Homology modeling of human histone deacetylase 10 and design of potential selective inhibitors

Abstract

Histone deacetylases (HDACs) are implicated in the pathology of various cancers, and their pharmacological blockade has proven to be promising in reversing the malignant phenotypes. However, lack of crystal structures of some of the human HDAC isoforms (e.g., HDAC10) hinders the design of the isoform-selective inhibitor. Here, the recently solved X-ray crystal structure of Danio rerio (zebrafish) HDAC10 (Protein Data Bank (PDB) ID; 5TD7, released on 24 May 2017) was retrieved from the PDB and used as a template structure to model the three-dimensional structure of human HDAC10. The overall quality of the best model (M0017) was assessed by computing its z-score—a measure of the deviation of the total energy of the structure with respect to an energy distribution derived from random conformations and by docking of known HDAC10 inhibitors to its catalytic cavity. Furthermore, to identify potential HDAC10-selective inhibitor ligand-based virtual screening was carried out against the ZINC database. The free modeled structure of HDAC10 and its complexes with quisinostat and the highest-ranked compound ZINC19749069 were submitted to molecular dynamics simulation. The comparative analysis of root-mean-squared deviation, root-mean-squared fluctuation, radius of gyration (Rg), and potential energy of these systems showed that HDAC10-ZINC19749069 complex remained the most stable over time. Thus, M0017 could be potentially used for structure-based inhibitor against HDAC10, and ZINC19749069 may provide a scaffold for further optimization.

Communicated by Ramaswamy H. Sarma     

Synthesis,activity evaluation,and docking analysis of barbituric acid aryl hydrazone derivatives as RSK2 inhibitors

The 90 kDa ribosomal S6 kinases (RSKs), especially RSK2, have attracted attention for the development of new anticancer agents. Through structural optimization of the hit compound 1 from our previous study, a series of barbituric acid aryl hydrazone analogues were designed and synthesized as potential RSK2 inhibitors. The most potent one, compound 9, showed a higher activity against RSK2 with an IC₅₀ value of 1.95 μM. To analyze and elucidate their structure-activity relationship, the homology model of RSK2 N-terminal kinase domain was built and molecular docking simulations were performed, which provide helpful clues to design new inhibitors with desired activities.     

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.     

In silico approach towards identification of potential inhibitors of Helicobacter pylori DapE

Helicobacter pylori is a gastric mucosal pathogen and is associated with diseases like peptic ulcer and gastric cancer. To combat H. pylori infection, there is an urgent need for new class of antibiotics due to the emergence of drug-resistant strains. Enzymes involved in bacterial lysine biosynthetic pathways may be potential targets for antibacterial drug development, since lysine is an essential component of the bacterial peptidoglycan cell wall. No pathway exists for lysine biosynthesis in humans; hence, the inhibitors targeting bacterial enzymes may have selective toxicity. dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) is a critical enzyme of this pathway and deletion of DapE gene is lethal to H. pylori, since the organism has no alternative pathway for lysine biosynthesis. In this study, we reported a 3D model structure of H. pylorie DapE, which consisted of a catalytic domain and a dimerization domain generated by MODELLER software. We also confirmed the stability of the modeled structure through 10 ns molecular dynamics simulation using GROMACS software. Next, to identify potential small molecule inhibitors of DapE, drug-like small molecule-screening library was generated. This was performed by Tanimoto-based similarity searching in the PubChem Database with DapE substrate L,L-SDAP as a query molecule, followed by fragment-based docking approach using GLIDE XP. This approach identified two potential substrate-competitive small molecule inhibitors of DapE. These new molecules may provide a starting point to search for novel therapeutics.     

On the modeling of snake venom serine proteinase interactions with benzamidine-based thrombin inhibitors

Pit viper venoms contain a number of serine proteinases that exhibit one or more thrombin-like activities on fibrinogen and platelets, this being the case for the kinin-releasing and fibrinogen-clotting KN-BJ from the venom of Bothrops jararaca. A three-dimensional structural model of the KN-BJ2 serine proteinase was built by homology modeling using the snake venom plasminogen activator TSV-PA as a major template and porcine kallikrein as additional structural support. A set of intrinsic buried waters was included in the model and its behavior under dynamic conditions was molecular dynamics simulated, revealing a most interesting similarity pattern to kallikrein. The benzamidine-based thrombin inhibitors alpha-NAPAP, 3-TAPAP, and 4-TAPAP were docked into the refined model, allowing for a more insightful functional characterization of the enzyme and a better understanding of the reported comparatively low affinity of KN-BJ2 toward those inhibitors.     

Structure based drug designing of a novel antiflaviviral inhibitor for nonstructural 3 protein

Literature shows that Flaviviruses cause a variety of diseases, including fevers, encephalitis, and hemorrhagic fevers. NS3 is a multifunctional protein with an Nterminal protease domain (NS3pro) that is responsible for proteolytic processing of the viral polyprotein, and a C-terminal region that contains an RNA triphosphatase, RNA helicase and RNA-stimulated NTPase domain that are essential for RNA replication. Therefore, NS3 protein is the preferential choice for inhibition to stop the proteolytic processing. Hence, the 3D structure of NS3 protein was modeled using homology modeling by MODELLER 9v7. Evaluation of the constructed NS3 protein models were done by PROCHECK, VERYFY3D and through ProSA calculations. Ligands for the catalytic triad were designed using LIGBUILDER. The NS3 protein's catalytic triad was explored to find out the critical interactions pattern for inhibitor binding using molecular docking methodology using AUTODOCK Vina. It should be noted that these predicted data should be validated using suitable assays for further consideration. ABBREVIATIONS: DOPE - Discrete optimized protein energy, WHO - World Health Organization, ADME/T - Absorption, Distribution, Metabolism, Excretion and Toxicity.     

In-silico analysis of Sirt2 from Schistosoma mansoni: structures,conformations, and interactions with inhibitors

Sirtuins are NAD+-dependent lysine deacetylases member of the class III HDAC family. These are demonstrated to be therapeutic targets in parasitic diseases like schistosomiasis. Observations suggested that sirtuin enzyme is necessary for the functionality of fe/male reproductive system, due to which SmSirt2 is treated as a potential therapeutic target. There are no structural and molecular features of SmSirt2 have been reported yet. In this study, homology modeling has been used to determine the three-dimensional features of the SmSITRT2. Further, structure validation has been performed by energy minimization and Ramachandran plot. Validated structures are further subjected to molecular docking and virtual screening to find the best lead molecules for downstream analysis. Ten lead molecules were selected while comparing virtual screening of hSirt2 and SmSirt2 both. These leads are further compared with AKG2 which is known inhibitor of hSirt2 (?8.8 kcal/mol). Out of selected 10 leads, four of them (ZINC23995485 (?9.5 kcal/mol), ZINC53298162 (?9.4 kcal/mol), ZINC70927268 (?10.0 kcal/mol), ZINC89878705 (?11.2 kcal/mol)) have shown better interaction with SmSirt2, in which ZINC89878705 (?11.2 kcal/mol) shows a more compact packing as compared to AKG2 and rest of ligands. These molecules could be further subject to in vitro study and model of SmSirt2 has been proposed for further structure-based drug design projects concerning sirtuins from Schistosoma mansoni.     

Design and synthesis of novel PARP-1 inhibitors based on pyridopyridazinone scaffold

Various pyridopyridazinone derivatives were designed as Poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors. The pyridopyridazinone scaffold was used as an isostere of the phthalazine nucleus of the lead compound Olaparib in addition to some modifications in the tail part of the molecule. Preliminary biological evaluation indicated that most compounds possessed inhibitory potencies comparable to Olaparib in nanomolar level. The best PARP-1 inhibitory activity was observed for compound 8a with (IC₅₀ = 36 nM) compared to Olaparib as a reference drug (IC₅₀ = 34 nM). Molecular modeling simulation revealed that, the designed compounds docked well into PARP-1 active site and their complexes are stabilized by three key hydrogen bond interactions with both Gly863 and Ser904 as well as other favorable π-π and hydrogen-π stacking interactions with Tyr907 and Tyr896, respectively. Computational ADME study predicted that the target compounds 8a and 8e have proper pharmacokinetic and drug-likeness properties. These outcomes afford a new structural framework for the design of novel inhibitors for PARP-1.     

Identification and characterization of ErbB4 kinase inhibitors for effective breast cancer therapy

The overexpression of ErbB4 is associated with aggressive disease biology and reduced the survival of breast cancer patients. We have used ErbB4 receptor as a novel drug target to spearhead the rational drug design. The present study is divided into two parts. In the first part, we have exploited the hidden information inside ErbB4 kinase receptor both at sequence and structural level. PSI-BLAST algorithm is used to search similar sequences against ErbB4 kinase sequence. Top 15 sequences with high identity were selected for finding conserved and variable regions among sequences using multiple sequence alignment. In the second part, available 3?D structure of ErbB4 kinase is curated using loop modeling, and anomalies in the modeled structure is improved by energy minimization. The resultant structure is validated by analyzing dihedral angles by Ramachandran plot analysis. Furthermore, the potential binding sites were detected by using DoGSite and CASTp server. The similarity-search criterion is used for the preparation of our in-house database of drugs from DrugBank database. In total, 409 drugs yet to be tested against ErbB4 kinase is used for screening purpose. Virtual screening results in identification of 11 compounds with better binding affinity than lapatinib and canertinib. Study of protein–ligand interactions reveals information about amino acid residues; Lys726, Thr771, Met774, Cys778, Arg822, Thr835, Asp836 and Phe837 at the binding pocket. The physicochemical properties and bioactivity score calculation of selected compounds suggest them as biological active. This study presents a rich array that assist in expediting new drug discovery for breast cancer.     

Structural basis for the design and synthesis of selective HDAC inhibitors

Histone Deacetylases are considered promising targets for cancer epigenetic therapy, and small molecules able to modulate their biological function have recently gained an increasing interest as potential anticancer agents. In spite of their potential application in cancer therapy, most HDAC inhibitors unselectively bind the several HDAC isoforms, giving rise to different side-effects. In this context, we have traced out the structural elements responsible of selective binding for the therapeutically relevant different HDAC isoforms. The structural analysis has been carried out by molecular modeling, docking in the binding pockets of HDAC1–4 and HDAC6–8, 36 inhibitors presenting a well defined selectivity for the different isoforms. As quick proof of evidence, we have designed, synthesized and experimentally tested three selective ligands. The experimental data suggest that the obtained structural guidelines can be useful tools for the rational design of new potent inhibitors against selected HDAC isoforms.     

Triazoloquinoxalines-based DNA intercalators-Topo II inhibitors: design,synthesis, docking,ADMET and anti-proliferative evaluations

Sixteen [1, 2, 4]triazolo[4,3-a]quinoxalines as DNA intercalators-Topo II inhibitors have been prepared and their anticancer actions evaluated towards three cancer cell lines. The new compounds affected on high percentage of MCF-7. Derivatives 7e, 7c and 7b exhibited the highest anticancer activities. Their activities were higher than that of doxorubicin. Molecular docking studies showed that the HBA present in the chromophore, the substituted distal phenyl moiety and the extended linkers enable our derivatives to act as DNA binders. Also, the pyrazoline moiety formed six H-bonds and improved affinities with DNA active site. Finally, 7e, 7c and 7b exhibited the highest DNA affinities and act as traditional intercalators of DNA. The most active derivatives 7e, 7c, 7b, 7g and 6e were subjected to evaluate their Topo II inhibition and DNA binding actions. Derivative 7e exhibited the highest binding affinity. It intercalates DNA at IC₅₀ = 29.06 µM. Moreover, compound 7e potently intercalates DNA at an IC₅₀ value of 31.24 µM. Finally, compound 7e demonstrated the most potent Topo II inhibitor at a value of 0.890 µM. Compound 7c exhibited an equipotent IC₅₀ value (0.940 µM) to that of doxorubicin. Furthermore, derivatives 7b, 7c, 7e and 7g displayed a high ADMET profile.     

2,3-Dihydrobenzofuran privileged structures as new bioinspired lead compounds for the design of mPGES-1 inhibitors

2,3-Dihydrobenzofurans are proposed as privileged structures and used as chemical platform to design small compound libraries. By combining molecular docking calculations and experimental verification of biochemical interference, we selected some potential inhibitors of microsomal prostaglandin E₂ synthase (mPGES)-1. Starting from low affinity natural product 1, by our combined approach we identified the compounds 19 and 20 with biological activity in the low micromolar range. Our data suggest that the 2,3-dihydrobenzofuran derivatives might be suitable bioinspired lead compounds for development of new generation mPGES-1 inhibitors with increased affinity.     

c-jun在癫痫发病机制及治疗中的作用

c-jun是立早基因家族（immediate early genes,IEGs）中的重要成员之一,在包括癫痫在内的许多应激过程中具有重要作用。近年来,关于c-jun在癫痫发病机制及治疗中作用的研究取得了较大的进展。多种有效的抗癫痫治疗措施可以下调癫痫动物模型中c-jun的过度表达,从而发挥神经保护及抗癫痫效能。     

Identification and validation of l-asparaginase as a potential metabolic target against Mycobacterium tuberculosis

Multidrug-resistant Mycobacterium tuberculosis (Mtb) has emerged as a major health challenge, necessitating the search for new molecular targets. A secretory amidohydrolase, l -asparaginase of Mtb (MtA), originally implicated in nitrogen assimilation and neutralization of acidic microenvironment inside human alveolar macrophages, has been proposed as a crucial metabolic enzyme. To investigate whether this enzyme could serve as a potential drug target, it was studied for structural details and active site–specific inhibitors were tested on cultured Mycobacterial strain. The structural details of MtA obtained through comparative modeling and molecular dynamics simulations provided insights about the orchestration of an alternate reaction mechanism at the active site. This was contrary to the critical Tyr flipping mechanism reported in other asparaginases. We report the novel finding of Tyr to Val replacement in catalytic triad I along with the structural reorganization of a β-hairpin loop upon substrate binding in MtA active site. Further, 5 MtA-specific, active-site–based inhibitors were obtained by following a rigorous differential screening protocol. When tested on Mycobacterium culture, 3 of these, M3 (ZINC 4740895), M26 (ZINC 33535), and doxorubicin showed promising results with inhibitory concentrations (IC ₅₀) of 431, 100, and 56 µM, respectively. Based on our findings and considering stark differences with human asparaginase, we project MtA as a promising molecular target against which the selected inhibitors may be used to counteract Mtb infection effectively.     

In silico characterization of binding mode of CCR8 inhibitor: homology modeling,docking and membrane based MD simulation study

Human CC-chemokine receptor 8 (CCR8) is a crucial drug target in asthma that belongs to G-protein-coupled receptor superfamily, which is characterized by seven transmembrane helices. To date, there is no X-ray crystal structure available for CCR8; this hampers active research on the target. Molecular basis of interaction mechanism of antagonist with CCR8 remains unclear. In order to provide binding site information and stable binding mode, we performed modeling, docking and molecular dynamics (MD) simulation of CCR8. Docking study of biaryl-ether-piperidine derivative (13C) was performed inside predefined CCR8 binding site to get the representative conformation of 13C. Further, MD simulations of receptor and complex (13C-CCR8) inside dipalmitoylphosphatidylcholine lipid bilayers were performed to explore the effect of lipids. Results analyses showed that the Gln91, Tyr94, Cys106, Val109, Tyr113, Cys183, Tyr184, Ser185, Lys195, Thr198, Asn199, Met202, Phe254, and Glu286 were conserved in both docking and MD simulations. This indicated possible role of these residues in CCR8 antagonism. However, experimental mutational studies on these identified residues could be effective to confirm their importance in CCR8 antagonism. Furthermore, calculated Coulombic interactions represented the crucial roles of Glu286, Lys195, and Tyr113 in CCR8 antagonism. Important residues identified in this study overlap with the previous non-peptide agonist (LMD-009) binding site. Though, the non-peptide agonist and currently studied inhibitor (13C) share common substructure, but they differ in their effects on CCR8. So, to get more insight into their agonist and antagonist effects, further side-by-side experimental studies on both agonist (LMD-009) and antagonist (13C) are suggested.     

Molecular modeling study for the design of novel acetyl-CoA carboxylase inhibitors using 3D QSAR,molecular docking and dynamic simulations

Acetyl-CoA carboxylase (ACC) enzyme plays an important role in the regulation of biosynthesis and oxidation of fatty acids. ACC is a recognized drug target for the treatment of obesity and diabetes. Combination of ligand and structure-based in silico methods along with activity and toxicity prediction provides best lead compounds in the drug discovery process. In this study, a data-set of 100 ACC inhibitors were used for the development of comparative molecular field analysis (CoMFA) and comparative molecular similarity index matrix analysis (CoMSIA) models. The generated contour maps were used for the design of novel ACC inhibitors. CoMFA and CoMSIA models were used for the predication of activity of designed compounds. In silico toxicity risk prediction study was carried out for the designed compounds. Molecular docking and dynamic simulations studies were performed to know the binding mode of designed compounds with the ACC enzyme. The designed compounds showed interactions with key amino acid residues important for catalysis, and good correlation was observed between binding free energy and inhibition of ACC.     

Three dimensional modeling of N-terminal region of galanin and its interaction with the galanin receptor

The neuropeptide galanin comes under the powerful and versatile modulators of classical neurotransmitters and is present in brain tissues, which are intimately involved in epileptogenesis. It acts as appealing targets for studying basic mechanisms of seizure initiation and arrest, and for the development of novel approaches for various neurodegenerative diseases. Galanin is widely distributed in the mammalian brain which controls various processes such as sensation of pain, learning, feeding, sexual behaviour, carcinogenesis, pathophysiology of neuroendocrine tumors and others. The function of galanin can be exploited through its interaction with three G-protein coupled receptors subtypes such as GalR1, GalR2 and GalR3. The N-terminal region of galanin comprises about highly conserved 15 amino acid residues, which act as the crucial region for agonist-receptor binding. We have constructed a theoretical structural model for the N-terminal region of galanin from Homo sapiens by homology modeling. The stereochemistry of the model was checked using PROCHECK. The functionally conserved regions were identified by surface mapping of phylogenetic information generated by online web algorithm ConSurf. The docking studies on the pharmacologically important galanin receptors with the theoretical model of N-terminal region of galanin predicted crucial residues for binding which would be useful in the development of novel leads for neurodegenerative disorders.     

Necrotic death of neurons following an excitotoxic insult is prevented by a peptide inhibitor of c-jun N-terminal kinase

Peptide inhibitors of c-Jun N-terminal kinase (JNK) have been shown to potently protect against cerebral ischemia. The protective effect has been ascribed to prevention of apoptosis, but cell death following cerebral ischemia is a consequence of both apoptotic and necrotic cell death. We evaluated whether a peptide inhibitor (TAT-TIJIP) of JNK could prevent necrotic cell death in an in vitro model of excitotoxic neuronal death. We find that TAT-TIJIP effectively prevented cell death by interfering with several processes which have been identified as leading to cell death by necrosis. In particular, reactive oxygen species production was reduced, as indicated by an 88% decrease in the rate of dihydroethidium fluorescence in the presence of TAT-TIJIP. Furthermore, TAT-TIJIP attenuated the increase in cytosolic calcium following the excitotoxic insult. The potent neuroprotective properties of JNK peptide inhibitors likely reflects their abilities to prevent cell death by necrosis as well as apoptosis.     

Probing the structure of falcipain-3, a cysteine protease from Plasmodium falciparum: comparative protein modeling and docking studies

Increasing resistance of malaria parasites to conventional antimalarial drugs is an important factor contributing to the persistence of the disease as a major health threat. The ongoing search for novel targets has resulted in identification and expression of several enzymes including cysteine proteases that are implicated in hemoglobin degradation. Falcipain-2 and falcipain-3 are considered to be the two principal cysteine proteases in this degradation, and hence, are potential drug targets. A homology model of falcipain-3 was built and validated by various structure/geometry verification tools as well as docking studies of known substrates. The correlation coefficient of 0.975 between interaction energies and K(m) values of these substrates provided additional support for the model. On comparison with the previously reported falcipain-2 homology model, the currently constructed falcipain-3 structure showed important differences between the S2 pockets that might explain the variations in the K(m) values of various substrates for these enzymes. Further, docking studies also provided insight into possible binding modes and interactions of ligands with falcipain-3. Results of the current study could be employed in de novo drug design leading to development of new antimalarial agents.