either domain. In contrast, substrate cleavage and DNA unwinding by these mutants were mostly enhanced compared with the wild‐type protein. Disrupting the interface did not significantly alter RNA unwinding activity; however, the full‐length protein was more efficient in RNA unwinding than the isolated protease domain, suggesting a more direct role in RNA processing independent of the interface. Our findings suggest that HCV NS3/4A adopts an “extended” catalytically active conformation, and interface formation acts as a switch to regulate activity. We propose a unifying model connecting HCV NS3/4A conformational states and protease and helicase function, where interface formation and the dynamic interplay between the two enzymatic domains of HCV NS3/4A potentially modulate the protease and helicase activities in vivo.     

A FRET-based assay for the discovery of West Nile Virus NS2B-NS3 protease inhibitors

The West Nile Virus (WNV) has been a worldwide epidemic since the early 1990s. Currently there are no therapeutic treatments for WNV infections. One particular avenue of treatment is inhibition of the NS2B-NS3 protease, an enzyme that is crucial for WNV replication. In our effort to increase the number of NS2B-NS3 protease inhibitors, we report a novel FRET-based high throughput assay for the discovery of WNV NS2B-NS3 protease inhibitors. For this assay, a FRET-based peptide substrate was synthesized and kinetically characterized with the NS2B-NS3 protease. The new substrate exhibits a K_m of 3.35 ± 0.31 μM, a k_cat of 0.0717 ± 0.0016 s^?1 and a k_cat/K_m of 21,400 ± 2000 M^?1 s^?1.     

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。     

A quantum mechanics/molecular mechanics study of the reaction mechanism of the hepatitis C virus NS3 protease with the NS5A/5B substrate

Combined quantum mechanics and molecular mechanics (QM/MM) calculations were carried out to characterize the reaction mechanism of the NS3 protease with its preferred substrate (NS5A/5B). The main purpose of this study was to locate the barrier states and intermediates along the distinguished coordinate path (DCP) involved in this process. These structures, and in particular the one corresponding to the first barrier state and intermediate (B1 and I1), could be a starting point for the synthesis of inhibitors of this protease, which could be used to treat hepatitis C. The two first steps of the reaction mechanism were studied, i.e., the acylation step and the breaking of the peptide bond. The first step takes place through a tetracoordinated intermediate, as suggested from previous works on other Serine proteases. The importance of the different amino acid residues was also considered (perturbation study where the MM charges of each residue were set to zero independently). The residues of the oxyanion hole were confirmed as the most important for the electrostatic stabilization of the tetracoordinate intermediate. Moreover, the role of other residues, e.g., Arg-155 and Asp-79, was also explained.     

Japanese Encephalitis Virus NS2B-3 Protein Complex Promotes Cell Apoptosis and Viral Particle Release by Down-Regulating the Expression of AXL

Virologica Sinica - Japanese encephalitis virus (JEV) is a flavivirus transmitted by mosquitoes that causes severe encephalitis in humans and animals. It has been suggested that AXL, a...