Identification of potential inhibitors for HCV NS5b of genotype 4a by combining dynamic simulation,protein–ligand interaction fingerprint, 3D pharmacophore,docking and 3D QSAR

Abstract

HCV NS5B polymerase has been one of the most attractive targets for developing new drugs for HCV infection and many drugs were successfully developed, but all of them were designed for targeting Hepatitis C Virus genotype 1 (HCV GT1). Hepatitis C virus genotype 4a (HCV GT4a) dominant in Egypt has paid less attention. Here, we describe our protocol of virtual screening in identification of novel potential potent inhibitors for HCV NS5B polymerase of GT4a using homology modeling, protein–ligand interaction fingerprint (PLIF), docking, pharmacophore, and 3D CoMFA quantitative structure activity relationship (QSAR). Firstly, a high-quality 3D model of HCV NS5B polymerase of GT4a was constructed using crystal structure of HCV NS5B polymerase of GT1 (PDB ID: 3hkw) as a template. Then, both the model and the template were simulated to compare conformational stability. PLIF was generated using five crystal structures of HCV NS5B (PDB ID: 4mia, 4mib, 4mk9, 4mka, and 4mkb), which revealed the most important residues and their interactions with the co-crystalized ligands. After that, a 3D pharmacophore model was developed from the generated PLIF data and then used as a screening filter for 17000328 drug-like zinc database compounds. 900 compounds passed the pharmacophore filter and entered the docking-based virtual screening stage. Finally, a 3D CoMFA QSAR was developed using 42 compounds as a training and 19 compounds as a test set. The 3D CoMFA QSAR was used to design and screen some potential inhibitors, these compounds were further evaluated by the docking stage. The highest ranked five hits from docking result (compounds (p1–p4) and compound q1) were selected for further analysis.

Communicated by Ramaswamy H. Sarma     

Self-organizing neural network for modeling 3D QSAR of colchicinoids

A novel scheme for modeling 3D QSAR has been developed. A method involving multiple self-organizing neural network adjusted to be analyzed by the PLS (partial least squares) analysis was used to model 3D QSAR of the selected colchicinoids. The model obtained allows the identification of some structural determinants of the biological activity of compounds.     

Novel small-molecule inhibitors of hepatitis C virus entry block viral spread and promote viral clearance in cell culture

Combinations of direct-acting anti-virals offer the potential to improve the efficacy, tolerability and duration of the current treatment regimen for hepatitis C virus (HCV) infection. Viral entry represents a distinct therapeutic target that has been validated clinically for a number of pathogenic viruses. To discover novel inhibitors of HCV entry, we conducted a high throughput screen of a proprietary small-molecule compound library using HCV pseudoviral particle (HCVpp) technology. We independently discovered and optimized a series of 1,3,5-triazine compounds that are potent, selective and non-cytotoxic inhibitors of HCV entry. Representative compounds fully suppress both cell-free virus and cell-to-cell spread of HCV in vitro. We demonstrate, for the first time, that long term treatment of an HCV cell culture with a potent entry inhibitor promotes sustained viral clearance in vitro. We have confirmed that a single amino acid variant, V719G, in the transmembrane domain of E2 is sufficient to confer resistance to multiple compounds from the triazine series. Resistance studies were extended by evaluating both the fusogenic properties and growth kinetics of drug-induced and natural amino acid variants in the HCVpp and HCV cell culture assays. Our results indicate that amino acid variations at position 719 incur a significant fitness penalty. Introduction of I719 into a genotype 1b envelope sequence did not affect HCV entry; however, the overall level of HCV replication was reduced compared to the parental genotype 1b/2a HCV strain. Consistent with these findings, I719 represents a significant fraction of the naturally occurring genotype 1b sequences. Importantly, I719, the most relevant natural polymorphism, did not significantly alter the susceptibility of HCV to the triazine compounds. The preclinical properties of these triazine compounds support further investigation of entry inhibitors as a potential novel therapy for HCV infection.     

Inhibitors of HCV NS5B polymerase: synthesis and structure-activity relationships of N-alkyl-4-hydroxyquinolon-3-yl-benzothiadiazine sulfamides

Substituted N-alkyl-4-hydroxyquinolon-3-yl-benzothiadiazine sulfamides were investigated as inhibitors of genotype 1 HCV polymerase. Structure-activity relationship patterns for this class of compounds are discussed.     

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.     

Efficient culture adaptation of hepatitis C virus recombinants with genotype-specific core-NS2 by using previously identified mutations

Hepatitis C virus (HCV) is an important cause of chronic liver disease, and interferon-based therapy cures only 40 to 80% of patients, depending on HCV genotype. Research was accelerated by genotype 2a (strain JFH1) infectious cell culture systems. We previously developed viable JFH1-based recombinants encoding the structural proteins (core, E1, E2), p7, and NS2 of prototype isolates of the seven major HCV genotypes; most recombinants required adaptive mutations. To enable genotype-, subtype-, and isolate-specific studies, we developed efficient core-NS2 recombinants from additional genotype 1a (HC-TN and DH6), 1b (DH1 and DH5), and 3a (DBN) isolates, using previously identified adaptive mutations. Introduction of mutations from isolates of the same subtype either led to immediate efficient virus production or accelerated culture adaptation. The DH6 and DH5 recombinants without introduced mutations did not adapt to culture. Universal adaptive effects of mutations in NS3 (Q1247L, I1312V, K1398Q, R1408W, and Q1496L) and NS5A (V2418L) were investigated for JFH1-based genotype 1 to 5 core-NS2 recombinants; several mutations conferred adaptation to H77C (1a), J4 (1b), S52 (3a), and SA13 (5a) but not to ED43 (4a). The mutations permitting robust virus production in Huh7.5 cells had no apparent effect on viral replication but allowed efficient assembly of intracellular infectious HCV for adapted novel or previously developed recombinants. In conclusion, previously identified mutations permitted development of novel HCV core-NS2 genotype recombinants. Mutations adapting several recombinants to culture were identified, but no mutations were universally adaptive across genotypes. This work provides tools for analysis of HCV genotype specificity and may promote the understanding of genotype-specific patterns in HCV disease and control.     

Domain 3 of NS5A protein from the hepatitis C virus has intrinsic alpha-helical propensity and is a substrate of cyclophilin A

Nonstructural protein 5A (NS5A) is essential for hepatitis C virus (HCV) replication and constitutes an attractive target for antiviral drug development. Although structural data for its in-plane membrane anchor and domain D1 are available, the structure of domains 2 (D2) and 3 (D3) remain poorly defined. We report here a comparative molecular characterization of the NS5A-D3 domains of the HCV JFH-1 (genotype 2a) and Con1 (genotype 1b) strains. Combining gel filtration, CD, and NMR spectroscopy analyses, we show that NS5A-D3 is natively unfolded. However, NS5A-D3 domains from both JFH-1 and Con1 strains exhibit a propensity to partially fold into an α-helix. NMR analysis identifies two putative α-helices, for which a molecular model could be obtained. The amphipathic nature of the first helix and its conservation in all genotypes suggest that it might correspond to a molecular recognition element and, as such, promote the interaction with relevant biological partner(s). Because mutations conferring resistance to cyclophilin inhibitors have been mapped into NS5A-D3, we also investigated the functional interaction between NS5A-D3 and cyclophilin A (CypA). CypA indeed interacts with NS5A-D3, and this interaction is completely abolished by cyclosporin A. NMR heteronuclear exchange experiments demonstrate that CypA has in vitro peptidyl-prolyl cis/trans-isomerase activity toward some, but not all, of the peptidyl-prolyl bonds in NS5A-D3. These studies lead to novel insights into the structural features of NS5A-D3 and its relationships with CypA.     

Design, synthesis, biological evaluation and QSAR studies of novel bisepipodophyllotoxins as cytotoxic agents

Two moieties of epipodophyllotoxin have been linked at C4-position to provide novel bisepipodophyllotoxin analogues. These have been evaluated for their anticancer potential and DNA-topoisomerase II poisoning activity. Most of these analogues have exhibited promising in vitro anticancer activity against different human tumour cell lines and interestingly 4(')-O-methylated analogues have shown increased cytotoxic activity. Similarly, the DNA-topo II poisoning activity tested for these compounds has not only exhibited the DNA cleavage potential comparable to etoposide, but for some compounds this cleavage potential is superior to etoposide. Further, an interesting structure-activity relationship of these epipodophyllotoxin dimers have been generated on the basis of GI(50) values. The equations indicated that GI(50) activity is strongly dependent on structural and thermodynamic properties. These QSAR results are discussed in conjunction with conformational analysis from molecular modelling studies. QSAR models developed in these studies will be helpful in the future to design novel potent bispodophyllotoxin analogues by minor structural modifications.     

Inhibitors of HCV NS5B polymerase: synthesis and structure-activity relationships of unsymmetrical 1-hydroxy-4,4-dialkyl-3-oxo-3,4-dihydronaphthalene benzothiadiazine derivatives

Substituted 1-hydroxy-4,4-dialkyl-3-oxo-3,4-dihydronaphthalene benzothiadiazine derivatives were investigated as inhibitors of genotype 1 HCV polymerase. Structure-activity relationship patterns for this class of compounds are discussed. It was found that the saturated alkane dialkyl units provided the most active analogs.     

Identification of potential inhibitors for HCV NS3 genotype 4a by combining protein–ligand interaction fingerprint, 3D pharmacophore,docking, and dynamic simulation

HCV NS3 protease domain has been one of the most attractive targets for developing new drugs for HCV infection and many drugs were successfully developed, but all of them were designed for targeting HCV genotype 1 infection. HCV genotype 4a dominant in Egypt has paid less attention. Here, we describe our protocol of virtual screening in identification of novel potential potent inhibitors for HCV NS3 of genotype 4a using homology modeling, PLIF (protein–ligand interaction fingerprint), docking, pharmacophore, and dynamic simulation. A high-quality 3D model of HCV NS3 protease of genotype 4a was constructed using crystal structure of HCV NS3 protease of genotype 1b (PDB ID: 4u01) as a template. PLIF was generated using five crystal structures of HCV NS3 (PDB ID: 4u01, 3kee, 4ktc, 4i33, and 5epn) which revealed the most important residues and their interactions with the co-crystalized ligands. A 3D pharmacophore model consisting of six features was developed from the generated PLIF data and then used as a screening filter for 11,244 compounds. Only 423 compounds passed the pharmacophore filter and entered the docking-based virtual screening stage. The highest ranked five hits from docking result (compound (C1–C5)) were selected for further analysis. They exhibited stronger interaction and higher binding affinity than HCV NS3 protease ligands. Dynamic simulation of the protein–best lead complex was performed to validate and augment the virtual screening results and it showed that these compounds have a strong binding affinity and could be very effective in treating HCV genotype 4a infections.     

Network based analysis of hepatitis C virus core and NS4B protein interactions

Hepatitis C virus (HCV) is a major cause of chronic liver disease worldwide. Here we attempt to further our understanding of the biological context of protein interactions in HCV pathogenesis, by investigating interactions between HCV proteins Core and NS4B and human host proteins. Using the yeast two-hybrid (Y2H) membrane protein system, eleven human host proteins interacting with Core and 45 interacting with NS4B were identified, most of which are novel. These interactions were used to infer overall protein interaction maps linking the viral proteins with components of the host cellular networks. Core and NS4B proteins contribute to highly compact interaction networks that may enable the virus to respond rapidly to host physiological responses to HCV infection. Analysis of the interaction networks highlighted enriched biological pathways likely influenced in HCV infection. Inspection of individual interactions offered further insights into the possible mechanisms that permit HCV to evade the host immune response and appropriate host metabolic machinery. Follow-up cellular assays with cell lines infected with HCV genotype 1b and 2a strains validated Core interacting proteins ENO1 and SLC25A5 and host protein PXN as novel regulators of HCV replication and viral production. ENO1 siRNA knockdown was found to inhibit HCV replication in both the HCV genotypes and viral RNA release in genotype 2a. PXN siRNA inhibition was observed to inhibit replication specifically in genotype 1b but not in genotype 2a, while SLC25A5 siRNA facilitated a minor increase in the viral RNA release in genotype 2a. Thus, our analysis can provide potential targets for more effective anti-HCV therapeutic intervention.     

QSAR studies on structurally similar 2-(4-methanesulfonylphenyl)pyran-4-ones as selective COX-2 inhibitors: a Hansch approach

QSAR analysis based on classical Hansch approach was adopted on two recently reported novel series of 2-phenylpyran-4-ones as selective cyclooxygenase-2 (COX-2) inhibitors. The 6-methyl derivatives of title compounds bifurcate as 3-phenoxypyran-4-ones (subset A) and 3-phenylpyran-4-ones (subset B) among series 1. Series 2 consists of 5-chloro derivatives of title compounds. Various regression equations were derived to study the influence of phenoxy and phenyl ring substituents of series 1 compounds on COX-2, COX-1 and selective COX-2 over COX-1 inhibitory activity. The best triparametric equation derived for 36 compounds of series 1 explains the hydrophobic, electronic and steric requirements for improved COX-2 inhibitory activity. QSAR model derived to explore the selective COX-2 over COX-1 inhibition showed that selectivity could be influenced by size and lipophilicity of substituents. The size of the first atom of 2 substituents appears to have negative effect on selectivity, whereas highly polar 3 substituents at R are favorable for improved selectivity. QSAR investigations on series 2 compounds revealed some interesting correlation of COX-2 inhibitory activity with calculated physicochemical properties of whole molecules. The positive logP confirms the hydrophobic interaction of series 2 compounds with COX-2 enzyme. The positive MR term indicates that an overall increase in size and polarizabilty of the molecules increases COX-2 inhibitory activity. The positive contribution of structural variable suggests biphenyl analogs are extremely potent COX-2 inhibitors.     

Identification of halosalicylamide derivatives as a novel class of allosteric inhibitors of HCV NS5B polymerase

Halosalicylamide derivatives were identified from high-throughput screening as potent inhibitors of HCV NS5B polymerase. The subsequent structure and activity relationship revealed the absolute requirement of the salicylamide moiety for optimum activity. Methylation of either the hydroxyl group or the amide group of the salicylamide moiety abolished the activity while the substitutions on both phenyl rings are acceptable. The halosalicylamide derivatives were shown to be non-competitive with respect to elongation nucleotide and demonstrated broad genotype activity against genotype 1-3 HCV NS5B polymerases. Inhibitor competition studies indicated an additive binding mode to the initiation pocket that is occupied by the thiadiazine class of compounds and an additive binding mode to the elongation pocket that is occupied by diketoacids, but a mutually exclusive binding mode with respect to the allosteric thumb pocket that is occupied by the benzimidazole class of inhibitors. Therefore, halosalicylamides represent a novel class of allosteric inhibitors of HCV NS5B polymerase.     

Novel pyridyl-fused 3-amino chroman derivatives with dual action at serotonin transporter and 5-HT1A receptor

Structural modifications of the initial lead, 3-aminochroman (4), led to the identification of a novel series of pyridyl-fused amino chroman derivatives (5-8) and the structural isomers (9-12). The compounds described were evaluated for dual 5-HT transporter inhibitory and 5-HT(1A) receptor activities. The design strategy, synthesis, and in vitro biological characterization for these novel compounds are described.