The pathogenic effect of SNPs on structure and function of human TLR4 using a computational approach.

The human toll-like receptor (hTLR) 4 single nucleotide polymorphisms (SNPs) are interconnected with cancer, multiple genetic disorders and other immune-related diseases. The detrimental effect of SNPs in hTLR4 with respect to structure and function has not been explored in depth. The present study concatenates the biological consequences of the SNPs along with structural modifications predicted at the hTLR4 gene. A total of 7910 SNPs of hTLR4 were screened, and 21 damage-causing SNPs were identified. Out of 21, seven are present in the extracellular region, of which three were detected as deleterious and the fourth one as moderate. These three mutations are located in a highly conserved region and influence conformational change. The change leads to the widening of the Leucine-rich repeat (LRR) arc to a maximum of 16.9 Å and a minimum of 8.7 Å. Expansion/shortening of LRR arc, never discussed before, would cause loss of myeloid differentiation factor 2 (MD-2) interactions in the interior and diminish lipopolysaccharide (LPS) responses. Similarly, in all mutant structures, the binding region for HMGB1 and LPS is deflating or in an unsupportive conformation. Thus, SNPs affect the regular signaling cascade and might result in human sepsis, genetic disorders, cancer and other immunological related diseases.Communicated by Ramaswamy H. Sarma.

Structural impact of pathogenic SNPs on ß-tubulin using molecular dynamics study.

Single nucleotide polymorphisms (SNPs) in the TUBB1 (ß-tubulin) gene have been implicated as the primary cause of macro thrombocytopenia. Therefore it is essential to identify the potential SNPs which are harmful to cause diseases such as macro thrombocytopenia. The impact caused by these variants on ß-tubulin is twofold, both structural and functional. Multiple in-silico tools were used to scrutinise the most deleterious nsSNPs (non-synonymous SNPs) via sequence and structure-based approaches. Further, the ß-tubulin protein model incorporating identified mutants was subjected to MD (molecular dynamic) simulations to analyse the impact on protein structure. A total of 2974 SNPs of TUBB1 were retrieved from various sources, and 32 nsSNPs were identified. By screening through sequence-based technique, 13 variants were detected as deleterious and further structure-based filtration was carried out to find thermally destabilising variants. Finally, three variants have been detected as highly destabilising by the mCSM server and chosen for the MD study. All three variants are present in the N-terminal, Intermediate, and C-terminal regions, breaking the spatial arrangement required for microtubule assembly. The spatial arrangement of these variants is in deviation with respect to WT (wild type) ß-tubulin. The protein model was subjected to a simulation period of 100 ns. The FEL analysis revealed multiple clusters with minor populations indicating the unstable conformation adapted by the ß-tubulin. The normal mode vector analysis exhibited high-intensity flexible motions at the C-terminal end, responsible for binding with MAPs (microtubule-associated proteins), an essential region in microtubule assembly. All these results reveal that the SNP's predicted eventually influence the spatial arrangement of ß-tubulin, which would disturb the stacking arrangement of αß tubulin dimer in microtubule assembly. The present study may set a path to cure the diseases like macro thrombocytopenia.Communicated by Ramaswamy H. Sarma.

Computational investigations of indanedione and indanone derivatives in drug discovery: Indanone derivatives inhibits cereblon, an E3 ubiquitin ligase component.

BACKGROUND: Cereblon, an extensively studied multifunctional protein, is a Cullin 4-RING E3 ubiquitin ligase complex component. Cereblon is a well-known target of thalidomide and its derivatives. Cereblon is involved in multiple myeloma cell apoptosis. When ligands such as thalidomide and lenalidomide bind to cereblon, it recognizes various neosubstrates based on the ligand shape and properties. We have identified novel CRBN inhibitors, namely DHFO and its analogs, with structural features that are slightly different from thalidomide but stronger cereblon-binding affinity. We selected indanedione and indanone derivatives from the literature to understand and compare their cereblon-mediated substrate recognition potential. METHODS: Computational investigations of possible CRBN inhibitors were investigated by molecular docking with Autodock Vina and DockThor programs. The properties of the compounds' ADME/T and drug-likeness were investigated. A molecular dynamics study was carried out for four selected molecules, and the molecular interactions were analyzed using PCA-based FEL methods. The binding affinity was calculated using the MM/PBSA method. RESULTS: We conducted computational investigations on 68 indanedione and indanone derivatives binding with cereblon. Ten molecules showed better CRBN binding affinity than thalidomide. We studied the drug-likeness properties of the selected ten molecules, and four of the most promising molecules (DHFO, THOH, DIMS, and DTIN) were chosen for molecular dynamics studies. The MM/PBSA calculations showed that the DHFO, already shown to be a 5-LOX/COX2 inhibitor, has the highest binding affinity of - 163.16 kJ/mol with cereblon. CONCLUSION: The selected CRBN inhibitor DHFO has demonstrated the highest binding affinity with cereblon protein compared to other molecules. Thalidomide and its derivatives have a new substitute in the form of DHFO, which produces an interaction hotspot on the surface of the cereblon. Ease of chemical synthesis, low toxicity, versatile therapeutic options, and pleiotropism of DHFO analogs provide an opportunity for exploring clinical alternatives with versatile therapeutic potential for a new category of indanedione molecules as novel modulators of E3 ubiquitin ligases.

In Silico Characterization of B Cell and T Cell Epitopes for Subunit Vaccine Design of Salmonella typhi PgtE: A Molecular Dynamics Simulation Approach.

Typhoid fever is an acute illness in humans, caused by Salmonella typhi, a gram-negative bacterium. Outer membrane proteins of S. typhi have strong potential for its use in the development of subunit vaccine against typhoid. In the current study, peptide-based subunit vaccine was constructed from outer membrane protease E (PgtE) against S. typhi. B cell and T cell epitopes were identified at fold level with a validated three-dimensional modeled structure. T cell epitopes from PgtE (IHPDTSANY) have 99.5% binding to a maximum number of major histocompatibility complex class I and class II alleles. They also bind to the typhoid-resistant human leukocyte antigen (HLA) alleles DRB1*0401. PgtE epitopes were docked with HLA-DR4 (PDB ID: 1D5M) and a contact map was constructed. A simulation search for the binding site for full flexibility of the peptide from CABS- (Cα, Cß, side-chain)-dock shows stable interactions. Molecular dynamics simulation studies revealed that the PgtE-epitope complex structure was more stable throughout the simulation (20 ns) and interaction did not change the radius of gyration. In conclusion, computational analysis, molecular docking, and molecular dynamics (MD) simulation of PgtE-epitope complex were used to elucidate the binding mode, and the dynamical changes of epitopes were more suitable for vaccine development against typhoid.

B-cell and T-cell epitope identification with stability analysis of AI-2 import ATP-binding cassette LsrA from S. typhiIn silico approach.

Typhoid fever is a severe illness in humans, caused by Salmonella typhi, a Gram-negative bacterium. Membrane proteins of S. typhi have strong potential for its use in development of subunit vaccine against typhoid. In current study, peptide-based subunit vaccine constructed from AI-2 import ATP-binding cassette transporter protein (LsrA) against S. typhi. B-cell and T-cell epitopes were identified at fold level with validated 3-D theoretical modelled structure. T-cell epitope from LsrA (LELPGSRPQ) has binds to maximum number (82.93%) of MHC class I and class II alleles. LsrA epitope was docked with HLA-DR4 and contact map were constructed to analyze molecular interaction (docking) studies. Simulation search for the binding site for full flexibility of the peptide from CABS-dock shows the stable interactions. MD simulation analysis reveals that LsrA epitope was binding and interacting firmly with the HLA-DR4. Hence, we are proposing that LsrA epitope would be a prominent epitope vaccine for human specific pathogen of S. typhi, which requires further steps to be elevated as a vaccine drug in near future.

Understanding Russell's viper venom factor V activator's substrate specificity by surface plasmon resonance and in-silico studies.

Blood coagulation factor V (FV) is activated either by Factor X or thrombin, cleaving at three different sites viz., Site I (Arg709-Ser710), site II (Arg1018-Thr1019), and site III (Arg1545-Ser1546). Russell's viper venom factor V activator (RVV-V) is a thrombin-like serine proteinase that activates FV with selective, single cleavage at site III. A long lasting effort is being pending in understanding the 'selective' binding specificity of the RVV-V towards site III. Here, we present the binding kinetic study of RVV-V with two designed peptides corresponding to the regions from site I (Gln699-Asn713) and site II (1008Lys-Pro1022), respectively, that include 15 amino acids. Our investigation for justifying the binding efficacy and kinetics of peptides includes SPR method, protein-peptide docking, molecular dynamics simulation, and principal component analysis (PCA). Surprisingly, the SPR experiment disclosed that the Peptide II showed a lower binding affinity with KD of 2.775 mM while the Peptide I showed none. Docking and simulation of both the peptides with RVV-V engaged either rooted or shallow binding for Peptide II and Peptide I respectively. The peptide binding resulted in global conformational changes in the native fold of RVV-V, whereas the similar studies for thrombin failed to make major changes in the native fold. In support, the PCA analysis for RVV-V showed the dislocation of catalytic triad upon binding both the peptides. Hence, RVV-V, a serine protease, is incompetent in cleaving these two sites. This study suggests a transition in RVV-V from the native rigid to the distorted flexible structure and paves a way to design a new peptide substrate/inhibitor.