Sunsetting Binding MOAD with its last data update and the addition of 3D-ligand polypharmacology tools.

Binding MOAD is a database of protein-ligand complexes and their affinities with many structured relationships across the dataset. The project has been in development for over 20 years, but now, the time has come to bring it to a close. Currently, the database contains 41,409 structures with affinity coverage for 15,223 (37%) complexes. The website BindingMOAD.org provides numerous tools for polypharmacology exploration. Current relationships include links for structures with sequence similarity, 2D ligand similarity, and binding-site similarity. In this last update, we have added 3D ligand similarity using ROCS to identify ligands which may not necessarily be similar in two dimensions but can occupy the same three-dimensional space. For the 20,387 different ligands present in the database, a total of 1,320,511 3D-shape matches between the ligands were added. Examples of the utility of 3D-shape matching in polypharmacology are presented. Finally, plans for future access to the project data are outlined.

Exploring the different states of wild-type T-cell receptor and mutant conformational changes towards understanding the antigen recognition.

Recognition of proteolytic peptide fragments presented by major histocompatibility complex (MHC) on target cells by T-cell receptor (TCR) is among the most important interactions in the adaptive immune system. Several computational studies have been performed to investigate conformational and dynamical properties of TCRs for enhanced immunogenicity. Here, we present the large-scale molecular dynamics (MD) simulation studies of the two comprehensive systems consisting of the wild-type and mutant IG4 TCR in complex with the tumor epitope NY-ESO peptide (SLLMWITQC) and analyzed for mapping conformational changes of TCR in the states prior to antigen binding, upon antigen binding and after the antigen was released. All of the simulations were performed with different states of TCRs for each 1000 ns of simulation time, providing six simulations for time duration of 6000 ns (6µs). We show that rather than undergoing most critical conformational changes upon antigen binding, the high proportion of complementarity-determining region (CDR) loops change by comparatively small amount. The hypervariable CDRα3 and CDRß3 loops showed significant structural changes. Interestingly, the TCR ß chain loops showed the least changes, which is reliable with recent implications that ß domain of TCR may propel antigen interaction. The mutant shows higher rigidity than wild-type even in released state; expose an induced fit mechanism occurring from the re-structuring of CDRα3 loop and can allow enhanced binding affinity of the peptide antigen. Additionally, we show that CDRα3 loop and peptide contacts are an adaptive feature of affinity enhanced mutant TCR.Communicated by Ramaswamy H. Sarma.

Genetic mutational analysis of ß-catenin gene affecting GSK-3ß phosphorylation plays a role in gallbladder carcinogenesis: Results from a case control study.

This manuscript has reported different mutations of ß-catenin gene in gallbladder cancer patients which affect GSK-3ß phosphorylation site. PURPOSE: Gallbladder carcinoma (GBC) is a relatively rare and fatal cancer with poor prognosis. The molecular mechanism of gallbladder carcinogenesis is still not clear. Wnt signaling pathway is a highly conserved pathway that regulates proliferation, differentiation, migration, genetic stability, apoptosis, and stem cell renewal. ß-catenin plays major role in Wnt signaling and aberrations in ß-catenin has found to be involved in several cancers pathogenesis. This study was carried out to document the mutations of ß-catenin gene in gallbladder cancer and to evaluate its possible role in gallbladder carcinogenesis. METHODS: PCR-SSCP (Single Stranded Conformation Polymorphism) for ctnnb1 was performed in 50 patients each of gallbladder cancer, cholelithiasis and 50 healthy controls. Samples that showed variation in banding pattern were sequenced. RESULTS: Variation in banding pattern was observed in 9 (18%) samples of GBC, 4 (8%) of cholelithiasis and 2 (4%) of control. Sequencing analysis showed 9 novel mutations of ctnnb1 in exon 3 in 18% of gallbladder cancer (χ2 = 5.778; p < 0.05). Six point mutations, 1 deletion and 1 insertion mutation were found in 9 cases of gallbladder cancer. All point mutations were mis-sense mutation that affected highly conserved serine or threonine region that is important for GSK-3ß phosphorylation. CONCLUSION: Findings of the study suggests that high frequency of non synonymous mutations of ß-catenin gene (ctnnb1) occurs in patients with gallbladder cancer. As these mutations mainly effect GSK 3ß phosphorylation, it may be concluded that this might be an important step in gallbladder carcinogenesis. These ß-catenin mutations lead to Wnt pathway activation and appear to have a role in progression from inflammation to cancer in gallbladder.

Structural insights into the lipid transfer mechanism of a non-specific lipid transfer protein.

The non-specific lipid transfer proteins (nsLTPs) are multifunctional seed proteins engaged in several different physiological processes. The nsLTPs are stabilized by four disulfide bonds and exhibit a characteristic hydrophobic cavity, which is the primary lipid binding site. While these proteins are known to transfer lipids between membranes, the mechanism of lipid transfer has remained elusive. Four crystal structures of nsLTP from Solanum melongena, one in the apo-state and three myristic acid bound states were determined. Among the three lipid bound states, two lipid molecules were bound on the nsLTP surface at different positions and one was inside the cavity. The lipid-dependent conformational changes leading to opening of the cavity were revealed based on structural and spectroscopic data. The surface-bound lipid represented a transient intermediate state and the lipid ultimately moved inside the cavity through the cavity gate as revealed by molecular dynamics simulations. Two critical residues in the loop regions played possible 'gating' role in the opening and closing of the cavity. Antifungal activity and membrane permeabilization effect of nsLTP against Fusarium oxysporum suggested that it could possibly involve in bleaching out the lipids. Collectively, these studies support a model of lipid transfer mechanism by nsLTP via intermediate states.

Variations in macrophage migration inhibitory factor gene are not associated with visceral leishmaniasis in India.

BACKGROUND: The host genetic factors play important role in determining the outcome of visceral leishmaniasis (VL). Macrophage migration inhibitory factor (MIF) is an important host cytokine, which is a key regulator of innate immune system. Genetic variants in MIF gene have been found to be associated with several inflammatory and infectious diseases. Role of MIF is well documented in leishmaniasis diseases, including Indian visceral leishmaniasis, where elevated level of serum MIF has been associated with VL phenotypes. However, there was no genetic study to correlate MIF variants in VL, therefore, we aimed to study the possible association of three reported MIF gene variants -794 CATT, -173G > C and non-coding RNA gene LOC284889 in Indian VL phenotype. METHODS: Study subjects comprised of 214 VL patients along with ethnically and demographically matched 220 controls from VL endemic regions of Bihar state in India. RESULTS: We found no significant difference between cases and controls in allelic, genotypic and haplotype frequency of the markers analysed [-794 CATT repeats (χ2=0.86; p=0.35; OR=0.85; 95% CI=0.61-1.19); -173 G>C polymorphism (χ2=1.11; p=0.29; OR=0.83; 95% CI=0.59-1.16); and LOC284889 (χ2=0.78; p=0.37; OR=0.86; 95% CI=0.61-1.20)]. CONCLUSION: Since we did not find any significant differences between case and control groups, we conclude that sequencing of complete MIF gene and extensive study on innate and adaptive immunity genes may help in identifying genetic variations that are associated with VL susceptibility/resistance among Indians.

Comparative Analysis of Mutational Profile of Sonic hedgehog Gene in Gallbladder Cancer.

BACKGROUND: Gallbladder cancer has high incidence in northeastern India; mortality too is high as the disease is often diagnosed late. Numerous studies have shown the role of sonic hedgehog (shh) in different cancers, an important ligand of the hedgehog signaling pathway. AIM: This study was carried out to evaluate the shh gene mutations in gallbladder cancer patients. METHODS: PCR-SSCP was performed for shh gene in 50 samples each of gallbladder cancer, cholelithiasis, and control. The samples showing aberration in banding pattern were sequenced. RESULTS: Variation in banding pattern was observed in 20% gallbladder cancer cases, 10% in cholelithiasis, and none of the control (χ 2 = 11.111; p < 0.05). Sequencing results revealed seven novel point mutations in GBC cases. These novel mutations were found to be associated with histopathology (p < 0.05) and stage (p < 0.05) of gallbladder cancer. CONCLUSION: This study reveals several novel individual and repetitive mutations of shh gene in GBC and cholelithiasis samples that may be used as diagnostic markers for gallbladder carcinogenesis.

Protein-Protein Interaction for the De Novo Design of Cyclin-Dependent Kinase Peptide Inhibitors.

The homology of the inhibitor binding site regions on the surface of cyclin-dependent kinases (CDKs) makes actual CDK inhibitors unable to bind specifically to their molecular targets. Most of them are ATP competitive inhibitors with low specificity that also affect the phosphorylation mechanisms of other nontarget kinases giving rise to harmful side effects. So, the search of specific and potent inhibitors able to bind to the desired CDK target is still a pending issue. Structure based drug design minimized the erroneous binding and increased the affinity of the inhibitor interaction. In the case of CDKs their activation and regulation mechanisms mainly depend on protein-protein interactions (PPIs). The design of drugs targeting these PPIs makes feasible and promising towards the discovery of new and specific CDK inhibitors. Development of peptide inhibitors for a target protein is an emerging approach in computer aided drug designing. This chapter describes in detail methodology for use of the VitAL-Viterbi algorithm for de novo peptide design of CDK2 inhibitors.

Fragment-Based De Novo Design of Cyclin-Dependent Kinase 2 Inhibitors.

Cyclin-dependent kinases (CDKs) are core components of the cell cycle machinery that govern the transition between phases during cell cycle progression. Abnormalities in CDKs activity and regulation are common features of cancer, making CDK family members attractive targets for the development of anticancer drugs. One of the main bottlenecks hampering the development of drugs for kinase is the difficulty to attain selectivity. A huge variety of small molecules have been reported as CDK inhibitors, as potential anticancer agents, but none of these has been approved for commercial use. Computer-based molecular design supports drug discovery by suggesting novel new chemotypes and compound modifications for lead candidate optimization. One of the methods known as de novo ligand design technique has emerged as a complementary approach to high-throughput screening. Several automated de novo software programs have been written, which automatically design novel structures to perfectly fit in known binding site. The de novo design supports drug discovery assignments by generating novel pharmaceutically active agents with desired properties in a cost as well as time efficient approach. This chapter describes procedure and an overview of computer-based molecular de novo design methods on a conceptual level with successful examples of CDKs inhibitors.

Targeting the cyclin-binding groove site to inhibit the catalytic activity of CDK2/cyclin A complex using p27(KIP1)-derived peptidomimetic inhibitors.

Functionally activated cyclin-dependent kinase 2 (CDK2)/cyclin A complex has been validated as an interesting therapeutic target to develop the efficient antineoplastic drug based on the cell cycle arrest. Cyclin A binds to CDK2 and activates the kinases as well as recruits the substrate and inhibitors using a hydrophobic cyclin-binding groove (CBG). Blocking the cyclin substrate recruitment on CBG is an alternative approach to override the specificity hurdle of the currently available ATP site targeting CDK2 inhibitors. Greater understanding of the interaction of CDK2/cyclin A complex with p27 (negative regulator) reveals that the Leu-Phe-Gly (LFG) motif region of p27 binds with the CBG site of cyclin A to arrest the malignant cell proliferation that induces apoptosis. In the present study, Replacement with Partial Ligand Alternatives through Computational Enrichment (REPLACE) drug design strategies have been applied to acquire LFG peptide-derived peptidomimetics library. The peptidomimetics function is equivalent with respect to substrate p27 protein fashion but does not act as an ATP antagonist. The combined approach of molecular docking, molecular dynamics (MD), and molecular electrostatic potential and ADME/T prediction were carried out to evaluate the peptidomimetics. Resultant interaction and electrostatic potential maps suggested that smaller substituent is desirable at the position of phenyl ring to interact with Trp217, Arg250, and Gln254 residues in the active site. The best docked poses were refined by the MD simulations which resulted in conformational changes. After equilibration, the structure of the peptidomimetic and receptor complex was stable. The results revealed that the various substrate protein-derived peptidomimetics could serve as perfect leads against CDK2 protein.

Homology modeling, molecular dynamics, and docking studies of pattern-recognition transmembrane protein-lipopolysaccharide and ß-1,3 glucan-binding protein from Fenneropenaeus indicus.

Lipopolysaccharide and ß-1,3 glucan-binding protein (LGBP) is a family of pattern-recognition transmembrane proteins (PRPs) which plays a vital role in the immune mechanism of crustaceans in adverse conditions. Fenneropenaeus indicus LGBP-deduced amino acid has conserved potential recognition motif for ß-1,3 linkages of polysaccharides and putative RGD (Arg-Gly-Asp) cell adhesion sites for the activation of innate defense mechanism. In order to understand the stimulating activity of ß-1,3 glucan (ß-glucan) and its interaction with LGBP, a 3D model of LGBP is generated. Molecular docking is performed with this model, and the results indicate Arg71 with strong hydrogen bond from RGD domain of LGBP. Moreover, from the docking studies, we also suggest that Arg34, Lys68, Val135, and Ala146 in LGBP are important amino acid residues in binding as they have strong bonding interaction in the active site of LGBP. In our in vitro studies, yeast agglutination results suggest that shrimp F. indicus LGBP possesses sugar binding and recognition sites in its structure, which is responsible for agglutination reaction. Our results were synchronized with the already reported evidence both in vivo and in vitro experiments. This investigation may be valuable for further experimental investigation in the synthesis of novel immunomodulator.

Insights into the structural basis of 3,5-diaminoindazoles as CDK2 inhibitors: prediction of binding modes and potency by QM-MM interaction, MESP and MD simulation.

The novel 3,5-diaminoindazole derivatives are well-known as potent and anti-proliferative cyclin-dependent kinase 2 inhibitors. We report a combined quantum mechanics/molecular mechanics study to determine the protein-ligand interaction energy, and some quantum chemical descriptors to successfully rank these inhibitors. The results in this work show that the QM-MM interaction energy is strongly correlated to the biological activity and can be used as a predictor, which was further validated by Spearman's rank correlation coefficient. An exhaustive analysis of the protein-ligand structures obtained from molecular dynamics simulations shows specific interactions within the active site. Furthermore, the docking study was supported by electronic property analysis using density functional theory at the B3LYP/3-21*G level. The results obtained from molecular docking and surface analysis shed some insight on steric and electronic complementarities of these molecules to CDK2. Aqueous solvation energy values give an indication of the solubility and can be used as a guide for the pharmacokinetic optimization of these molecules. Furthermore, ADME/T properties calculated are in the desirable range, so these compounds are predicted to be drug like with low toxicity potential. Overall, the approach was successful in the cases considered, and it could be useful for the design of inhibitors in the lead optimization phase of drug discovery against CDK2.

Temporal variations in basicranium dimorphism of North Indians.

In recent years, several investigations have documented notable secular changes in human skull morphology. The present study is the next link in the series, addressing its possible effects in the cranial base in North Indian populations. The present study aims to examine the sexual dimorphism in cranial base of two temporally different samples of North Indian population to provide updated population specific osteometric standards. The contemporary and the subrecent samples are comprised of 158 (M 110; F 48) and 325 adult crania (M 206; F 119), respectively. Five variables of cranial base were measured, and data were subjected to discriminant function analysis using SPSS 16.0. The t-tests between the two temporally different samples showed significant sexual dimorphism and population variation. Contemporary females showed comparatively greater dimensions than subrecent. Contemporary population exhibited less sexual dimorphism than subrecent population. Factors associated with the changes in sexual dimorphism are likely to be a consequence of improved nutrition and population admixture in last 50 years. The accuracy of sex prediction ranged from 48.1 to 70.0 % in contemporary sample and 60.9 % to 77.2 % in subrecent sample. Overall, these findings support the conclusion that sexual dimorphism shows variation within few decades, so sex discriminating osteometric standards must be updated regularly.

Extra precision docking, free energy calculation and molecular dynamics simulation studies of CDK2 inhibitors.

Molecular docking, free energy calculation and molecular dynamics (MD) simulation studies have been performed, to explore the putative binding modes of 3,5-diaminoindazoles, imidazo(1,2-b)pyridazines and triazolo(1,5-a) pyridazines series of Cyclin-dependent kinase (CDK2) inhibitors. To evaluate the effectiveness of docking protocol in flexible docking, we have selected crystallographic bound compound to validate our docking procedure as evident from root mean square deviations (RMSDs). We found different binding sites namely catalytic, inhibitory phosphorylation, cyclin binding and CKS-binding site of the CDK2 contributing towards the binding of these compounds. Moreover, correlation between free energy of binding and biological activity yielded a statistically significant correlation coefficient. Finally, three representative protein-ligand complexes were subjected to molecular dynamics simulation to determine the stability of the predicted conformations. The low value of the RMSDs between the initial complex structure and the energy minimized final average complex structure suggests that the derived docked complexes are close to equilibrium. We suggest that the phenylacetyl type of substituents and cyclohexyl moiety make the favorable interactions with a number of residues in the active site, and show better inhibitory activity to improve the pharmacokinetic profile of compounds against CDK2. The structure-based drug design strategy described in this study will be highly useful for the development of new inhibitors with high potency and selectivity.

Shape and pharmacophore-based virtual screening to identify potential cytochrome P450 sterol 14α-demethylase inhibitors.

Sterol 14α-demethylase (CYP51) is a cytochrome P450 heme thiolate containing enzyme involved in biosynthesis of membrane sterols, including sterol in animals, ergosterol in fungi, and a variety of C24-modified sterols in plants and protozoa. Several clinical drugs have been developed to reduce the impact of fungal diseases, but their clinical uses have been limited by the emergence of drug resistance and insufficiencies in their antifungal activity. Therefore, in order to identify potential CYP51 inhibitors, we have implemented a virtual screening (VS) protocol by using both phase shape and pharmacophore model (AHHRR) against Asinex, ChemBridge and Maybridge databases. A filtering protocol, including Lipinski filter, number of rotatable bonds and different precisions of molecular docking was applied in hits selection. The results indicated that both shape-based and pharmacophore-based screening yielded the best result with potential inhibitors. The searched compounds were also evaluated with ADME properties, which show excellent pharmacokinetic properties under the acceptable range. We identified potential CYP51 inhibitors for further investigation, they could also be employed to design ligands with enhanced inhibitory potencies and to predict the potencies of analogs to guide synthesis/or prepare synthetic antifungal analogs against CYP51.

Sexual dimorphism in ulna: an osteometric study from India.

Determination of sex constitutes the most important element during the identification process of human skeletal remains. Several sex-specific features of human skeleton have been exploited for sex determination with varying reliability. This study aims to obtain sexual dimorphic standards for ulnae of the north Indian population. Eight measurements were obtained on a sample of 106 ulnae (males--80, females--26) in the age range of 25-65 years. The sexual dimorphism index and demarking points were calculated for all the variables. The data were then subjected to stepwise and direct discriminant function analysis. The best discriminator of sex was the maximum length (84.9%) followed by radial notch width (84%). In stepwise analysis, these two variables were selected and provided an accuracy of 88.7% (M-87.5%, F-92.3%). The proximal end provided a classification rate of 81.1% (M-80%, F-84.6%) with selection of the notch length and olecranon width.

Exploring the selectivity of a ligand complex with CDK2/CDK1: a molecular dynamics simulation approach.

Cyclin-dependent kinases (CDKs) are core components of the cell cycle machinery that govern the transition between phases during cell cycle progression. Abnormalities in CDKs activity and regulation are common features of cancer, making CDK family members attractive targets for the development of anticancer drugs. Their inhibitors have entered in clinical trials to treat cancer. Very recently, Heathcote et al. (J. Med. Chem. 2010, 53:8508-8522) have found a ligand BS194 that has a high affinity with CDK2 (IC(50) = 3 nM) but shows low affinity with CDK1 (IC(50) = 30 nM). To understand the selectivity, we used homology modeling, molecular docking, molecular dynamics, and free-energy calculation to analyze the interactions. A rational three-dimensional model of the CDK1/BS194 complex is built. We found that Leu83 is a key residue that recognizes BS194 more effectively with CDK2 with good binding free energies rather than CDK1. Energetic analysis reveals that van der Waals interaction and non-polar contributions to solvent are favorable in the formation of complexes and amine group of the ligand, which plays a crucial role for binding selectivity between CDK2 and CDK1.

Pharmacophore modelling and atom-based 3D-QSAR studies on N-methyl pyrimidones as HIV-1 integrase inhibitors.

Pharmacophore modelling and atom-based 3D-QSAR studies were carried out for a series of compounds belonging to N-methyl pyrimidones as HIV-1 integrase inhibitors. Based on the ligand-based pharmacophore model, we got 5-point pharmacophore model AADDR, with two hydrogen bond acceptors (A), two hydrogen bond donors (D) and one aromatic ring (R). The generated pharmacophore-based alignment was used to derive a predictive atom-based 3D-QSAR model for the training set (r(2) = 0.92, SD = 0.16, F = 84.8, N = 40) and for test set (Q(2) = 0.71, RMSE = 0.06, Pearson R = 0.90, N = 10). From these results, AADDR pharmacophore feature was selected as best common pharmacophore hypothesis, and atom-based 3D-QSAR results also support the outcome by means of favourable and unfavourable regions of hydrophobic and electron-withdrawing groups for the most potent compound 30. These results can be useful for further design of new and potent HIV-1 IN inhibitors.

Isolation of Salmonella typhi from apparently healthy liver.

It is suggested that Salmonella typhi resides mostly in hepatobiliary system especially in gallbladder in chronic typhoid carriers. It is not very clear whether in gallbladder lumen or on its wall or in liver. However, we had been successful in detecting S. typhi in liver by PCR targeting flagellin gene sequences. Therefore, in the present study, we tried to isolate the bacterium from liver tissue collected from dead bodies brought for post mortem examination. We could isolate S. typhi in 2 of 20 such liver tissues examined by using conventional isolation techniques. The isolates were identified by routine phenotypic characters and were confirmed by amplification and sequencing of two conserved genes i.e. 16S rDNA and flagellin (fliC) gene followed by blasting on www.ncbi.nlm.nih.gov.

Sex determination using mandibular ramus flexure: a preliminary study on Indian population.

Determination of sex by morphological assessment has been one of the oldest approaches in forensic anthropology. Loth and Henneberg(6) introduced a morphological trait "Mandibular Ramus Flexure" for sex identification with a high accuracy of 99% in African Blacks. However, the population specificity of sexually dimorphic features is well known. The purpose of this study is to test the reliability of this trait in Indian population. A total number of 112 adult mandibles (88 males and 24 females) were studied from the Department of Forensic Medicine, Institute of Medical Sciences, Banaras Hindu University, India. The mandibles were scored according to the original method by two observers in three different sessions. This was done to test inter and intra-observer errors in identifying the trait. The result shows that this trait can be used to diagnose sex with an average accuracy of upto 82%. Though, inter and intra-observer errors were present but could be minimized with extended practice. So, the trait has the potential to be relied upon as a single morphological trait for determination of sex in Indian population.