Design of inhibitors of the HIV-1 integrase core domain using virtual screening

Acquired immunodeficiency syndrome (AIDS) is a disease of the human immune system caused by the human immunodeficiency virus (HIV). The integrase (IN) enzyme of HIV interacts with several cellular and viral proteins during the integration process. Thus, it represents an appropriate target for antiretroviral drugs (ARVs). We performed virtual screening of database compounds and designed analogues using Elvitegravir (EVG) as a standard compound. The 378 screened compounds were retrieved from ZINC, ChemSpider, PubChem, and ChemBank Chemical Databases based on chemical similarity and literature searches related to the structure of EVG. The Physiochemical properties, Bioactivity, Toxicity and Absorption, Distribution, Metabolism and Excretion of Molecules (ADME) of these compounds were predicted and docking Experiments were conducted using Molegro Virtual Docker software. The docking and ADME suggested very significant results in regard to EVG. The MolDock and Rerank scores were used to analyze the results. The compounds ZINC26507991 (-84.22), Analogue 9 (-68.49), ZINC20731658 (-66.79), ZINC00210363 (-43.44) showed better binding orientation with IN receptor model with respect to EVG (182.52). The ZINC26507991 has showed significant ADME result.     

Identification of potent virtual leads to design novel PLK1 inhibitors: pharmacophore modelling,virtual screening and molecular docking studies

The aim of this study was to identify novel scaffolds and utilise them in designing potent PLK1 inhibitors. Three-dimensional pharmacophore models on the basis of chemical features were developed for PLK1 on the basis of the known inhibitors. The best pharmacophore model, Hypo 1, which has the highest correlation (0.96), the highest cost difference (75.7494), the lowest total cost and RMSD (75.7494, 0.5458), contains two hydrophobics, one ring aromatic and one hydrogen donor. Hypo 1 was validated by the test set, decoy set and the Fischer's randomisation method. Then it was used for chemical database virtual screening. The hit compounds were filtered by Lipinski's rule of five and absorption, distribution, metabolism, elimination and toxicity properties. Finally, 24 compounds with good estimated activity values were used for docking studies. These results will be used to develop new inhibitors of PLK1 as leads.     

Identifying the novel inhibitors of lysine-specific demethylase 1 (LSD1) combining pharmacophore-based and structure-based virtual screening

Abstract

Lysine-specific demethylase 1 (LSD1) has been reported to connect with a range of solid tumors. Thus, the exploration of LSD1 inhibitors has emerged as an effective strategy for cancer treatment. In this study, we constructed a pharmacophore model based on a series of flavin adenine dinucleotide (FAD)-competing inhibitors bearing triazole???dithiocarbamate scaffold combining docking, structure–activity relationship (SAR) study, and molecular dynamic (MD) simulation. Meanwhile, another pharmacophore model was also constructed manually, relying on several speculated substrate-competing inhibitors and reported putative vital interactions with LSD1. On the basis of the two pharmacophore models, multi-step virtual screenings (VSs) were performed against substrate-binding pocket and FAD-binding pocket, respectively, combining pharmacophore-based and structure-based strategy to exploit novel LSD1 inhibitors. After bioassay evaluation, four compounds among 21 hits with diverse and novel scaffolds exhibited inhibition activity at the range of 3.63–101.43?μM. Furthermore, substructure-based enrichment was performed, and four compounds with a more potent activity were identified. After that, the time-dependent assay proved that the most potent compound with IC₅₀ 2.21?μM inhibits LSD1 activity in a manner of time-independent. In addition, the compound exhibited a cellular inhibitory effect against LSD1 in MGC-803 cells and may inhibit cell migration and invasion by reversing EMT in cultured gastric cancer cells. Considering the binding mode and SAR of the series of compounds, we could roughly deem that these compounds containing 3-methylxanthine scaffold act through occupying substrate-binding pocket competitively. This study presented a new starting point to develop novel LSD1 inhibitors.     

结合分子相似性、药效团和分子对接筛选新的HIV-1蛋白酶抑制剂

结合分子相似性、药效团和分子对接建立兼顾计算效率和预测准确度的HIV-1蛋白酶抑制剂筛选方法。首先通过对现有HIV-1蛋白酶抑制剂分子进行相似性分析,选取代表性的HIV-1蛋白酶抑制剂作为模板分子,构建和优化药效团模型,并从1万个化合物中优先筛选出500个化合物。而后采用分子对接方法进一步考察化合物与HIV-1蛋白酶结合情况,得到4个新的活性候选化合物,并进行其结合自由能计算和抗突变性分析。结果表明新候选化合物ST025723和HIV-1蛋白酶表现出较好的相互作用和抗突变性,具有深入研究的价值,同时也证明分子相似性、药效团和分子对接相结合能够快速有效地发现新颖活性候选化合物。     

Kinetic and thermodynamic characterization of HIV-1 protease inhibitors

Interaction kinetic and thermodynamic analyses provide information beyond that obtained in general inhibition studies, and may contribute to the design of improved inhibitors and increased understanding of molecular interactions. Thus, a biosensor-based method was used to characterize the interactions between HIV-1 protease and seven inhibitors, revealing distinguishing kinetic and thermodynamic characteristics for the inhibitors. Lopinavir had fast association and the highest affinity of the tested compounds, and the interaction kinetics were less temperature-dependent as compared with the other inhibitors. Amprenavir, indinavir and ritonavir showed non-linear temperature dependencies of the kinetics. The free energy, enthalpy and entropy (DeltaG, DeltaH, DeltaS) were determined, and the energetics of complex association (DeltaG(on), DeltaH(on), DeltaS(on)) and dissociation (DeltaG(off), DeltaH(off), DeltaS(off)) were resolved. In general, the energetics for the studied inhibitors was in the same range, with the negative free energy change (DeltaG < 0) due primarily to increased entropy (DeltaS > 0). Thus, the driving force of the interaction was increased degrees of freedom in the system (entropy) rather than the formation of bonds between the enzyme and inhibitor (enthalpy). Although the DeltaG(on) and DeltaG(off) were in the same range for all inhibitors, the enthalpy and entropy terms contributed differently to association and dissociation, distinguishing these phases energetically. Dissociation was accompanied by positive enthalpy (DeltaH(off) > 0) and negative entropy (DeltaS(off) < 0) changes, whereas association for all inhibitors except lopinavir had positive entropy changes (DeltaS(on) > 0), demonstrating unique energetic characteristics for lopinavir. This study indicates that this type of data will be useful for the characterization of target-ligand interactions and the development of new inhibitors of HIV-1 protease.     

Identification of InhA inhibitors: A combination of virtual screening,molecular dynamics simulations and quantum chemical studies

In the present work, multiple pharmacophore-based virtual screening of the SPECS natural product database was carried out to identify novel inhibitors of the validated biological target, InhA. The pharmacophore models were built from the five different groups of the co-crystallized ligands present within the active site. The generated models with the same features from each group were pooled and subjected to the test set validation, receiver–operator characteristic analysis and Güner–Henry studies. A set of five hypotheses with sensitivity > 0.5, specificity > 0.5, area under curve (AUC) > 0.7, and goodness of hit score > 0.7 were retrieved and exploited for the virtual screening. The common hits (87 molecules) obtained from these hypotheses were processed via drug-likeness filters. The filtered molecules (27 molecules) were compared for the binding modes and the top scored molecules (12 molecules) along with the reference (triclosan (TCL), docking score = ?11.65 kcal/mol) were rescored and reprioritized via molecular mechanics-generalized Born surface area approach. Eventually, the stability of reprioritized (10 molecules) docked complexes was scrutinized via molecular dynamics simulations. Moreover, the quantum chemical studies of the dynamically stable compounds (9 molecules) were performed to understand structural features essential for the activity. Overall, the protocol resulted in the recognition of nine lead compounds that can be targeted against InhA.     

Effectiveness of commercial inhibitors against subtype F HIV-1 protease

Subtype F wild type HIV protease has been kinetically characterized using six commercial inhibitors (amprenavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir) commonly used for HIV/AIDS treatment, as well as inhibitor TL-3 and acetyl-pepstatin. We also obtained kinetic parameters for two multi-resistant proteases (one of subtype B and one of subtype F) harboring primary and secondary mutations selected by intensive treatment with ritonavir/nelfinavir. This newly obtained biochemical data shows that all six studied commercially available protease inhibitors are significantly less effective against subtype F HIV proteases than against HIV proteases of subtype B, as judged by increased K_i and biochemical fitness (vitality) values. Comparison with previously reported kinetic values for subtype A and C HIV proteases show that subtype F wild type proteases are significantly less susceptible to inhibition. These results demonstrate that the accumulation of natural polymorphisms in subtype F proteases yields catalytically more active enzymes with a large degree of cross-resistance, which thus results in strong virus viability.     

Critical differences in HIV-1 and HIV-2 protease specificity for clinical inhibitors

Clinical inhibitor amprenavir (APV) is less effective on HIV‐2 protease (PR₂) than on HIV‐1 protease (PR₁). We solved the crystal structure of PR₂ with APV at 1.5 Å resolution to identify structural changes associated with the lowered inhibition. Furthermore, we analyzed the PR₁ mutant (PR_1M) with substitutions V32I, I47V, and V82I that mimic the inhibitor binding site of PR₂. PR_1M more closely resembled PR₂ than PR₁ in catalytic efficiency on four substrate peptides and inhibition by APV, whereas few differences were seen for two other substrates and inhibition by saquinavir (SQV) and darunavir (DRV). High resolution crystal structures of PR_1M with APV, DRV, and SQV were compared with available PR₁ and PR₂ complexes. Val/Ile32 and Ile/Val47 showed compensating interactions with SQV in PR_1M and PR₁, however, Ile82 interacted with a second SQV bound in an extension of the active site cavity of PR_1M. Residues 32 and 82 maintained similar interactions with DRV and APV in all the enzymes, whereas Val47 and Ile47 had opposing effects in the two subunits. Significantly diminished interactions were seen for the aniline of APV bound in PR_1M and PR₂ relative to the strong hydrogen bonds observed in PR₁, consistent with 15‐ and 19‐fold weaker inhibition, respectively. Overall, PR_1M partially replicates the specificity of PR₂ and gives insight into drug resistant mutations at residues 32, 47, and 82. Moreover, this analysis provides a structural explanation for the weaker antiviral effects of APV on HIV‐2.     

Discovery of the first SecA inhibitors using structure-based virtual screening

Bacterial protein secretion is a critical and complex process. The Sec machinery provides a major pathway for protein translocation across and integration into the cellular membrane in bacteria. Small molecule probes that perturb the functions of individual member proteins within the Sec machinery will be very important research tools as well as leads for future antimicrobial agent development. Herein we describe the discovery of inhibitors, through virtual screening, that specifically act on SecA ATPase, which is a critical member of the Sec system. These are the very first inhibitors reported for intrinsic SecA ATPase.     

Lack of synergy for inhibitors targeting a multi-drug-resistant HIV-1 protease

The three-dimensional structures of indinavir and three newly synthesized indinavir analogs in complex with a multi-drug-resistant variant (L63P, V82T, I84V) of HIV-1 protease were determined to approximately 2.2 A resolution. Two of the three analogs have only a single modification of indinavir, and their binding affinities to the variant HIV-1 protease are enhanced over that of indinavir. However, when both modifications were combined into a single compound, the binding affinity to the protease variant was reduced. On close examination, the structural rearrangements in the protease that occur in the tightest binding inhibitor complex are mutually exclusive with the structural rearrangements seen in the second tightest inhibitor complex. This occurs as adaptations in the S1 pocket of one monomer propagate through the dimer and affect the conformation of the S1 loop near P81 of the other monomer. Therefore, structural rearrangements that occur within the protease when it binds to an inhibitor with a single modification must be accounted for in the design of inhibitors with multiple modifications. This consideration is necessary to develop inhibitors that bind sufficiently tightly to drug-resistant variants of HIV-1 protease to potentially become the next generation of therapeutic agents.     

Discovery of potential novel microsomal triglyceride transfer protein inhibitors via virtual screening of pharmacophore modelling and molecular docking

In order to identify potential natural inhibitors against the microsomal triglyceride transfer protein (MTP), HipHop models were generated using 20 known inhibitors from the Binding Database. Using evaluation indicators, the best hypothesis model, Hypo1, was selected and utilised to screen the Traditional Chinese Medicine Database, which resulted in a hit list of 58 drug-like compounds. A homology model of MTP was built by MODELLER and was minimised by CHARMm force field. It was then validated by Ramachandran plot and Verify-3D so as to obtain a stable structure, which was further used to refine the 58 hits using molecular docking studies. Then, five compounds with higher docking scores which satisfied the docking requirements were discovered. Among them, Ginkgetin and Dauricine were most likely to be candidates that exhibition inhibiting effect on MTP. The screening strategy in this study is relatively new to the discovery of MTP inhibitors in medicinal chemistry. Moreover, it is important to note that, lomitapide, an approved MTP inhibitor, fits well with Hypo1 as well as our homology model of MTP, which confirmed the rationality of our studies. The results indicated the applicability of molecular modeling for the discovery of potential natural MTP inhibitors from traditional Chinese herbs.     

Evaluation of the substrate envelope hypothesis for inhibitors of HIV-1 protease

Crystallographic data show that various substrates of HIV protease occupy a remarkably uniform region within the binding site; this region has been termed the substrate envelope. It has been suggested that an inhibitor that fits within the substrate envelope should tend to evade viral resistance because a protease mutation that reduces the affinity of the inhibitor will also tend to reduce the affinity of substrate, and will hence decrease the activity of the enzyme. Accordingly, inhibitors that fit the substrate envelope better should be less susceptible to clinically observed resistant mutations, since these must also allow substrates to bind. The present study describes a quantitative measure of the volume of a bound inhibitor falling outside the substrate envelope, and observes that this quantity correlates with the inhibitor's losses in affinity to clinically relevant mutants. This measure may thus be useful as a penalty function in the design of robust HIV protease inhibitors.     

Discovery of novel HCV polymerase inhibitors using pharmacophore-based virtual screening

We report the use of pharmacophore-based virtual screening as an efficient tool for the discovery of novel HCV polymerase inhibitors. A three-dimensional pharmacophore model for the HCV-796 binding site, NNI site IV inhibitor, to the enzyme was built by means of the structure-based focusing module in Cerius2 program. Using these models as a query for virtual screening, we produced a successful example of using pharmacophore-based virtual screening to identify novel compounds with HCV replicon assay through inhibition of HCV polymerization. Among the hit compounds, compounds 1 and 2 showed 56% and 48% inhibition of NS5B polymerization activity at 20 μM, respectively. In addition, compound 1 also exhibited replicon activity with EC₅₀ value of 2.16 μM. Following up the initial hit, we obtained derivatives of compound 1 and evaluated polymerization inhibition activity and HCV replicon assay. These results provide information necessary for the development of more potent NS5B inhibitors.     

Insight into the structural similarity between HIV protease and secreted aspartic protease-2 and binding mode analysis of HIV-Candida albicans inhibitors

The analysis of the structural similarity between Candida albicans Sap2 and HIV-1 aspartic proteases by molecular modeling gave insight into the common requirements for inhibition of both targets. Structure superimposition of Sap2 and HIV-1 protease confirmed the similarity between their active sites and flap regions. HIV-1 protease inhibitors herein investigated can fit the active site of Sap2, adopting very similar ligand-backbone conformations. In particular, key anchoring sites consisting of Gly85 in Sap2 and Ile50 in HIV-1 protease, both belonging to their corresponding flap regions, were found as elements of a similar binding-mode interaction. The knowledge of the molecular basis for binding to both Sap2 and HIV-1 proteases may ultimately lead to the development of single inhibitor acting on both targets.