Evaluation of the amino acid binding site of Mycobacterium tuberculosis glutamine synthetase for drug discovery

A combination of a literature survey, structure-based virtual screening and synthesis of a small library was performed to identify hits to the potential antimycobacterial drug target, glutamine synthetase. The best inhibitor identified from the literature survey was (2S,5R)-2,6-diamino-5-hydroxyhexanoic acid (4, IC(50) of 610+/-15microM). In the virtual screening 46,400 compounds were docked and subjected to a pharmacophore search. Of these compounds, 29 were purchased and tested in a biological assay, allowing three novel inhibitors containing an aromatic scaffold to be identified. Based on one of the hits from the virtual screening a small library of 15 analogues was synthesized producing four compounds that inhibited glutamine synthetase.     

Discovery of novel inhibitors of Trypanosoma cruzi trans-sialidase from in silico screening

trans-Sialidase from Trypanosoma cruzi (TcTS) has emerged as a potential drug target for treatment of Chagas disease. Here, we report the results of virtual screening for the discovery of novel TcTS inhibitors, which targeted both the sialic acid and sialic acid acceptor sites of this enzyme. A library prepared from the Evotec database of commercially available compounds was screened using the molecular docking program GOLD, following the application of drug-likeness filters. Twenty-three compounds selected from the top-scoring ligands were purchased and assayed using a fluorimetric assay. Novel inhibitor scaffolds, with IC₅₀ values in the submillimolar range were discovered. The 3-benzothiazol-2-yl-4-phenyl-but-3-enoic acid scaffold was studied in more detail, and TcTS inhibition was confirmed by an alternative sialic acid transfer assay. Attempts to obtain crystal structures of these compounds with TcTS proved unsuccessful but provided evidence of ligand binding at the active site.     

Modeling of human CCR5 as Target for HIV-I and Virtual Screening with Marine Therapeutic compounds

Marine derivatives are of great pharmaceutical interest as inhibitory compound and search of bioactive compounds from Marine organism which is relatively new to medicinal chemistry. Our main aim in the study is to screen possible inhibitors against CCR5 which acts as co-receptor M-tropic HIV-1, through virtual screening of 122 Marine derived compounds from various organisms known to have biological activity. Homology Model of CCR5 was constructed using MODELLER and the Model was energy minimized and validated using PROCHECK to obtain a stable structure, which was further used for virtual screening of Marine derived compounds through molecular Docking studies using GOLD. The Docked complexes were validated and Enumerated based on the GOLD Scoring function to pick out the best Marine inhibitor based on GOLD score. Thus from the entire 122 Marine compounds which were Docked, we got best 4 of them with optimal GOLD Score. (LAMIVUDINE: 45.0218, BATZELLINE-D: 44.3852.ACYCLOVIR: 43.1362 and THIIOACETAMIDE: 42.7412) Further the Complexes were analyzed through LIGPLOT for their interaction for the 4 best docked Marine compounds. Thus from the Complex scoring and binding ability its deciphered that these Marine compounds could be promising inhibitors for M-tropic HIV-1 using CCR5 as Drug target yet pharmacological studies have to confirm it.     

Novel GSK-3beta inhibitors from sequential virtual screening

Glycogen synthase kinase-3 (GSK-3beta) has been emerging as a key therapeutic target for type-2 diabetics, Alzheimer's disease, cancer, and chronic inflammation. For the purpose of finding biologically active and novel compounds and providing new idea for drug-design, we performed virtual screening using commercially available database. Three-dimensional common feature pharmacophore model was developed by using HipHop program provided in Catalyst software and it was used as a query for screening database. Recursive partitioning (RP) model was developed as a filtering system, which was able to classify active and inactive compounds. Eventually, a sequential virtual screening procedure (SQSP) was conducted by applying the common feature pharmacophore and RP model in succession to discover novel potent GSK-3beta inhibitors. The final 56 hit compounds were carefully selected considering predicted docking mode in crystal structures. Subsequent enzyme assay for human GSK-3beta protein confirmed that three compounds of these hit compounds exhibit micromolar inhibitory activity. Here, we report novel hit compounds and their binding mode in the active site of GSK-3beta crystal structure.     

A Combination of Receptor-Based Pharmacophore Modeling & QM Techniques for Identification of Human Chymase Inhibitors

Inhibition of chymase is likely to divulge therapeutic ways for the treatment of cardiovascular diseases, and fibrotic disorders. To find novel and potent chymase inhibitors and to provide a new idea for drug design, we used both ligand-based and structure-based methods to perform the virtual screening(VS) of commercially available databases. Different pharmacophore models generated from various crystal structures of enzyme may depict diverse inhibitor binding modes. Therefore, multiple pharmacophore-based approach is applied in this study. X-ray crystallographic data of chymase in complex with different inhibitors were used to generate four structure–based pharmacophore models. One ligand–based pharmacophore model was also developed from experimentally known inhibitors. After successful validation, all pharmacophore models were employed in database screening to retrieve hits with novel chemical scaffolds. Drug-like hit compounds were subjected to molecular docking using GOLD and AutoDock. Finally four structurally diverse compounds with high GOLD score and binding affinity for several crystal structures of chymase were selected as final hits. Identification of final hits by three different pharmacophore models necessitates the use of multiple pharmacophore-based approach in VS process. Quantum mechanical calculation is also conducted for analysis of electrostatic characteristics of compounds which illustrates their significant role in driving the inhibitor to adopt a suitable bioactive conformation oriented in the active site of enzyme. In general, this study is used as example to illustrate how multiple pharmacophore approach can be useful in identifying structurally diverse hits which may bind to all possible bioactive conformations available in the active site of enzyme. The strategy used in the current study could be appropriate to design drugs for other enzymes as well.     

In silico screening and biological evaluation of inhibitors of Src-SH3 domain interaction with a proline-rich ligand

Src signalling and transduction are directly involved in cell growth, cell cycle, malignant transformation and cell migration, providing therapeutic opportunities through inhibition of Src. Here we report virtual screening for novel compounds that inhibit the Src-SH3 protein-protein interaction with a proline-rich peptide ligand. Computational docking of the ZINC compound database was performed using GOLD. Top-scoring compounds were assayed using a fluorescence polarization-based assay. A benzoquinoline derivative showed micromolar inhibition of binding between Src-SH3 and the proline-rich peptide. Several analogues were subsequently assayed showing the requirement of a linker between the benzoquinoline and phenyl rings, and electron donating substituents on the phenyl ring.     

Computational approach to the identification of novel Aurora-A inhibitors

Aurora kinase A has been emerging as a key therapeutic target for the design of anticancer drugs. For the purpose of finding biologically active and novel compounds and providing new ideas for drug-design, we performed virtual screening using commercially available databases. A three-dimensional common feature pharmacophore model was developed with the HipHop program provided in the Catalyst software package, and this model was used as a query for screening the databases. A recursive partitioning (RP) model was developed as a filtering system, which was able to classify active and inactive compounds. Eventually, a step-wise virtual screening procedure was conducted by applying the common feature pharmacophore and the RP model in succession to discover novel potent Aurora-A inhibitors. A total of 68 compounds were selected for testing of their in vitro anticancer activities against various human cancer cell lines. Based on the activity data, we have identified fifteen compounds that warrant further investigation. Several compounds have a high inhibition rate (above 80% at 10 ??M) and a GI₅₀ lower than 5 ??M for the cell lines DU145 and HT29. Enzyme assay for these compounds identified hits with micro molar activity. Compound C11 has the highest activity (IC₅₀ = 5.09 ??M). The hits obtained from this screening scheme could be potential drug candidates after further optimization.     

Scoring functions and enrichment: a case study on Hsp90

Background  

The need for fast and accurate scoring functions has been driven by the increased use of in silico virtual screening twinned with high-throughput screening as a method to rapidly identify potential candidates in the early stages of drug development. We examine the ability of some the most common scoring functions (GOLD, ChemScore, DOCK, PMF, BLEEP and Consensus) to discriminate correctly and efficiently between active and non-active compounds among a library of ~3,600 diverse decoy compounds in a virtual screening experiment against heat shock protein 90 (Hsp90).     

Novel human mPGES-1 inhibitors identified through structure-based virtual screening

Microsomal prostaglandin E synthase-1 (mPGES-1) is an inducible prostaglandin E synthase after exposure to pro-inflammatory stimuli and, therefore, represents a novel target for therapeutic treatment of acute and chronic inflammatory disorders. It is essential to identify mPGES-1 inhibitors with novel scaffolds as new leads or hits for the purpose of drug design and discovery that aim to develop the next-generation anti-inflammatory drugs. Herein we report novel mPGES-1 inhibitors identified through a combination of large-scale structure-based virtual screening, flexible docking, molecular dynamics simulations, binding free energy calculations, and in vitro assays on the actual inhibitory activity of the computationally selected compounds. The computational studies are based on our recently developed three-dimensional (3D) structural model of mPGES-1 in its open state. The combined computational and experimental studies have led to identification of new mPGES-1 inhibitors with new scaffolds. In particular, (Z)-5-benzylidene-2-iminothiazolidin-4-one is a promising novel scaffold for the further rational design and discovery of new mPGES-1 inhibitors. To our best knowledge, this is the first time a 3D structural model of the open state mPGES-1 is used in structure-based virtual screening of a large library of available compounds for the mPGES-1 inhibitor identification. The positive experimental results suggest that our recently modeled trimeric structure of mPGES-1 in its open state is ready for the structure-based drug design and discovery.     

Rational design of bis-indolylmethane-oxadiazole hybrids as inhibitors of thymidine phosphorylase

Inhibition of Thymidine phosphorylase (TP) is continuously studied for the design and development of new drugs for the treatment of neoplastic diseases. As a part of our effort to identify TP inhibitors, we performed a structure-based virtual screening (SBVS) of our compound collection. Based on the insights gained from structures of virtual screening hits, a scaffold was designed using 1,3,4-oxadiazole as the basic structural feature and SAR studies were carried out for the optimization of this scaffold. Twenty-five novel bis-indole linked 1,3,4-oxadiazoles (7–31) were designed, synthesized and tested in vitro against E. coli TP (EcTP). Compound 7 emerged as potent TP inhibitor with an IC₅₀ value of 3.50?±?0.01?μM. Docking studies were carried out using GOLD software on thymidine phosphorylase from human (hTP) and E. coli (EcTP). Various hydrogen bonding, hydrophobic interactions, and π-π stacking were observed between designed molecules and the active site amino acid residues of the studied enzymes.     

Oxidative modifications and down-regulation of ubiquitin carboxyl-terminal hydrolase L1 associated with idiopathic Parkinson's and Alzheimer's diseases

Alzheimer's disease (AD) and Parkinson's disease (PD) are the two most common neurodegenerative diseases that occur either in relatively rare, familial forms or in common, sporadic forms. The genetic defects underlying several monogenic familial forms of AD and PD have recently been identified, however, the causes of other AD and PD cases, particularly sporadic cases, remain unclear. To gain insights into the pathogenic mechanisms involved in AD and PD, we used a proteomic approach to identify proteins with altered expression levels and/or oxidative modifications in idiopathic AD and PD brains. Here, we report that the protein level of ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1), a neuronal de-ubiquitinating enzyme whose mutation has been linked to an early-onset familial PD, is down-regulated in idiopathic PD as well as AD brains. By using a combination of two-dimensional gel electrophoresis and mass spectrometry, we have identified three human brain UCH-L1 isoforms, a full-length form and two amino-terminally truncated forms. Our proteomic analyses reveal that the full-length UCH-L1 is a major target of oxidative damage in AD and PD brains, which is extensively modified by carbonyl formation, methionine oxidation, and cysteine oxidation. Furthermore, immunohistochemical studies show that prominent UCH-L1 immunostaining is associated with neurofibrillary tangles and that the level of soluble UCH-L1 protein is inversely proportional to the number of tangles in AD brains. Together, these results provide evidence supporting a direct link between oxidative damage to the neuronal ubiquitination/de-ubiquitination machinery and the pathogenesis of sporadic AD and PD.     

Combining pharmacophore, docking and substructure search approaches to identify and optimize novel B-Raf(V600E) inhibitors

In this study for searching novel B-Raf(V600E) inhibitors, pharmacophore-based virtual screening identified 1 as a hit bearing 5-benzylidene-2-thioxodihydropyrimidine-4,6(1H,5H)-dione. Based on 1, scaffold hopping inspired by molecular docking discovered 5-(furan-2-ylmethylene)-2-thioxodihydropyrimidine-4,6(1H,5H)-dione as a new and better scaffold. Substructure search with the new scaffold identified 28 active compounds, among which 12 compounds (42.9%) showed IC(50) less than 1μM. Especially, compound 3o, which is 10-fold more potent than the hit 1, is a potent inhibitor comparable to that of the marketed drug vemurafenib.     

Identification of novel drug scaffolds for inhibition of SARS-CoV 3-Chymotrypsin-like protease using virtual and high-throughput screenings

We have used a combination of virtual screening (VS) and high-throughput screening (HTS) techniques to identify novel, non-peptidic small molecule inhibitors against human SARS-CoV 3CLpro. A structure-based VS approach integrating docking and pharmacophore based methods was employed to computationally screen 621,000 compounds from the ZINC library. The screening protocol was validated using known 3CLpro inhibitors and was optimized for speed, improved selectivity, and for accommodating receptor flexibility. Subsequently, a fluorescence-based enzymatic HTS assay was developed and optimized to experimentally screen approximately 41,000 compounds from four structurally diverse libraries chosen mainly based on the VS results. False positives from initial HTS hits were eliminated by a secondary orthogonal binding analysis using surface plasmon resonance (SPR). The campaign identified a reversible small molecule inhibitor exhibiting mixed-type inhibition with a K_i value of 11.1 μM. Together, these results validate our protocols as suitable approaches to screen virtual and chemical libraries, and the newly identified compound reported in our study represents a promising structural scaffold to pursue for further SARS-CoV 3CLpro inhibitor development.     

A discovery of novel Mycobacterium tuberculosis pantothenate synthetase inhibitors based on the molecular mechanism of actinomycin D inhibition

Mycobacterium tuberculosis pantothenate synthetase is a potential anti-tuberculosis target, and a high-throughput screening system was previously developed to identify its inhibitors. Using a similar system, we screened a small library of compounds and identified actinomycin D (ActD) as a weak inhibitor of pantothenate synthetase. A new method was established to discover more effective inhibitors by determining the molecular mechanism of ActD inhibition followed by structure-based virtual screening. The molecular interaction of inhibition was determined by circular dichroism and tryptophan fluorescence quenching. The structure-based search and virtual screening were performed using the Molecular Operating Environment (MOE) program and SYBYL 7.5, respectively. Two inhibitors were identified with an IC₅₀ for pantothenate synthetase that was at least ten times better than that of ActD.     

In silico high-throughput virtual screening and molecular dynamics simulation study to identify inhibitor for AdeABC efflux pump of Acinetobacter baumannii

Emergence of multi-drug resistant strains of Acinetobacter baumannii has caused significant health problems and is responsible for high morbidity and mortality. Overexpression of AdeABC efflux system is one of the major mechanisms. In this study, we have focused on overcoming the drug resistance by identifying inhibitors that can effectively bind and inhibit integral membrane protein, AdeB of this efflux pump. We performed homology modeling to generate structure of AdeB using MODELLER v9.16 followed by model refinement using 3D-Refine tool and validated using PSVS, ProsaWeb, ERRAT, etc. The energy minimization of modeled protein was done using Protein preparation wizard application included in Schrodinger suite. High-throughput virtual screening of 159,868 medicinal compounds against AdeB was performed using three sequential docking modes (i.e. HTVS, SP and XP). Furthermore, absorption, distribution, metabolism, excretion, and toxicity (ADMET) analysis was done using QIKPROP. The selected 123 compounds were further analyzed for binding free energy by molecular mechanics (using prime MM-GBSA). We have also performed enrichment study (ROC curve analysis) to validate our docking results. The selected molecule and its interaction with AdeB were validated by molecular dynamics simulation (MDS) using GROMACS v5.1.4. In silico high-throughput virtual screening and MDS validation identified ZINC01155930 ((4R)-3-(cycloheptoxycarbonyl)-4-(4-etochromen-3-yl)-2-methyl-4,6,7,8-tetrahydroquinolin-5-olate) as a possible inhibitor for AdeB. Hence, it might be a suitable efflux pump inhibitor worthy of further investigation in order to be used for controlling infections caused by Acinetobacter baumannii.     

Computational exploration of maternal embryonic leucine zipper kinase (MELK) as a cancer drug target

Maternal embryonic leucine zipper kinase (MELK) is of vital importance due to its significant role in cancer development and its association with poor prognosis in different cancers. Here, we employed several computer aided drug design approaches to shortlist potential binding molecules of MELK. For virtual screening, asinex oncology library (containing 6334 drugs) and comprehensive marine natural products database (containing approximately 32,000 drugs) were used. The study identified two drug molecules: Top-2 and Top-3 as high affinity binding MELK molecules compared to the control co-crystalized Top-1 inhibitor. Both the shortlisted compounds and the control showed high stable binding free energy and high GOLD score. The compounds and control also reported stable dynamics with root mean square deviations (RMSD) value ～ 2 Å in 500 ns. Similarly, the MELK active site residues were observed in good stability with the compounds. Further, it was noticed the compounds/control formed multiple hydrogen bonds with the MELK active pocket residues which is the main reason of high intermolecular stability. Atomic level binding free energies determined van der Waals and electrostatic energies to play vital role in stable complex formation. From drug likeness and pharmacokinetics perspective, the compounds are ideal molecules for further investigation. Overall, the results are promising and might be tested in in vivo and in vitro studies against MELK.     

Inhibitors of human tyrosyl-DNA phospodiesterase (hTdp1) developed by virtual screening using ligand-based pharmacophores

Human tyrosyl-DNA phosphodiesterase (hTdp1) inhibitors have become a major area of drug research and structure-based design since they have been shown to work synergistically with camptothecin (CPT) and selectively in cancer cells. The pharmacophore features of 14 hTdp1 inhibitors were used as a filter to screen the ChemNavigator iResearch Library of about 27 million purchasable samples. Docking of the inhibitors and hits obtained from virtual screening was performed into a structural model of hTdp1 based on a high resolution X-ray crystal structure of human Tdp1 in complex with vanadate, DNA and a human topoisomerase I (TopI)-derived peptide (PDB code: 1NOP). A total of 46 compounds matching the three-dimensional arrangement of the pharmacophoric features were assayed. Using a high-throughput screening assay, we have identified an 1H-indol-3-yl-acetic acid derivative as a potent Tdp1 inhibitor with an IC₅₀ value of 7.94 μM. The obtained novel chemotype may provide a new scaffold for developing inhibitors of Tdp1.     

New inhibitor of 3-phosphoinositide dependent protein kinase-1 identified from virtual screening

3-Phosphoinositide-dependent protein kinase-1 (PDK1) has been recognized as a promising anticancer target. Thus, it is interesting to identify new inhibitors of PDK1 for anticancer drug discovery. Through a combined use of virtual screening and wet experimental activity assays, we have identified a new PDK1 inhibitor with IC(50)=~200 nM. The anticancer activities of this compound have been confirmed by the anticancer activity assays using 60 cancer cell lines. The obtained new PDK1 inhibitor and its PDK1-inhibitor binding mode should be valuable in future de novo design of novel, more potent and selective PDK1 inhibitors for future development of anticancer therapeutics.     

Computational modeling-based discovery of novel classes of anti-inflammatory drugs that target lanthionine synthetase C-like protein 2

Background

Lanthionine synthetase component C-like protein 2 (LANCL2) is a member of the eukaryotic lanthionine synthetase component C-Like protein family involved in signal transduction and insulin sensitization. Recently, LANCL2 is a target for the binding and signaling of abscisic acid (ABA), a plant hormone with anti-diabetic and anti-inflammatory effects.

Methodology/Principal Findings

The goal of this study was to determine the role of LANCL2 as a potential therapeutic target for developing novel drugs and nutraceuticals against inflammatory diseases. Previously, we performed homology modeling to construct a three-dimensional structure of LANCL2 using the crystal structure of lanthionine synthetase component C-like protein 1 (LANCL1) as a template. Using this model, structure-based virtual screening was performed using compounds from NCI (National Cancer Institute) Diversity Set II, ChemBridge, ZINC natural products, and FDA-approved drugs databases. Several potential ligands were identified using molecular docking. In order to validate the anti-inflammatory efficacy of the top ranked compound (NSC61610) in the NCI Diversity Set II, a series of in vitro and pre-clinical efficacy studies were performed using a mouse model of dextran sodium sulfate (DSS)-induced colitis. Our findings showed that the lead compound, NSC61610, activated peroxisome proliferator-activated receptor gamma in a LANCL2- and adenylate cyclase/cAMP dependent manner in vitro and ameliorated experimental colitis by down-modulating colonic inflammatory gene expression and favoring regulatory T cell responses.

Conclusions/Significance

LANCL2 is a novel therapeutic target for inflammatory diseases. High-throughput, structure-based virtual screening is an effective computational-based drug design method for discovering anti-inflammatory LANCL2-based drug candidates.     

Identification of type I and type II inhibitors of c-Yes kinase using in silico and experimental techniques

c-Yes kinase is considered as one of the attractive targets for anti-cancer drug design. The DFG (Asp-Phe-Gly) motif present in most of the kinases will adopt active and inactive conformations, known as DFG-in and DFG-out and their inhibitors are classified into type I and type II, respectively. In the present study, two screening protocols were followed for identification of c-Yes kinase inhibitors. (i) Structure-based virtual screening (SBVS) and (ii) Structure-based (SB) and Pharmacophore-based (PB) tandem screening. In SBVS, the c-Yes kinase structure was obtained from homology modeling and seven ensembles with different active site scaffolds through molecular dynamics (MD) simulations. For SB-PB tandem screening, we modeled ligand bound active and inactive conformations. Physicochemical properties of inhibitors of Src kinase family and c-Yes kinase were used to prepare target focused libraries for screenings. Our screening procedure along with docking showed 520 probable hits in SBVS and tandem screening (120 and 400, respectively). Out of 5000 compounds identified from different computational methods, 2410 were examined using kinase inhibition assays. It includes 266 compounds (5.32%) identified from our method. We observed that 14 compounds (12%) are identified by the present method out of 168 that showed > 30% inhibition. Among them, three compounds are novel, unique, and showed good inhibition. Further, we have studied the binding of these compounds at the DFG-in and DFG-out conformations and reported the probable class (type I or type II). Hence, we suggest that these compounds could be novel drug leads for regulation of colorectal cancer.