Understanding the structural basis of the binding specificity of c-di-AMP to M. smegmatis RecA using computational biology approach.

Mycobacterium tuberculosis RecA (MtRecA), a protein involved in DNA repair, homologous recombination and SOS pathway, contributes to the development of multidrug resistance. ATP binding-site in RecA has been a drug target to disable RecA dependent DNA repair. For the first time, experiments have shown the existence and binding of c-di-AMP to a novel allosteric site in the C-terminal-Domain (CTD) of Mycobacterium smegmatis RecA (MsRecA), a close homolog of MtRecA. In addition, it was observed that the c-di-AMP was not binding to Escherichia coli RecA (EcRecA). This article analyses the possible interactions of the three RecA homologs with the various c-di-AMP conformations to gain insights into the structural basis of the natural preference of c-di-AMP to MsRecA and not to EcRecA, using the structural biology tools. The comparative analysis, based on amino acid composition, homology, motifs, residue types, docking, molecular dynamics simulations and binding free energy calculations, indeed, conclusively indicates strong binding of c-di-AMP to MsRecA. Having very similar results as MsRecA, it is highly plausible for c-di-AMP to strongly bind MtRecA as well. These insights from the in-silico studies adds a new therapeutic approach against TB through design and development of novel allosteric inhibitors for the first time against MtRecA.Communicated by Ramaswamy H. Sarma.

Conformational variation of site specific glycated albumin: A Molecular dynamics approach.

Human serum albumin (HSA) is a major cargo protein, which undergoes glycation in hyperglycaemic conditions and results in impaired function. In physiological conditions, HSA plays a crucial role in pharmacological activities such as drug transport or delivery through its binding capacity and also by its enzymatic activity, which enables the translation of pro-drugs into active drugs. In this study, the impact of the methylglyoxal-mediated glycation on dynamic behaviour of inter-domain motion, Cys34 reactivity, binding site residual interaction and secondary structure transition were investigated through molecular dynamics simulation. The alteration in inter-domain motion reflects the effect of glycation-mediated changes on the structural conformation of albumin. The binding site residue interactions and volume analysis revealed the impact of glycation on the geometry of the binding site. We also found the correlation of Cys34 reactivity with increase of turns in the region between Ia-h4 and Ia-h5. The rise in turn formation in that region keeps Tyr84 farther away from Cys34 which could lead to higher Cys34 reactivity. In parallel, significant alterations in alpha helical content of helices in the binding sites were observed. These structural and conformational changes in glycated albumin could be the causative agents for functional impairment which leads to diabetic complications.

S-adenosyl-l-methionine interaction signatures in methyltransferases.

The switching on or off of methylation, a change from a normal methylation to hyper or hypo methylation is implicated in many diseases that include cancers, infectious, neurodegenerative diseases and others. Methyltransferases are one of the most sought targets that have diversified for the methylation of a variety of substrates. However, without S-adenosyl-l-methionine (SAM), the universal methyl donor, the majority of the methyltransferases remain functionally inactive. In this article, we did a comprehensive analysis of all available SAM-receptor crystal structures at atom, moiety and structure levels to gain deeper insights into the structure and function of SAM. SAM demonstrated flexibility in binding to a variety of receptors irrespective of the size of the binding pockets. Further analysis of the binding pockets resulted in all SAM conformations clustering into four natural shapes. The conserved interaction analysis provides an unambiguous orientation of SAM binding to receptors which has been elusive till now. SAM peptide moiety (SPM) and SAM nucleobase moiety (SNM) show up to 89% interactions with receptors whereas only 11% interactions with SAM ribose moiety (SRM). It is found that SPM and SNM terminal atoms anchor to the highly conserved receptor subsites creating a workbench for catalysis. It is seen that every interacting atom and its position is crucial in the methyl transfer phenomenon. A very unique observation is that the methyl group of SAM does not have even one interaction with the receptor. The deep insights gained help in the design and development of novel drugs against the methyltransferases.Communicated by Ramaswamy H. Sarma.

Estimated sensitivity profiles of lung cancer specific uncommon BRAF mutants towards experimental and clinically approved kinase inhibitors.

Current experimental and clinical data are inadequate to conclusively predict the oncogenicity of uncommon BRAF mutants and their sensitivity towards kinase inhibitors. Therefore, the present study aims at estimating sensitivity profiles of uncommon lung cancer specific BRAF mutations towards clinically approved as well as experimental therapeutics based on computationally derived direct binding energies. Based on the data derived from cBioportal, BRAF mutants displayed significant mutual exclusivity with KRAS and EGFR mutants indicating them as potential drivers in lung cancer. Predicted sensitivity of BRAF-V600E conformed to published experimental and clinical data thus validating the usefulness of computational approach. The BRAF-V600K displayed higher sensitivity to most inhibitors as compared to that of the BRAF-V600E. All the uncommon mutants displayed higher sensitivity than both the wild type and BRAF-V600E towards PLX 8394 and LSN3074753. While V600K, G469R and N581S displayed favorable sensitivity profiles to most inhibitors, V600L/M, G466A/E/V and G469A/V displayed resistance profiles to a variable degree. Notably, molecular dynamic (MD) simulation revealed that increased number of interactions caused enhanced sensitivity of G469R and N581S towards sorafenib. RAF kinase inhibitors were further classified into two groups as per their selectivity (Group I: BRAF-V600E-selective and Group II: CRAF-selective) based on which potential mutation-wise combinations of RAF kinase inhibitors were proposed to overcome resistance. Based on computational inhibitor sensitivity profiles, appropriate treatment strategies may be devised to prevent or overcome secondary drug resistance in lung cancer patients with uncommon mutations.

An in silico approach to study the role of epitope order in the multi-epitope-based peptide (MEBP) vaccine design.

With different countries facing multiple waves, with some SARS-CoV-2 variants more deadly and virulent, the COVID-19 pandemic is becoming more dangerous by the day and the world is facing an even more dreadful extended pandemic with exponential positive cases and increasing death rates. There is an urgent need for more efficient and faster methods of vaccine development against SARS-CoV-2. Compared to experimental protocols, the opportunities to innovate are very high in immunoinformatics/in silico approaches, especially with the recent adoption of structural bioinformatics in peptide vaccine design. In recent times, multi-epitope-based peptide vaccine candidates (MEBPVCs) have shown extraordinarily high humoral and cellular responses to immunization. Most of the publications claim that respective reported MEBPVC(s) assembled using a set of in silico predicted epitopes, to be the computationally validated potent vaccine candidate(s) ready for experimental validation. However, in this article, for a given set of predicted epitopes, it is shown that the published MEBPVC is one among the many possible variants and there is high likelihood of finding more potent MEBPVCs than the published candidates. To test the same, a methodology is developed where novel MEBP variants are derived by changing the epitope order of the published MEBPVC. Further, to overcome the limitations of current qualitative methods of assessment of MEBPVC, to enable quantitative comparison and ranking for the discovery of more potent MEBPVCs, novel predictors, Percent Epitope Accessibility (PEA), Receptor specific MEBP vaccine potency (RMVP), MEBP vaccine potency (MVP) are introduced. The MEBP variants indeed showed varied MVP scores indicating varied immunogenicity. Further, the MEBP variants with IDs, SPVC_446 and SPVC_537, had the highest MVP scores indicating these variants to be more potent MEBPVCs than the published MEBPVC and hence should be preferred candidates for immediate experimental testing and validation. The method enables quicker selection and high throughput experimental validation of vaccine candidates. This study also opens the opportunity to develop new software tools for designing more potent MEBPVCs in less time.

16S rRNA Methyltransferases as Novel Drug Targets Against Tuberculosis.

Tuberculosis (TB) is an airborne infectious disease caused by Mycobacterium tuberculosis (M.tb) whose natural history traces back to 70,000 years. TB remains a major global health burden. Methylation is a type of post-replication, post-transcriptional and post-translational epi-genetic modification involved in transcription, translation, replication, tissue specific expression, embryonic development, genomic imprinting, genome stability and chromatin structure, protein protein interactions and signal transduction indicating its indispensable role in survival of a pathogen like M.tb. The pathogens use this epigenetic mechanism to develop resistance against certain drug molecules and survive the lethality. Drug resistance has become a major challenge to tackle and also a major concern raised by WHO. Methyltransferases are enzymes that catalyze the methylation of various substrates. None of the current TB targets belong to methyltransferases which provides therapeutic opportunities to develop novel drugs through studying methyltransferases as potential novel targets against TB. Targeting 16S rRNA methyltransferases serves two purposes simultaneously: a) translation inhibition and b) simultaneous elimination of the ability to methylate its substrates hence stopping the emergence of drug resistance strains. There are ~ 40 different rRNA methyltransferases and 13 different 16S rRNA specific methyltransferases which are unexplored and provide a huge opportunity for treatment of TB.

Designing a next generation multi-epitope based peptide vaccine candidate against SARS-CoV-2 using computational approaches.

COVID-19 caused by SARS-CoV-2 was declared a global pandemic by WHO (World Health Organization) in March, 2020. Within 6 months, nearly 750,000 deaths are claimed by COVID-19 across the globe. This called for immediate social, scientific, technological, public and community interventions. Considering the severity of infection and the associated mortalities, global efforts are underway to develop preventive measures against SARS-CoV-2. Among the SARS-CoV-2 target proteins, Spike (S) glycoprotein (a.k.a S Protein) is the most studied target known to trigger strong host immune response. A detailed analysis of S protein-based epitopes enabled us to design a novel B-cell-derived T-cell Multi-epitope-based peptide (MEBP) vaccine candidate. This involved a systematic and comprehensive computational protocol consisting of prediction of dual-purpose epitopes and designing an MEBP vaccine construct. This was followed by 3D structure validation, MEBP complex interaction studies, in silico cloning and vaccine dose-based immune response simulation to evaluate the immunogenic potency of the vaccine construct. The dual-purpose epitope prediction protocol was designed such that the same epitope elicits both humoral and cellular immune response unlike the earlier designs. Further, the epitopes predicted were screened against stringent criteria to ensure selection of a potent candidate with maximum antigen coverage and best immune response. The vaccine dose-based immune response simulation studies revealed a rapid antigen clearance through antibody generation and elevated levels of cell-mediated immunity during repeated exposure of the vaccine. The favourable results of the analysis strongly indicate that the vaccine construct is indeed a potent vaccine candidate and ready to proceed to the next steps of experimental validation and efficacy studies. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-020-02574-x.