Exploring the molecular basis for selective binding of Mycobacterium tuberculosis Asp kinase toward its natural substrates and feedback inhibitors: A docking and molecular dynamics study

Tuberculosis (TB) is still a major public health problem, compounded by the human immunodeficiency virus (HIV)-TB co-infection and recent emergence of multidrug-resistant (MDR) and extensively drug resistant (XDR)-TB. In this context, aspartokinase of mycobacterium tuberculosis has drawn attention for designing novel anti-TB drugs. Asp kinase is an enzyme responsible for the synthesis of 4-phospho-L-aspartate from L-aspartate and involved in the branched biosynthetic pathway leading to the synthesis of amino acids lysine, threonine, methionine and isoleucine. An intermediate of lysine biosynthetic branch, mesodiaminopimelate is also a component of the peptidoglycan which is a component of bacterial cell wall. To interfere with the production of all these amino acids and cell wall, it is possible to inhibit Asp kinase activity. This can be achieved using Asp kinase inhibitors. In order to design novel Asp kinase inhibitors as effective anti-TB drugs, it is necessary to have an understanding of the binding sites of Asp kinase. As no crystal structure of the enzyme has yet been published, we built a homology model of Asp kinase using the crystallized Asp kinase from M. Jannaschii, as template structures (2HMF and 3C1M). After the molecular dynamics refinement, the optimized homology model was assessed as a reliable structure by PROCHECK, ERRAT, WHAT-IF, PROSA2003 and VERIFY-3D. The results of molecular docking studies with natural substrates, products and feedback inhibitors are in agreement with the published data and showed that ACT domain plays an important role in binding to ligands. Based on the docking conformations, pharmacophore model can be developed by probing the common features of ligands. By analyzing the results, ACT domain architecture, certain key residues that are responsible for binding to feedback inhibitors and natural substrates were identified. This would be very helpful in understanding the blockade mechanism of Asp kinase and providing insights into rational design of novel Asp kinase inhibitors for M.tuberculosis.     

TagSNP approach for HLA risk allele genotyping of Saudi celiac disease patients: effectiveness and pitfalls

Background: Celiac disease (CD) is a genetically complex autoimmune disease which is triggered by dietary gluten. Human leukocyte antigen (HLA) class II genes are known to act as high-risk markers for CD, where >95% of CD patients carry (HLA), DQ2 and/or DQ8 alleles. Therefore, the present study was conducted to investigate the distribution of HLA haplotypes among Saudi CD patients and healthy controls by using the tag single nucleotide polymorphisms (SNP).Methods: HLA-tag SNPs showing strong linkage value (r²>0.99) were used to predict the HLA DQ2 and DQ8 genotypes in 101 Saudi CD patients and in 103 healthy controls by using real-time polymerase chain reaction technique. Genotype calls were further validated by Sanger sequencing method.Results: A total of 63.7% of CD cases and of 60.2% of controls were predicted to carry HLA-DQ2 and DQ8 heterodimers, either in the homozygous or heterozygous states. The prevalence of DQ8 in our CD patients was predicted to be higher than the patients from other ethnic populations (35.6%). More than 32% of the CD patients were found to be non-carriers of HLA risk haplotypes as predicted by the tag SNPs.Conclusion: The present study highlights that the Caucasian specific HLA-tag SNPs would be of limited value to accurately predict CD specific HLA haplotypes in Saudi population, when compared with the Caucasian groups. Prediction of risk haplotypes by tag SNPs in ethnic groups is a good alternate approach as long as the tag SNPs were identified from the local population genetic variant databases.     

Design and evaluation of new chemotherapeutics of aloe-emodin (AE) against the deadly cancer disease: an in silico study

The Bcl-2 family proteins include pro- and antiapoptotic factors acting as critical arbiters of apoptotic cell death decisions in most circumstances. Evasion of apoptosis is one of the hallmarks of cancer, relevant to tumorigenesis as well as resistance to cytotoxic drugs, and deregulation of Bcl-2 proteins was observed in many cancers. Since Bax-mediated induction of apoptosis is a crucial mechanism in cancerous cells, we aimed at conducting in silico analysis on Bax in order to predict the possible interactions for anticancer agents. The present report depicts the binding mode of aloe-emodin and its structurally modified derivatives onto Bax. The structural information about the binding site of Bax for docked compounds obtained from this study could aid in screening and designing new anticancer agents or selective inhibitors for chemotherapy against Bax.     

In-Silico Analysis of Inflammatory Bowel Disease (IBD) GWAS Loci to Novel Connections

Genome-wide association studies (GWASs) for many complex diseases, including inflammatory bowel disease (IBD), produced hundreds of disease-associated loci—the majority of which are noncoding. The number of GWAS loci is increasing very rapidly, but the process of translating single nucleotide polymorphisms (SNPs) from these loci to genomic medicine is lagging. In this study, we investigated 4,734 variants from 152 IBD associated GWAS loci (IBD associated 152 lead noncoding SNPs identified from pooled GWAS results + 4,582 variants in strong linkage-disequilibrium (LD) (r² ≥0.8) for EUR population of 1K Genomes Project) using four publicly available bioinformatics tools, e.g. dbPSHP, CADD, GWAVA, and RegulomeDB, to annotate and prioritize putative regulatory variants. Of the 152 lead noncoding SNPs, around 11% are under strong negative selection (GERP++ RS ≥2); and ~30% are under balancing selection (Tajima’s D score >2) in CEU population (1K Genomes Project)—though these regions are positively selected (GERP++ RS <0) in mammalian evolution. The analysis of 4,734 variants using three integrative annotation tools produced 929 putative functional SNPs, of which 18 SNPs (from 15 GWAS loci) are in concordance with all three classifiers. These prioritized noncoding SNPs may contribute to IBD pathogenesis by dysregulating the expression of nearby genes. This study showed the usefulness of integrative annotation for prioritizing fewer functional variants from a large number of GWAS markers.     

Probing ligand binding modes of Mycobacterium tuberculosis MurC ligase by molecular modeling, dynamics simulation and docking

Multi drug resistance capacity for Mycobacterium tuberculosis (MDR-Mtb) demands the profound need for developing new anti-tuberculosis drugs. The present work is on Mtb-MurC ligase, which is an enzyme involved in biosynthesis of peptidoglycan, a component of Mtb cell wall. In this paper the 3-D structure of Mtb-MurC has been constructed using the templates 1GQQ and 1P31. Structural refinement and energy minimization of the predicted Mtb-MurC ligase model has been carried out by molecular dynamics. The streochemical check failures in the energy minimized model have been evaluated through Procheck, Whatif ProSA, and Verify 3D. Further torsion angles for the side chains of amino acid residues of the developed model were determined using Predictor. Docking analysis of Mtb-MurC model with ligands and natural substrates enabled us to identify specific residues viz. Gly125, Lys126, Arg331, and Arg332, within the Mtb-MurC binding pocket to play an important role in ligand and substrate binding affinity and selectivity. The availability of Mtb-MurC ligase built model, together with insights gained from docking analysis will promote the rational design of potent and selective Mtb-MurC ligase inhibitors as antituberculosis therapeutics.     

Targeted Molecular Sequencing Revealed Allelic Heterogeneity of <Emphasis Type="Italic">BRAF</Emphasis> and <Emphasis Type="Italic">PTPN11</Emphasis> Genes among Arab Noonan Syndrome Patients

Noonan syndrome (NS) is a very rare heterogenous genetic disorder often characterized by short stature, facial dysmorphisms, congenital heart defects and learning disabilities in affected children. In the current study, we sought to discover the disease causal mutations, inherited or de novo, for Noonan Syndrome among Arab patients. We screened the coding regions and splice sites of 10 known RAS/MAP Kinase pathway genes in 17 NS-trios and 100 random healthy volunteers by oilgonucleotide chip testing and Sanger sequencing methods. We found pathogenic heteroallelic de novo mutations in BRAF or PTPN11 gene in 7/17 (41.17%) of NS patients. None of the 200 chromosomes of healthy volunteers had those pathogenic mutations. Genotype-phenotype analysis showed positive correlation between BRAF and PTPN11 gene mutations and classical NS clinical manifestations. Characteristic facies is the major observed clinical manifestation among PTPN11-mutation positive cases (c.236A>G, c.854T>C, c.923A>G), whereas both characteristic facies and ectodermal manifestations are seen as dominant clinical features among BRAF-mutation positive cases (c.730A>C, c.770A>G, c.1406G>A). In addition to genotyping and clinical phenotyping, we performed computational structural analysis to examine the impact of amino acid substitution mutations on the conformation and folding of BRAF and PTPN11 proteins. Our results suggested that BRAF (c.730A>C, c.770A>G, c.1406G>A) and PTPN11 (c.236A>G, c.854T>C, c.923A>G) gene mutations elicits structural and functional alterations at protein level, which would eventually lead to dysregulation of RAS-MAPK signaling cascade, which plays critical a role in cell cycle and senescence. In conclusion, our study suggest that molecular screening of BRAF and PTPN11 genetic mutations in Arab NS patients may assist in deriving competitive outcomes related to clinical phenotyping and disease diagnosis.