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.     

Structure-based and shape-complemented pharmacophore modeling for the discovery of novel checkpoint kinase 1 inhibitors

Checkpoint kinase 1 (Chk1), a member of the serine/threonine kinase family, is an attractive therapeutic target for anticancer combination therapy. A structure-based modeling approach complemented with shape components was pursued to develop a reliable pharmacophore model for ATP-competitive Chk1 inhibitors. Common chemical features of the pharmacophore model were derived by clustering multiple structure-based pharmacophore features from different Chk1-ligand complexes in comparable binding modes. The final model consisted of one hydrogen bond acceptor (HBA), one hydrogen bond donor (HBD), two hydrophobic (HY) features, several excluded volumes and shape constraints. In the validation study, this feature-shape query yielded an enrichment factor of 9.196 and performed fairly well at distinguishing active from inactive compounds, suggesting that the pharmacophore model can serve as a reliable tool for virtual screening to facilitate the discovery of novel Chk1 inhibitors. Besides, these pharmacophore features were assumed to be essential for Chk1 inhibitors, which might be useful for the identification of potential Chk1 inhibitors.     

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。     

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     

Computer based design, synthesis and biological evaluation of novel indole derivatives as HCV NS3-4A serine protease inhibitors

A series of novel indoles were designed and their molecular modeling simulation study including fitting to a 3D pharmacophore model using CATALYST program and their docking into the NS3 active site was examined as HCV NS3 protease inhibitor. Several compounds showed significant high simulation docking score and fit values. The designed compounds were synthesized and biologically evaluated in vitro using an NS3 protease binding assay, where compounds 10a-k showed significant inhibitory activity (> or =67% inhibition at 100 microg/mL). Of these, compounds 10c and 10f demonstrated potent HCV NS3 protease inhibitors with IC(50) values of 15 and 13 microM, respectively. Enantio-selective Michael addition of an indole derivative in the presence of catalytic amount of AlCl(3) and quinine at room temperature afforded the adduct 7e in excellent yield with 73% ee. The product was converted into 10l, which showed lower activity than the mixture of the corresponding diastereoisomers.     

Computer aided screening of Accacia nilotica phytochemicals against HCV NS3/4a

Background: HCV has become a leading cause of liver cirrhosis and hepatocellular carcinoma and is a major health concern worldwide. To date, there is no vaccine available in the market to tackle this disease, therefore there is a strong need to develop antiviral compounds that can target all genotypes of HCV with the same efficiency. Medicinal plants have low cost and are less toxic therefore, extracts of medicinal plants can serve as important antiviral agents against HCV. This study was designed to screen phytochemicals of Accacia nilotica to find a potent drug candidate that can inhibit HCV infection effectively.Results: Docking of NS3/4A protease and Flavonoids of Accacia nilotica revealed that most of the flavonoids bound deeply with the active site of NS3/4A protease. Compound 01 showed a high ranking on docking score. All other compounds also showed reliable docking scores and had interactions with the binding cavity of NS3/4A protease, suggesting them as a potent drug candidate to block HCV replication.Conclusion: To recognize binding interactions of Accacia nilotica phytochemicals with NS3/4A protease, molecular docking was performed to find potential inhibitor against NS3/4A protease of HCV. After post docking analysis, important interactions were found between active compounds and active site of NS3/4A protease. It can be concluded from the study that phytochemicals of Accacia nilotica may serve as a potential drug candidate with relatively simple structural changes against HCV NS3/4A protease.     

Toll-Like Receptor 7 Agonists: Chemical Feature Based Pharmacophore Identification and Molecular Docking Studies

Chemical feature based pharmacophore models were generated for Toll-like receptors 7 (TLR7) agonists using HypoGen algorithm, which is implemented in the Discovery Studio software. Several methods tools used in validation of pharmacophore model were presented. The first hypothesis Hypo1 was considered to be the best pharmacophore model, which consists of four features: one hydrogen bond acceptor, one hydrogen bond donor, and two hydrophobic features. In addition, homology modeling and molecular docking studies were employed to probe the intermolecular interactions between TLR7 and its agonists. The results further confirmed the reliability of the pharmacophore model. The obtained pharmacophore model (Hypo1) was then employed as a query to screen the Traditional Chinese Medicine Database (TCMD) for other potential lead compounds. One hit was identified as a potent TLR7 agonist, which has antiviral activity against hepatitis virus in vitro. Therefore, our current work provides confidence for the utility of the selected chemical feature based pharmacophore model to design novel TLR7 agonists with desired biological activity.     

Ligand-based and structure-based approaches in identifying ideal pharmacophore against c-Jun N-terminal kinase-3

Structure and ligand based pharmacophore modeling and docking studies carried out using diversified set of c-Jun N-terminal kinase-3 (JNK3) inhibitors are presented in this paper. Ligand based pharmacophore model (LBPM) was developed for 106 inhibitors of JNK3 using a training set of 21 compounds to reveal structural and chemical features necessary for these molecules to inhibit JNK3. Hypo1 consisted of two hydrogen bond acceptors (HBA), one hydrogen bond donor (HBD), and a hydrophobic (HY) feature with a correlation coefficient (r²) of 0.950. This pharmacophore model was validated using test set containing 85 inhibitors and had a good r² of 0.846. All the molecules were docked using Glide software and interestingly, all the docked conformations showed hydrogen bond interactions with important hinge region amino acids (Gln155 and Met149) and these interactions were compared with Hypo1 features. The results of ligand based pharmacophore model (LBPM) and docking studies are validated each other. The structure based pharmacophore model (SBPM) studies have identified additional features, two hydrogen bond donors and one hydrogen bond acceptor. The combination of these methodologies is useful in designing ideal pharmacophore which provides a powerful tool for the discovery of novel and selective JNK3 inhibitors.     

Molecular Docking Investigation of the Binding Interactions of Macrocyclic Inhibitors with HCV NS3 Protease and its Mutants (R155K, D168A and A156V)

Hepatitis C Virus (HCV) non-structural protein 3 (NS3) protease drug resistance poses serious challenges on the design of an effective treatment. Substrate Envelope Hypothesis, “the substrates of HCV NS3/4A protease have a consensus volume inside the active site called substrate envelope” is used to design potent and specific drugs to overcome this problem. Using molecular docking, we studied the binding interaction of the different inhibitors and protein and evaluated the effect of three different mutations (R155K, D168A and A156V) on the binding of inhibitors. P2–P4 macrocycles of 5A/5B and modified 5A/5B hexapeptide sequences have the best scores against the wild-type protein ?204.506 and ?206.823 kcal/mole, respectively. Also, charged P2–P4 macrocycles of 3/4A and 4A/4B hexapeptide sequences have low scores with the wild-type protein ?200.467 and ?203.186 kcal/mole, respectively. R155K mutation greatly affects the conformation of the compounds inside the active site. It inverts its orientations, and this is because the large and free side chain of K155 which restricts the conformation of the large P2–P4 macrocycle. The conformation of charged P2–P4 macrocycle of 3/4A hexapeptide sequence in wild-type, A156V and D168A proteins is nearly equal; while that of charged P2–P4 macrocycle of 4A/4B hexapeptide sequence is different. Nevertheless, these compounds have a slight increase of Van der Waals volume compared to that of substrates, they are potent against mutations and have good scores. Therefore, the suggested drugs can be used as an effective treatment solving HCV NS3/4A protease drug resistance problem.     

Identification of Novel HIV 1- Protease Inhibitors: Application of Ligand and Structure Based Pharmacophore Mapping and Virtual Screening

A combined ligand and structure-based drug design approach provides a synergistic advantage over either methods performed individually. Present work bestows a good assembly of ligand and structure-based pharmacophore generation concept. Ligand-oriented study was accomplished by employing the HypoGen module of Catalyst in which we have translated the experimental findings into 3-D pharmacophore models by identifying key features (four point pharmacophore) necessary for interaction of the inhibitors with the active site of HIV-1 protease enzyme using a training set of 33 compounds belonging to the cyclic cyanoguanidines and cyclic urea derivatives. The most predictive pharmacophore model (hypothesis 1), consisting of four features, namely, two hydrogen bond acceptors and two hydrophobic, showed a correlation (r) of 0.90 and a root mean square of 0.71 and cost difference of 56.59 bits between null cost and fixed cost. The model was validated using CatScramble technique, internal and external test set prediction. In the second phase of our study, a structure-based five feature pharmacophore hypothesis was generated which signifies the importance of hydrogen bond donor, hydrogen bond acceptors and hydrophobic interaction between the HIV-1 protease enzyme and its inhibitors. This work has taken a significant step towards the full integration of ligand and structure-based drug design methodologies as pharmacophoric features retrieved from structure-based strategy complemented the features from ligand-based study hence proving the accuracy of the developed models. The ligand-based pharmacophore model was used in virtual screening of Maybridge and NCI compound database resulting in the identification of four structurally diverse druggable compounds with nM activities.     

Exploration and validation of diphosphate‐based Plasmodium LytB inhibitors using computational approaches

LytB or IspH is an indispensable enzyme and a suitable drug target of Plasmodium falciparum that participate in isoprenoid biosynthesis of nonmevalonate pathway (MEP). Recently, we have investigated the structural dynamics of Plasmodium LytB and proposed some novel diphosphate‐based inhibitors using molecular modeling and docking studies. Here, we have tried to characterize those previously screened molecules by quantitative structure activity relationships and pharmacophore‐based analyses, as well as validated the dynamics of their interactions with LytB protein. Five total compounds having PubChem CID 516 , 125696 , 165275 , 448012 , and 9921431 were predicted with significant inhibitory activity by quantitative structure activity relationships and pharmacophore models. Again, the molecular dynamics simulation results showed that these five compounds are able to form stable complexes with the receptor through many direct and water mediated interactions. The binding free energies calculated by Poisson‐Boltzmann surface area method resulted within the range between ?99.77 and ? 43.74 kcal/mol, which favoured their profound inhibitory affinity. Residues of LytB like His41, His74, Ser222, Ser223, and Asn224 in LytB were the main protagonists in contributing the majority of interaction energies to the ligands. Finally, the ADMET, toxicity, and drug‐likeness scores also affirm these compounds to be considered for further development of new antimalarial inhibitor in the future.     

Substrate specificity of the ileal and the hepatic Na(+)/bile acid cotransporters of the rabbit. II. A reliable 3D QSAR pharmacophore model for the ileal Na(+)/bile acid cotransporter

To design a reliable 3D QSAR model of the intestinal Na(+)/bile acid cotransporter, we have used a training set of 17 inhibitors of the rabbit ileal Na(+)/bile acid cotransporter. The IC(50) values of the training set of compounds covered a range of four orders of magnitude for inhibition of [(3)H]cholyltaurine uptake by CHO cells expressing the rabbit ileal Na(+)/bile acid cotransporter allowing the generation of a pharmacophore using the CATALYST algorithm. After thorough conformational analysis of each molecule, CATALYST generated a pharmacophore model characterized by five chemical features: one hydrogen bond donor, one hydrogen bond acceptor, and three hydrophobic features. The 3D pharmacophore was enantiospecific and correctly estimated the activities of the members of the training set. The predicted interactions of natural bile acids with the pharmacophore model of the ileal Na(+)/bile acid cotransporter explain exactly the experimentally found structure;-activity relationships for the interaction of bile acids with the ileal Na(+)/bile acid cotransporter (Kramer et al. 1999. J. Lipid. Res. 40: 1604;-1617). The natural bile acid analogues cholyltaurine, chenodeoxycholyltaurine, or deoxycholyltaurine were able to map four of the five features of the pharmacophore model: a) the five-membered ring D and the methyl group at position 18 map one hydrophobic site and the 21-methyl group of the side chain maps a second hydrophobic site; b) one of the alpha-oriented hydroxyl groups at position 7 or 12 fits the hydrogen bond donor feature; c) the negatively charged side chain acts as hydrogen bond acceptor; and d) the hydroxy group at position 3 does not specifically map any of the five binding features of the pharmacophore model. The 3-hydroxy group of natural bile acids is not essential for interactions with ileal or hepatic Na(+)/bile acid cotransporters. A modification of the 3-position of a natural bile acid molecule is therefore the preferred position for drug targeting strategies using bile acid transport pathways.     

Inhibitors of hepatitis C virus NS3.4A protease. Part 3: P2 proline variants

We recently described the identification of an optimized alpha-ketoamide warhead for our series of HCV NS3.4A inhibitors. We report herein a series of HCV protease inhibitors incorporating 3-alkyl-substituted prolines in P(2). These compounds show exceptional enzymatic and cellular potency given their relatively small size. The marked enhancement of activity of these 3-substituted proline derivatives relative to previously reported 4-hydroxyproline derivatives constitutes additional evidence for the importance of the S(2) binding pocket as the defining pharmacophore for inhibition of the NS3.4A enzyme.     

Primuline Derivatives That Mimic RNA to Stimulate Hepatitis C Virus NS3 Helicase-catalyzed ATP Hydrolysis

ATP hydrolysis fuels the ability of helicases and related proteins to translocate on nucleic acids and separate base pairs. As a consequence, nucleic acid binding stimulates the rate at which a helicase catalyzes ATP hydrolysis. In this study, we searched a library of small molecule helicase inhibitors for compounds that stimulate ATP hydrolysis catalyzed by the hepatitis C virus (HCV) NS3 helicase, which is an important antiviral drug target. Two compounds were found that stimulate HCV helicase-catalyzed ATP hydrolysis, both of which are amide derivatives synthesized from the main component of the yellow dye primuline. Both compounds possess a terminal pyridine moiety, which was critical for stimulation. Analogs lacking a terminal pyridine inhibited HCV helicase catalyzed ATP hydrolysis. Unlike other HCV helicase inhibitors, the stimulatory compounds differentiate between helicases isolated from various HCV genotypes and related viruses. The compounds only stimulated ATP hydrolysis catalyzed by NS3 purified from HCV genotype 1b. They inhibited helicases from other HCV genotypes (e.g. 1a and 2a) or related flaviviruses (e.g. Dengue virus). The stimulatory compounds interacted with HCV helicase in the absence of ATP with dissociation constants of about 2 μm. Molecular modeling and site-directed mutagenesis studies suggest that the stimulatory compounds bind in the HCV helicase RNA-binding cleft near key residues Arg-393, Glu-493, and Ser-231.     

3D-QSAR pharmacophore-based virtual screening,molecular docking and molecular dynamics simulation toward identifying lead compounds for NS2B–NS3 protease inhibitors

NS2B–NS3 protease has been identified to serve as lead drug design target due to its significant role in West Nile viral (WNV) and dengue virus (DENV) reproduction and replication. There are currently no approved chemotherapeutic drugs and effective vaccines to inhibit DENV and WNV infections. In this work, 3D-QSAR pharmacophore model has been developed to discover potential inhibitory candidates. Validation through Fischer’s model and decoy test indicate that the developed 3D pharmacophore model is highly predictive for DENV inhibitors, which was then employed to screen ZINC chemical library to obtain reasonable hits. Following ADMET filtering, 15 hits were subjected to further filter through molecular docking and CoMFA modeling. Finally, top three hits were identified as lead compounds or potential inhibitory candidates with IC₅₀ values of ～0.4637?µM and fitness of ～57.73. It is implied from CoMFA modeling that substituents at the side site of benzotriazole such as a p-nitro group (e.g. biphenyl head) and a carbonyl (e.g. carboxylate function) at the side site of furan or amino group may improve bioactivity of ZINC85645245, respectively. Molecular dynamics simulations (MDS) were performed to discover new interactions and reinforce the binding modes from docking for the hits also. The QSAR and MDS results obtained from this work should be useful in determining structural requirements for inhibitor development as well as in designing more potential inhibitors for NS2B–NS3 protease.     

Homology modeling, molecular dynamics, e-pharmacophore mapping and docking study of Chikungunya virus nsP2 protease

To date, no suitable vaccine or specific antiviral drug is available to treat Chikungunya viral (CHIKV) fever. Hence, it is essential to identify drug candidates that could potentially impede CHIKV infection. Here, we present the development of a homology model of nsP2 protein based on the crystal structure of the nsP2 protein of Venezuelan equine encephalitis virus (VEEV). The protein modeled was optimized using molecular dynamics simulation; the junction peptides of a nonstructural protein complex were then docked in order to investigate the possible protein–protein interactions between nsP2 and the proteins cleaved by nsP2. The modeling studies conducted shed light on the binding modes, and the critical interactions with the peptides provide insight into the chemical features needed to inhibit the CHIK virus infection. Energy-optimized pharmacophore mapping was performed using the junction peptides. Based on the results, we propose the pharmacophore features that must be present in an inhibitor of nsP2 protease. The resulting pharmacophore model contained an aromatic ring, a hydrophobic and three hydrogen-bond donor sites. Using these pharmacophore features, we screened a large public library of compounds (Asinex, Maybridge, TOSLab, Binding Database) to find a potential ligand that could inhibit the nsP2 protein. The compounds that yielded a fitness score of more than 1.0 were further subjected to Glide HTVS and Glide XP. Here, we report the best four compounds based on their docking scores; these compounds have IDs of 27943, 21362, ASN 01107557 and ASN 01541696. We propose that these compounds could bind to the active site of nsP2 protease and inhibit this enzyme. Furthermore, the backbone structural scaffolds of these four lead compounds could serve as building blocks when designing drug-like molecules for the treatment of Chikungunya viral fever.     

Cyclic amide bioisosterism: Strategic application to the design and synthesis of HCV NS5B polymerase inhibitors

Conformational modeling has been successfully applied to the design of cyclic bioisosteres used to replace a conformationally rigid amide bond in a series of thiophene carboxylate inhibitors of HCV NS5B polymerase. Select compounds were equipotent with the original amide series. Single-point mutant binding studies, in combination with inhibition structure–activity relationships, suggest this new series interacts at the Thumb-II domain of NS5B. Inhibitor binding at the Thumb-II site was ultimately confirmed by solving a crystal structure of 8b complexed with NS5B.     

Discovery of a hepatitis C target and its pharmacological inhibitors by microfluidic affinity analysis

More effective therapies are urgently needed against hepatitis C virus (HCV), a major cause of viral hepatitis. We used in vitro protein expression and microfluidic affinity analysis to study RNA binding by the HCV transmembrane protein NS4B, which plays an essential role in HCV RNA replication. We show that HCV NS4B binds RNA and that this binding is specific for the 3' terminus of the negative strand of the viral genome with a dissociation constant (Kd) of approximately 3.4 nM. A high-throughput microfluidic screen of a compound library identified 18 compounds that substantially inhibited binding of RNA by NS4B. One of these compounds, clemizole hydrochloride, was found to inhibit HCV RNA replication in cell culture that was mediated by its suppression of NS4B's RNA binding, with little toxicity for the host cell. These results yield new insight into the HCV life cycle and provide a candidate compound for pharmaceutical development.     

Molecular screening of phytochemicals from Amelanchier Alnifolia against HCV NS3 protease/helicase using computational docking techniques

Hepatitis C is serious health concern worldwide caused by HCV. It causes liver cirrhosis and hepato-cellular carcinoma. Development of prevention solutions is under progress. Meanwhile, the treatment of the viral disease using compounds isolated from natural medicinal plants is promising. The traditional use of photo-chemicals from medicinal plants like Amelanchier alnifolia for viral treatment is hopeful. Therefore, it is of interest to screen for flavonoids from Amelanchier alnifolia against protein targets of HCV. Hence, we assessed the binding of flavonoids to HCV NS3/4A protease and helicase proteins. Results show that Quercitin 3- galactoside and 3-glucosideshowed good binding score with protease and helicase respectively. Their interaction/binding sites are documented in this report. This data provide insights for the consideration of flavonoids as potential inhibitors of HCV/NS3/4A protease and helicase.     

Molecular Modeling Comparison of the Performance of NS5b Polymerase Inhibitor (PSI-7977) on Prevalent HCV Genotypes

The current available treatment for hepatitis C virus (HCV)—the causative of liver cirrhosis and development of liver cancer—is a dual therapy using modified interferon and ribavirin. While this regimen increases the sustained viral response rate up to 40–80 % in different genotypes, unfortunately, it is poorly tolerated by patients. PSI-7977, a prodrug for PSI-7409, is a Non-Structural 5b (NS5b) polymerase nucleoside inhibitor that is currently in phase III clinical trials. The activated PSI-7977 is a direct acting antiviral (DAA) drug that acts on NS5b polymerase of HCV through a coordination bond with the two Mg⁺² present at the GDD active site motif. The present work utilizes a molecular modeling approach for studying the interaction between the activated PSI-7977 and the 12 amino acids constituting a 5 Å region surrounding the GDD active triad motif for HCV genotypes 1a, 2b, 3b and 4a. The analysis of the interaction parameters suggests that PSI-7977 is probably a better DAA drug for HCV genotypes 1a and 3b rather than genotypes 2b and 4a.