RankProt: A multi criteria-ranking platform to attain protein thermostabilizing mutations and its in vitro applications - Attribute based prediction method on the principles of Analytical Hierarchical Process.

Attaining recombinant thermostable proteins is still a challenge for protein engineering. The complexity is the length of time and enormous efforts required to achieve the desired results. Present work proposes a novel and economic strategy of attaining protein thermostability by predicting site-specific mutations at the shortest possible time. The success of the approach can be attributed to Analytical Hierarchical Process and the outcome was a rationalized thermostable mutation(s) prediction tool- RankProt. Briefly the method involved ranking of 17 biophysical protein features as class predictors, derived from 127 pairs of thermostable and mesostable proteins. Among the 17 predictors, ionic interactions and main-chain to main-chain hydrogen bonds were the highest ranked features with eigen value of 0.091. The success of the tool was judged by multi-fold in silico validation tests and it achieved the prediction accuracy of 91% with AUC 0.927. Further, in vitro validation was carried out by predicting thermostabilizing mutations for mesostable Bacillus subtilis lipase and performing the predicted mutations by multi-site directed mutagenesis. The rationalized method was successful to render the lipase thermostable with optimum temperature stability and Tm increase by 20°C and 7°C respectively. Conclusively it can be said that it was the minimum number of mutations in comparison to the number of mutations incorporated to render Bacillus subtilis lipase thermostable, by directed evolution techniques. The present work shows that protein stabilizing mutations can be rationally designed by balancing the biophysical pleiotropy of proteins, in accordance to the selection pressure

Nature to Nurture- Identifying Phytochemicals from Indian Medicinal Plants as Prophylactic Medicine by Rational Screening to Be Potent Against Multiple Drug Targets of SARS-CoV-2

The COVID-19 pandemic resulted in millions of people being quarantined, impacting the world economy and health sector. There is no existing proven treatment for this disease. It may takea long time until a good candidate vaccine or a potent drug is made available in the market. Therefore, there is a need to search for alternative therapy. In the context, this work explored natural compounds from Indian medicinal plants to develop a prophylactic treatment regimen that will be instrumentalin controlling the spread of the deadly virus. In this work 1916 phytochemicals from 55 Indian medicinal plants, reported to possess anti-viral properties, were subjected to virtual screening on 8 structural and non-structural SARS-CoV-2 protein targets. Docking interactions, ADME and toxicity profiles of the 66 screened phytochemicals were correlated with 21 repurposed drugs that have been most cited in literature to be effective against SARS-CoV-2. Steroidal lactones from Withaniasomnifera and triterpenoids from Azadirachtaindica- with docking score ranging from -13 kcal/mol upto -6 kcal/mol were identified to occupy the top scoring virtually screened phytochemicals against the various targets of SARS-CoV-2. Importantly this work proposes that a concoction of these phytochemicals can act as prophylactic anti-viral medicine to control the spread of SARS-CoV-2 and also enhance natural immunity as the first line of defence towards such a deadly virus

High Throughput in Silico Identification of Novel Phytochemical Inhibitors for the Master Regulator of Inflammation (TNFα)

The over expression of Tumor necrosis factor-α (TNFα) has been implicated in a variety of disease and is classified as a therapeutic target for inflammatory diseases (Crohn disease, psoriasis, psoriatic arthritis, rheumatoid arthritis).Commercially available therapeutics are biologics which are associated with several risks and limitations. Small molecule inhibitors and natural compounds (saponins) were identified by researchers as lead molecules against TNFα, however, they were often associated with high IC50 values which can lead to their failure in clinical trials. This warrants research related to identification of better small molecule inhibitors by screening of large compound libraries. Recent developments have demonstrated power of natural compounds as safe therapeutics, hence, in this work, we have identified TNFα phytochemical inhibitors using high throughput in silico screening approaches of 6000 phytochemicals followed by 200 ns molecular dynamics simulations and relative binding free energy calculations. The work yielded potent hits that bind to TNFα at its dimer interface. The mechanism targeted was inhibition of oligomerization of TNFα upon phytochemical binding to restrict its interaction with TNF-R1 receptor. MD simulation analysis resulted in identification of two phytochemicals that showed stable protein-ligand conformations over time. The two compounds were triterpenoids: Momordicilin and Nimbolin A with relative binding energy- calculated by MM/PBSA to be -190.5 kJ/Mol and -188.03 kJ/Mol respectively. Therefore, through this work it is being suggested that these phytochemicals can be used for further in vitro analysis to confirm their inhibitory action against TNFα or can be used as scaffolds to arrive at better drug candidates.</p

Natures Therapy for COVID-19: Targeting the Vital Non-Structural Proteins (NSP) from SARS-CoV-2 with Phytochemicals from Indian Medicinal Plants

Containing COVID-19 is still a global challenge. It has affected the "normal" world bytargeting its economy and health sector. Research is more focused on finding a cure to thisdisease and is less concerned about other life threatening diseases like cancer. Thus we need todevelop a medical solution at the earliest. In this context the present work aimed to understandthe efficacy of 22 rationally screened phytochemicals from Indian medicinal plants obtainedfrom our previous work, following drug-likeness properties, against 6 non-structural-proteins(NSP) from SARS-CoV-2. 100 ns molecular dynamics simulations were performed andrelative binding free energies were computed by MM/PBSA. Further, principal componentanalysis, dynamic cross correlation and hydrogen bond occupancy were analyzed tocharacterize protein–ligand interactions. Biological pathway enrichment analysis was alsocarried out to elucidate the therapeutic targets of the phytochemicals in comparison to SARS-CoV-2. The potential binding modes and favourable molecular interaction profile of 9phytochemicals, majorly from Withania sominifera with lowest free binding energies, againstthe SARS-CoV-2 NSP targets were identified. It was understood that phytochemicals andrepurposed drugs with steroidal moieties in their chemical structures formed stable interactionswith the NSPs. Additionally, human target pathway analysis for SARS-CoV-2 andphytochemicals showed that cytokine mediated pathway and phosphorylation pathways werewith the most significant p-value. To summarize this work, we suggest a global approach oftargeting multiple proteins of SARS-CoV-2 with phytochemicals as a natural alternativetherapy for COVID-19. We also suggest that these phytochemicals need to be testedexperimentally to confirm their efficacy

Free Energy Landscapes and Residue Network Analysis for Six SARS-CoV-2 Targets in Complex with Plausible Phytochemical Inhibitors from Withania Somnifera: 1 µS Molecular Dynamics Simulations

The pandemic is here to stay- evident from the second wave that is severely affecting global population. Though vaccination is now available, the population size restricts its efficacy, especially in the third world countries. Therefore, to avoid a third wave, natural preventive therapeutics are the need of the hour. In this work the efficiency of phytochemicals from Withania somnifera to bind to a total of six SARS-CoV-2 targets have been shown.1 µs molecular dynamics simulations and essential dynamic analyses shed light on the changes induced by the phytochemicals and highlights their multipotent capabilities- 27-Hydroxywithanolide B was able to bind to three targets. Relative free energy of binding for all the phytochemicals were calculated by MM/PBSA. Minimum energy structures were extracted from their free energy landscapes and were subjected to PSN-ENM-NMA and network centrality analysis. Results showed that the phytochemical binding changes the residue-residue interaction network. Network communities increase while hubs and links decrease. Metapath rewiring occurs through residues Phe456 in spike protein, Thr26 and Tyr118 in main protease, Val49 and Phe156 in NSP3, Leu98 in NSP9, Leu4345 in NSP10, Phe440 and Phe843 in NSP12. This work tries to understand the mechanism of possible inhibition by the phytochemicals to combat SARS-CoV-2 with their capability of targeting multiple proteins. The insight from this study can be of great relevance to explore the changes in network properties induced by reported potential inhibitors against SARS-CoV-2 targets

The Natural Way Forward: Molecular Dynamics Simulation Analysis of Phytochemicals from Indian Medicinal Plants as Potential Inhibitors of SARS-CoV-2 Targets

The natural way forward: Molecular dynamics simulation analysis of phytochemicals from Indian medicinal plants as potential inhibitors of SARS-CoV-2 targetsPratap Kumar Parida 1#, Dipak Paul 1#, Debamitra Chakravorty 2*# 1 Noor Enzymes Private Limited, 37-B, Darga Road, Kolkata - 700 017, India2 Novel Techsciences (OPC) Private Limited, 37-B, Darga Road, 1st Floor, Kolkata - 700 017, India * Corresponding author:Debamitra Chakravorty, PhD (Project Lead - Computational Biology)Novel Techsciences (OPC) Private Limited, 37-B, Darga Road, 1st Floor, Kolkata - 700 017, IndiaE-mail: [email protected]#All the authors have contributed equally to the paper.AbstractThe pandemic COVID-19 has become a global panic and health issue forcing our lives towards a compromised "new normal". Research is still ongoing to develop effective antiviral drugs and vaccines against SARS-CoV-2. Thus, to address the current outbreak, development of natural inhibitors as a prophylactic measure is an attractive strategy due to their natural diversity and safety. Phytochemicals that target viral entry (Spike glycoprotein) and replication (3CLPro) are lucrative in terms of both economy and health for the treatment of the deadly virus. In this context, this work explored natural compounds from Indian medicinal plants as potential inhibitors for containing the spread SARS-CoV-2. The phytochemicals were rationally screened from 55 Indian medicinal plants in our previous work. All atom 100 ns molecular dynamics simulations were performed using high performance computing for 8 top scoring rationally screened phytochemicals from Withania somnifera and Azadirachta indica and two repurposed drugs against the spike glycoprotein and the main protease of SARS-CoV-2. MM/PBSA, Principal component analysis and hydrogen bond occupancy were analysed to characterize protein–ligand interactions and to find the binding free energy. Biological pathway enrichment analysis was also carried out to observe the therapeutic efficacy of these phytochemicals. The results revealed that Withanolide R (-141.96 KJ/Mol) and 2,3-Dihydrowithaferin A (-87.60 KJ/Mol) were with the lowest relative free energy of binding for main protease and the spike proteins respectively. It was also observed that the phytochemicals exhibit a remarkable multipotency with the ability to modulate various human biological pathways especially pathways in cancer. Conclusively we suggest that these compounds need further detailed in vivo experimental evaluation and clinical validation for implementation as potent therapeutic agent for combating SARS-CoV-2.</p

Insights on the disruption of the complex between human positive coactivator 4 and p53 by small molecules

Interaction between human positive coactivator 4 (PC4), an abundant nuclear protein, and the tumor suppressor protein p53 plays a crucial role in initiating apoptosis. In certain neurodegenerative diseases PC4 assisted-p53-dependent apoptosis may play a central role. Thus, disruption of p53-PC4 interaction may be a good drug target for certain disease pathologies. A p53-derived short peptide (AcPep) that binds the C-terminal domain of PC4 (C-PC4) is known to disrupt PC4-p53 interaction. To fully characterize its binding mode and binding site on PC4, we co-crystallized C-PC4 with the peptide and determined its structure. The crystal, despite exhibiting mass spectrometric signature of the peptide, lacked peptide electron density and showed a novel crystal lattice, when compared to C-PC4 crystals without the peptide. Using peptide-docked models of crystal lattices, corresponding to our structure and the peptide-devoid structure we show the origin of the novel crystal lattice to be dynamically bound peptide at the previously identified putative binding site. The weak binding is proposed to be due to the lack of the N-terminal domain of PC4 (N-PC4), which we experimentally show to be disordered with no effect on PC4 stability. Taking cue from the structure, virtual screening of ∼18.6 million small molecules from the ZINC15 database was performed, followed by toxicity and binding free energy filtering. The novel crystal lattice of C-PC4 in presence of the peptide, the role of the disordered N-PC4 and the high throughput identification of potent small molecules will allow a better understanding and control of p53-PC4 interaction