Ligand based virtual screening and biological evaluation of inhibitors of chorismate mutase (Rv1885c) from Mycobacterium tuberculosis H37Rv

We have identified new lead candidates that possess inhibitory activity against Mycobacterium tuberculosis H37Rv chorismate mutase by a ligand-based virtual screening optimized for lead evaluation in combination with in vitro enzymatic assay. The initial virtual screening using a ligand-based pharmacophore model identified 95 compounds from an in-house small molecule database of 15,452 compounds. The obtained hits were further evaluated by molecular docking and 15 compounds were short listed based on docking scores and the other scoring functions and subjected to biological assay. Chorismate mutase activity assays identified four compounds as inhibitors of M. tuberculosis chorismate mutase (MtCM) with low K(i) values. The structural models for these ligands in the chorismate mutase binding site will facilitate medicinal chemistry efforts for lead optimization against this protein.     

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.     

Dynamic and Multi-Pharmacophore Modeling for Designing Polo-Box Domain Inhibitors

The polo-like kinase 1 (Plk1) is a critical regulator of cell division that is overexpressed in many types of tumors. Thus, a strategy in the treatment of cancer has been to target the kinase activity (ATPase domain) or substrate-binding domain (Polo-box Domain, PBD) of Plk1. However, only few synthetic small molecules have been identified that target the Plk1-PBD. Here, we have applied an integrative approach that combines pharmacophore modeling, molecular docking, virtual screening, and in vitro testing to discover novel Plk1-PBD inhibitors. Nine Plk1-PBD crystal structures were used to generate structure-based hypotheses. A common pharmacophore model (Hypo1) composed of five chemical features was selected from the 9 structure-based hypotheses and used for virtual screening of a drug-like database consisting of 159,757 compounds to identify novel Plk1-PBD inhibitors. The virtual screening technique revealed 9,327 compounds with a maximum fit value of 3 or greater, which were selected and subjected to molecular docking analyses. This approach yielded 93 compounds that made good interactions with critical residues within the Plk1-PBD active site. The testing of these 93 compounds in vitro for their ability to inhibit the Plk1-PBD, showed that many of these compounds had Plk1-PBD inhibitory activity and that compound Chemistry_28272 was the most potent Plk1-PBD inhibitor. Thus Chemistry_28272 and the other top compounds are novel Plk1-PBD inhibitors and could be used for the development of cancer therapeutics.     

Identification of novel CYP2A6 inhibitors by virtual screening

The human CYP2A6 enzyme metabolises several xenobiotics including nicotine, the addictive component in tobacco. Reduced activity of CYP2A6, either for genetic reasons or by administering inhibitors of CYP2A6, reduces tobacco smoking. The aim was to design novel inhibitors of CYP2A6 using 3D-QSAR analysis combined with virtual screening. A 3D-QSAR model was utilised to identify the most important features of the inhibitors, and this knowledge was used to design inhibitors for CYP2A6. Chemical database screening yielded several potent inhibitor candidates such as alkylamine derivatives (compound no. 5, IC(50)=0.1 μM) and 1-benzothiophene-3-carbaldehyde that can be used as lead compounds in the development of drugs for smoking reduction therapy.     

Pharmacophore modeling and hybrid virtual screening for the discovery of novel IκB kinase 2 (IKK2) inhibitors

IKK2 (IκB kinase 2) inhibitors have been identified as potential drug candidates in the treatment of various immune/inflammatory disorders as well as cancer. So far more than one hundred small molecule inhibitors against IKK2 have been reported publicly. In this investigation, pharmacophore modeling was carried out to clarify the essential structure-activity relationship for the known IKK2 inhibitors. One of the established pharmacophore hypotheses, namely Hypo8, which has the best prediction ability to an external test data set, was suggested as a template for virtual screening. Evaluation of the performances of Hypo8 and a hybrid method (Hypo81docking) in virtual screening indicated that the use of the hybrid virtual screening considerably increased the hit rate and enrichment factor. The hybrid method was therefore adopted for screening several commercially available chemical databases, including Specs, NCI, Maybridge and Chinese Nature Product Database (CNPD), for novel potent IKK2 inhibitors. The hit compounds were subsequently subjected to filtering by Lipinski's rule of five. Finally some of the final hit compounds were selected and suggested for further experimental investigations.     

Crystal structures of proto-oncogene kinase Pim1: a target of aberrant somatic hypermutations in diffuse large cell lymphoma

Pim1, a serine/threonine kinase, is involved in several biological functions including cell survival, proliferation, and differentiation. While pim1 has been shown to be involved in several hematopoietic cancers, it was also recently identified as a target of aberrant somatic hypermutation in diffuse large cell lymphoma (DLCL), the most common form of non-Hodgkin's lymphoma. The crystal structures of Pim1 in apo form and bound with AMPPNP have been solved and several unique features of Pim1 were identified, including the presence of an extra beta-hairpin in the N-terminal lobe and an unusual conformation of the hinge connecting the two lobes of the enzyme. While the apo Pim1 structure is nearly identical with that reported recently, the structure of AMPPNP bound to Pim1 is significantly different. Pim1 is unique among protein kinases due to the presence of a proline residue at position 123 that precludes the formation of the canonical second hydrogen bond between the hinge backbone and the adenine moiety of ATP. One crystal structure reported here shows that changing P123 to methionine, a common residue that offers the backbone hydrogen bond to ATP, does not restore the ATP binding pocket of Pim1 to that of a typical kinase. These unique structural features in Pim1 result in novel binding modes of AMP and a known kinase inhibitor scaffold, as shown by co-crystallography. In addition, the kinase activities of five Pim1 mutants identified in DLCL patients have been determined. In each case, the observed effects on kinase activity are consistent with the predicted consequences of the mutation on the Pim1 structure. Finally, 70 co-crystal structures of low molecular mass, low-affinity compounds with Pim1 have been solved in order to identify novel chemical classes as potential Pim1 inhibitors. Based on the structural information, opportunities for optimization of one specific example are discussed.     

Synthesis and biological testing of novel pyridoisothiazolones as histone acetyltransferase inhibitors

We present a combination of database screening, synthesis and in vitro testing to identify novel histone acetyltransferase (HAT) inhibitors. The National Cancer Institute compound collection (NCI) and several commercial databases were filtered by similarity-based virtual screening to find new HAT inhibitors. Employing the recombinant HAT p300/CBP-associated factor (PCAF) and two different histone substrates for screening, pyridoisothiazolones were identified as inhibitors of human PCAF. Due to the limited solubility of the initial hits, we synthesized and tested them on PCAF. The compounds inhibit the proliferation of cancer cells. In summary, valuable chemical tools and potential lead candidates for new anticancer agents directed against HATs as new targets have been identified.     

Structure-based virtual screening and biological evaluation of potent and selective ADAM12 inhibitors

We describe a series of potent and selective inhibitors of ADAM12 that were discovered using computational screening of a focused virtual library. The initial structure-based virtual screening selected 64 compounds from a 3D database of 67,062 molecules. Being evaluated by a cell-based ADAM12 activity assay, compounds 5, 11, 14, 16 were further identified as the potent and selective inhibitors of ADAM12 with low nanomolar IC₅₀ values. The mechanism underlying the potency and selectivity of a representative compound, 5, was investigated through molecular docking studies.     

HIV-1 integrase pharmacophore model derived from diverse classes of inhibitors

A three-dimensional pharmacophore model has been generated for HIV-1 integrase (HIV-1 IN) from known inhibitors. A dataset consisting of 26 inhibitors was selected on the basis of the information content of the structures and activity data as required by the catalyst/HypoGen program. Our model was able to predict the activity of other known HIV-1 IN inhibitors not included in the model generation, and can be further used to identify structurally diverse compounds with desired biological activity by virtual screening.     

Discovery of new GSK-3β inhibitors through structure-based virtual screening

Glycogen synthase kinase-3β (GSK-3β) is an attractive therapeutic target for human diseases, such as diabetes, cancer, neurodegenerative diseases, and inflammation. Thus, structure-based virtual screening was performed to identify novel scaffolds of GSK-3β inhibitors, and we observed that conserved water molecules of GSK-3β were suitable for virtual screening. We found 14 hits and D1 (IC₅₀ of 0.71?μM) were identified. Furthermore, the neuroprotection activity of D1–D3 was validated on a cellular level. 2D similarity searches were used to find derivatives of high inhibitory compounds and an enriched structure–activity relationship suggested that these skeletons were worthy of study as potent GSK-3β inhibitors.     

High performance screening, structural and molecular dynamics analysis to identify H1 inhibitors from TCM Database@Taiwan

New-type oseltamivir-resistant H1N1 influenza viruses have been a major threat to human health since the 2009 flu pandemic. To resolve the drug resistance issue, we aimed to identify a new type of inhibitors against H1 from traditional Chinese medicine (TCM) by employing the world's largest TCM database () for virtual screening and molecular dynamics (MD). From the virtual screening results, sodium (+)-isolaricireinol-2 alpha-sulfate, sodium 3,4-dihydroxy-5-methoxybenzoic acid methyl ester-4-sulfate, sodium (E)-7-hydroxy-1,7-bis(4-hydroxyphenyl)hept-5-ene-3S-sulfonate, and 3-methoxytyramine-betaxanthin were identified as potential drug-like compounds. MD simulation of the binding poses with the key residues Asp103 and Glu83, as well as other binding site residues, identified higher numbers of hydrogen bonds than N-Acetyl-D-Glucosamine (NAG), the natural ligand of the esterase domain in H1. Ionic bonds, salt bridges, and electrostatic energy also contribute to binding stability. Key binding residues include Lys71, Glu83, Asp103, and Arg238. Structural moieties promoting H-bond or salt bridge formations at these locations greatly contribute to a stable ligand-protein complex. An available sodium atom for ionic interactions with Asp103 can further stabilize the ligands. Based on virtual screening, MD simulation, and interaction energy evaluation, TCM candidates demonstrate good potential as novel H1 inhibitors. In addition, the identified stabilizing features can provide insights for designing highly stable H1 inhibitors.     

In silico approaches to identify novel myeloid cell leukemia-1 (Mcl-1) inhibitors for treatment of cancer

Myeloid cell leukemia-1 (Mcl-1) has been a validated and attractive target for cancer therapy. Over-expression of Mcl-1 in many cancers allows cancer cells to evade apoptosis and contributes to the resistance to current chemotherapeutics. Here, we identified new Mcl-1 inhibitors using a multi-step virtual screening approach. First, based on two different ligand-receptor complexes, 20 pharmacophore models were established by simultaneously using ‘Receptor-Ligand Pharmacophore Generation’ method and manual build feature method, and then carefully validated by a test database. Then, pharmacophore-based virtual screening (PB-VS) could be performed by using the 20 pharmacophore models. In addition, docking study was used to predict the possible binding poses of compounds, and the docking parameters were optimized before performing docking-based virtual screening (DB-VS). Moreover, a 3D QSAR model was established by applying the 55 aligned Mcl-1 inhibitors. The 55 inhibitors sharing the same scaffold were docked into the Mcl-1 active site before alignment, then the inhibitors with possible binding conformations were aligned. For the training set, the 3D QSAR model gave a correlation coefficient r² of 0.996; for the test set, the correlation coefficient r² was 0.812. Therefore, the developed 3D QSAR model was a good model, which could be applied for carrying out 3D QSAR-based virtual screening (QSARD-VS). After the above three virtual screening methods orderly filtering, 23 potential inhibitors with novel scaffolds were identified. Furthermore, we have discussed in detail the mapping results of two potent compounds onto pharmacophore models, 3D QSAR model, and the interactions between the compounds and active site residues.     

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.     

Kinase inhibitory potencies and in vitro antiproliferative activities of N-10 substituted pyrrolo[2,3-a]carbazole derivatives

Development of potent and selective Pim kinase inhibitors has recently emerged as an important field for the design of new anti-cancer drugs. We report the synthesis of new N-10-substituted pyrrolo[2,3-a]carbazole derivatives and their evaluation as Pim kinase inhibitors. Moreover, in vitro antiproliferative activity of these compounds was evaluated toward a human fibroblast primary culture and three human solid cancer cell lines (PA1, PC3 and DU145). Compounds 3, 7 and 10 showed inhibitory potencies toward Pim-1 and Pim-3 in the nanomolar range. Additionally, dimethylamino analog 10 also demonstrated interesting sub-micromolar antiproliferative activities toward the cell lines tested.     

Designing and optimization of novel human LMTK3 inhibitors against breast cancer – a computational approach

Estrogen receptor-α (ERα) is expressed more in patients with breast cancer and its level correlated with endocrine resistance. LMTK3 is reported as breast cancer target with regulation of estrogen receptor-α (ERα) through phosphorylation. In this computational study, structure-based inhibitor screening was performed on human LMTK3 using ZINC database. ATP-binding cavity with critical residues involved in the LMTK3 phosphorylation was used as target site for the screening. From the large ligand library, the best compounds were screen with three-phase virtual screening methods in Dockblaster, AutoDock Vina and AutoDock, respectively. The evaluation of ligands was carried out by binding energy and weak interactions, such as hydrogen bond interactions and hydrophobic contacts, in the target site that favors LMTK3 inhibition. Top compounds were found to be more effective in druglikeness activity by ADME prediction. The stability and binding affinity of ligand complexes were optimized by trajectory analysis such as RMSD, Rg, SASA and interhydrogen bonds from molecular dynamics simulations. The behavior of protein motion after ligand binding was illustrated by eigenvectors from principal component analysis (PCA). In addition, binding free energy of the LMTK3–ligand complexes were calculated by MM/PBSA methods and results supported the strong binding in dynamic system. Thus, the computational studies illustrated the structural insights on LMTK3 inhibition mechanism by ligands ZINC04670539, ZINC05607079 and ZINC04344028, also proposed as potent lead candidates. Our findings step towards developing novel LMTK3 inhibitors and identified lead candidates can be future breast cancer drugs with further experimental studies.     

In silico discovery and biophysical evaluation of novel 5-(2-hydroxybenzylidene) rhodanine inhibitors of DNA gyrase B

Bacterial DNA gyrase is an established and validated target for the development of novel antibacterials. In our previous work, we identified a novel series of bacterial gyrase inhibitors from the class of 4-(2,4-dihydroxyphenyl) thiazoles. Our ongoing effort was designated to search for synthetically more available compounds with possibility of hit to lead development. By using the virtual screening approach, new potential inhibitors were carefully selected from the focused chemical library and tested for biological activity. Herein we report on a novel class of 5-(2-hydroxybenzylidene) rhodanines as gyrase B inhibitors with activity in low micromolar range and moderate antibacterial activity. The binding of the two most active compounds to the enzyme target was further characterised using surface plasmon resonance (SPR) and differential scanning fluorimetry methods (DSF).     

Identification of novel chemical inhibitors for ubiquitin C-terminal hydrolase-L3 by virtual screening

UCH-L3 (ubiquitin C-terminal hydrolase-L3) is a de-ubiquitinating enzyme that is a component of the ubiquitin-proteasome system and known to be involved in programmed cell death. A previous study of high-throughput drug screening identified an isatin derivative as a UCH-L3 inhibitor. In this study, we attempted to identify a novel inhibitor with a different structural basis. We performed in silico structure-based drug design (SBDD) using human UCH-L3 crystal structure data (PDB code; 1XD3) and the virtual compound library (ChemBridge CNS-Set), which includes 32,799 chemicals. By a two-step virtual screening method using DOCK software (first screening) and GOLD software (second screening), we identified 10 compounds with GOLD scores of over 60. To address whether these compounds exhibit an inhibitory effect on the de-ubiquitinating activity of UCH-L3, we performed an enzymatic assay using ubiquitin-7-amido-4-methylcoumarin (Ub-AMC) as the substrate. As a result, we identified three compounds with similar basic dihydro-pyrrole skeletons as UCH-L3 inhibitors. These novel compounds may be useful for the research of UCH-L3 function, and in drug development for UCH-L3-associated diseases.     

Identification of novel inhibitors of HCV RNA-dependent RNA polymerase by pharmacophore-based virtual screening and in vitro evaluation

Hepatitis C virus (HCV) is the major etiological agent of non-A, non-B hepatitis where no effective treatment is available. The HCV NS5B with RNA-dependent RNA polymerase (RdRp) activity is a key target for the treatment of HCV infection. Here we report novel NS5B polymerase inhibitors identified by virtual screening and in vitro evaluation of their inhibitory activities. On the basis of a newly identified binding pocket of NS5B, distinct from the nucleotide binding site but highly conserved among various HCV isolates, we performed virtual screening of compounds that fit this binding pocket from the available chemical database of 3.5 million compounds. The inhibitory activities of the in silico selected 119 compounds were estimated with in vitro RdRp assay. Three compounds with IC50 values of about 20 μM were identified, and their kinetic analyses suggest that these compounds are noncompetitive inhibitors with respect to the ribonucleotide substrate. Furthermore, the single-point mutations of the conserved residues in the binding pocket of NS5B resulted in the significant decrease of the RdRp activity, indicating that the binding pocket presented here might be important for the therapeutic intervention of HCV. These novel inhibitors would be useful for the development of effective anti-HCV agents.