MD simulations indicate Omicron P132H of SARS-CoV-2 Mpro is a potential allosteric mutant involved in modulating the dynamics of catalytic site entry loop.

The SARS-CoV-2 main protease Mpro, essential for viral replication is an important drug target. It plays a critical role in processing viral polyproteins necessary for viral replication assembly. One of the predominant SARS-CoV-2 Mpro mutations of Omicron variant is Pro132His. Structurally, this mutation site is located ∼22 Å away from the catalytic site. The solved crystal structure of this mutant in complex with inhibitors as well as its reported catalytic efficiency did not show any difference with respect to the wild type. Thus, the mutation was concluded to be non-allosteric. Based on microsecond long MD simulation of the Pro132His mutant and wild type, we show that Pro132His mutation affects the conformational equilibrium with more population of conformational substates having open catalytic site, modulated by the dynamics of the catalytic site entry loop, implying the allosteric nature of this mutation. The structural analysis indicates that rearrangement of hydrogen bonds between His132 and adjacent residues enhances the dynamics of the linker, which in turn is augmented by the inherent dynamic flexibility of the catalytic pocket entry site due to the presence of charged residues. The altered dynamics leading to loss of secondary structures corroborate well with the reported compromised thermal stability.

Over-representation analysis of angiogenic factors in immunosuppressive mechanisms in neoplasms and neurological conditions during COVID-19.

BACKGROUND: Recent studies emphasized the necessity to identify key (human) biological processes and pathways targeted by the Coronaviridae family of viruses, especially Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Coronavirus Disease (COVID-19) caused up to 33-55 % death rates in COVID-19 patients with malignant neoplasms and Alzheimer's disease. Given this scenario, we identified biological processes and pathways involved in various diseases which are most likely affected by COVID-19. METHODS: The COVID-19 DisGeNET data set (v4.0) contains the associations between various diseases and human genes known to interact with viruses from Coronaviridae family and were obtained from the IntAct Coronavirus data set annotated with DisGeNET data. We constructed the disease-gene network to identify genes that are involved in various comorbid diseased states. Communities from the disease-gene network were identified using Louvain method and functional enrichment through over-representation analysis methodology was used to discover significant biological processes and pathways shared between COVID-19 and other diseases. RESULT: The COVID-19 DisGeNET data set (v4.0) comprised of 828 human genes and 10,473 diseases (including various phenotypes) that together constituted nodes in the disease-gene network. Each of the 70,210 edges connects a human gene with an associated disease. The top 10 genes linked to most number of diseases were VEGFA, BCL2, CTNNB1, ALB, COX2, AGT, HLA-A, HMOX1, FGF2 and COMT. The most vulnerable group of patients thus discovered had comorbid conditions such as carcinomas, malignant neoplasms and Alzheimer's disease. Finally, we identified 15 potentially useful biological processes and pathways for improved therapies. Vascular endothelial growth factor (VEGF) is the key mediator of angiogenesis in cancer. It is widely distributed in the brain and plays a crucial role in brain inflammation regulating the level of angiopoietins. With a degree of 1899, VEGFA was associated with maximum number of diseases in the disease-gene network. Previous studies have indicated that increased levels of VEGFA in the blood results in dyspnea, Pulmonary Edema (PE), Acute Lung Injury (ALI) and Acute Respiratory Distress Syndrome (ARDS). In case of COVID-19 patients with neoplasms and other neurological symptoms, our results indicate VEGFA as a therapeutic target for inflammation suppression. As VEGFs are known to disproportionately affect cancer patients, improving endothelial permeability and vasodilation with anti-VEGF therapy could lead to suppression of inflammation and also improve oxygenation. As an outcome of our study, we make case for clinical investigations towards anti-VEGF therapies for such comorbid conditions affected by COVID-19 for better therapeutic outcomes.

Community detection in Epstein-Barr virus associated carcinomas and role of tyrosine kinase in etiological mechanisms for oncogenesis.

BACKGROUND: Epstein-Barr virus (EBV) affects more than 90% of global population. The role of the virus in causing infectious mononucleosis (IM) affecting B-cells and epithelial cells and in the development of EBV associated cancers is well documented. Investigating the associated interactions can pave way for the discovery of novel therapeutic targets for EBV associated lymphoproliferative (Burkitt's Lymphoma and Hodgkin's Lymphoma) and non-lymphoproliferative diseases (Gastric cancer and Nasopharyngeal cancer). METHODS: Based on the DisGeNET (v7.0) data set, we constructed a disease-gene network to identify genes that are involved in various carcinomas, viz. Gastric cancer (GC), Nasopharyngeal cancer (NPC), Hodgkin's lymphoma (HL) and Burkitt's lymphoma (BL). We identified communities in the disease-gene network and performed functional enrichment using over-representation analysis to detect significant biological processes/pathways and the interactions between them. RESULT: We identified the modular communities to explore the relation of this common causative pathogen (EBV) with different carcinomas such as GC, NPC, HL and BL. Through network analysis we identified the top 10 genes linked with EBV associated carcinomas as CASP10, BRAF, NFKBIA, IFNA2, GSTP1, CSF3, GATA3, UBR5, AXIN2 and POLE. Further, the tyrosine-protein kinase (ABL1) gene was significantly over-represented in 3 out of 9 critical biological processes, viz. in regulatory pathways in cancer, the TP53 network and the Imatinib and chronic myeloid leukemia biological processes. Consequently, the EBV pathogen appears to target critical pathways involved in cellular growth arrest/apoptosis. We make our case for BCR-ABL1 tyrosine-kinase inhibitors (TKI) for further clinical investigations in the inhibition of BCR-mediated EBV activation in carcinomas for better prognostic and therapeutic outcomes.

Physiological models to study the effect of molecular crowding on multi-drug bound proteins: insights from SARS-CoV-2 main protease.

Molecular Dynamics simulations are often used in drug design. However, such simulations do not account for the physiological environment of the receptor; hence overlook its impact on biomolecular interactions. To address this lacuna, we identified three objectives to pursue - develop models of physiological environment, study a drug-receptor complex in such environments, and identify methods to analyze these complicated simulations. Two novel physiological models were developed and studied. The first, called 'm10', comprises of 10 of the most abundant cytoplasmic metabolites at physiological concentrations. The second, called 'phy', supplements m10 with an additional crowder protein to elicit macromolecular crowding effect. The main protease (Mpro) of SARS-CoV-2, being essential for viral replication, is an attractive drug target for COVID-19. Hence, we chose Mpro docked with multiple drugs as our model drug-receptor system. With a plethora of compounds, physiological systems can be exceedingly large and complex. A novel Spark-based software (SparkTraj) was developed to rapidly analyze non-specific contacts and water interactions. Our study shows that crowding enhances the difference in the dynamics of apo- vs drug-bound complexes. Metabolites, at times as a cluster, were seen interacting with the protease, drugs, and binding sites in drug-free receptor. Except one that crawled to an adjacent pocket in phy, the drugs remained in their respective pockets in all simulations. Given these observations, we hope that the models and approach presented here would help the optimization, evaluation, and selection of potential drugs. Generic biomolecular dynamics could also benefit from such models and tools.Communicated by Ramaswamy H. Sarma.

Targeting allosteric pockets of SARS-CoV-2 main protease Mpro.

Repurposing of antivirals is an attractive therapeutic option for the treatment of COVID-19. Main protease (Mpro), also called 3 C-like protease (3CLpro) is a key protease of SARS-CoV-2 involved in viral replication, and is a promising drug target for antivirals. A major challenge to test the efficacy of antivirals is the conformational plasticity of Mpro and its future mutation prone flexibility. Suitable choice of drugs in catalytic and allosteric pockets appear to be essential for combination therapy. Current study, based on docking and extensive set of MD simulations, finds the combination of Elbasvir, Glecaprevir and Ritonavir to be a viable candidate for further experimental drug testing/pharmacophore design for Mpro.Communicated by Ramaswamy H. Sarma.

Molecular crowding and conserved interface interactions of human argonaute protein-miRNA-target mRNA complex.

RNA interference (RNAi) has been of interest given its role in genetic interference. More significantly, recent studies provided evidence of it being one of the antiviral response mechanisms in humans. Argonaute (Ago) protein plays a central role in the RNA-induced silencing complex (RISC) that cleaves mRNA. Molecular crowding in cellular systems is known to impact dynamics and interactions of biomolecules. We present here the results from our molecular dynamics simulations based study on the interfaces between Ago, miRNA and Target RNA in presence of molecular crowders. 6 simulations at 3 crowder concentrations, including the aqueous condition, were performed. Our results indicate that crowding changes the dynamics, makes the complex stabler and aids binding free energy. More importantly, features conserved across the three systems and amino acid residues with crowding resilient interactions with RNA are identified.Communicated by Ramaswamy H. Sarma.

Insights into the effect of molecular crowding on the structure, interactions and functions of siRNA-PAZ complex through molecular dynamics studies.

siRNA molecules are well known to be involved in the post-transcriptional regulation of gene expression and play a key role in understanding the intricacies of eukaryotic gene regulation. While it is widely known that 3' end of siRNA binds to the PAZ domain of Argonaute proteins, it remains unclear whether the molecular crowding facilitates or hinders the overall siRNA-protein interactions during RNA interference. The biological interaction networks controlling the cellular functions of any biological cell may behave very differently in crowded environment as compared to the dilute conditions. Therefore, it is of interest to study the siRNA-protein interactions under more physiologically relevant conditions. In our previous work, we studied the role of hydrogen bond interactions and water network interactions towards the structural integrity of siRNA-PAZ complex. We also described the motions relevant for the functioning of the complex and analyzed the biphasic interaction of the 3' end of siRNA within the PAZ domain under aqueous condition. In the present work, we studied the dynamics of siRNA-PAZ complex in the presence of water-soluble crowding agent. We observed significant changes in interactions and dynamics of siRNA-PAZ complex in the presence of crowder. The conserved H-bond interactions were destabilized by ≈12%, while interfacial water networks were stabilized in the presence of crowder. The analysis of siRNA-PAZ dynamics revealed the stabilizing role of ARG52, ARG172, LYS53 and LYS173 in siRNA-PAZ complex. Interestingly, despite increase in flexibility as measured by RMSD, crowding stabilized top modes. Communicated by Ramaswamy H. Sarma.

Dynamics of the native and the ligand-bound structures of eosinophil cationic protein: network of hydrogen bonds at the catalytic site.

Eosinophil Cationic Protein (ECP) is sequentially and structurally similar to ribonuclease A (RNase A). It belongs to the RNase A family of proteins and the RNA catalysis is essential to its biological function. In the present study, we have generated the dinucleotide-bound structures of ECP by docking the dinucleotides to a number of molecular dynamics (MD) generated ECP structures. The stability of the docked enzyme-ligand complexes was ascertained by extensive MD simulations. The modes of ligand binding are explored by essential dynamics studies. The role of water molecules in the stability of the complex and in the catalysis was investigated. The active site residues form a complex network of connections with the ligand and with a water molecule. The catalytic mechanism of the RNA cleavage is examined on the basis of the active site geometry obtained by the simulations.

Conformational transitions in eosinophil cationic protein: a molecular dynamics study in aqueous environment.

Extensive molecular dynamics simulations have been performed on eosinophil cationic protein (ECP). The two structures found in the crystallographic dimer (ECPA and ECPB) have been independently simulated. A small difference in the pattern of the sidechain hydrogen bonds in the starting structure has resulted in interesting differences in the conformations accessed during the simulations. In one simulation (ECPB), a stable equilibrium conformation was obtained and in the other (ECPA), conformational transitions at the level of sidechain interactions were observed. The conformational transitions exhibit the involvement of the solvent (water) molecules with a pore-like construct in the equilibrium conformation and an opening for a large number of water molecules during the transition phase. The details of these transitions are examined in terms of intra-protein hydrogen bonds, protein-water networks and the residence times of water molecules on the polar atoms of the protein. These properties show some significant differences in the region between the N-terminal helix and the loop before the C-terminal strand as a function of different conformations accessed during the simulations. However, the stable hydrogen bonds, the protein-water networks, and the hydration patterns in most part of the protein including the active site are very much similar in both the simulations, indicating the fact that these are intrinsic properties of proteins.

Protein-water interactions in ribonuclease A and angiogenin: a molecular dynamics study.

It is known that water molecules play an important role in the biological functioning of proteins. The members of the ribonuclease A (RNase A) family of proteins, which are sequentially and structurally similar, are known to carry out the obligatory function of cleaving RNA and individually perform other diverse biological functions. Our focus is on elucidating whether the sequence and structural similarity lead to common hydration patterns, what the common hydration sites are and what the differences are. Extensive molecular dynamics simulations followed by a detailed analysis of protein-water interactions have been carried out on two members of the ribonuclease A superfamily-RNase A and angiogenin. The water residence times are analyzed and their relationship with the characteristic properties of the protein polar atoms, such as their accessible surface area and mean hydration, is studied. The capacity of the polar atoms to form hydrogen bonds with water molecules and participate in protein-water networks are investigated. The locations of such networks are identified for both proteins.

Stability and dynamics of domain-swapped bovine-seminal ribonuclease.

The proteins of the ribonuclease-A (RNase-A) family are monomeric, with the exception of bovine-seminal ribonuclease (BS-RNase). BS-RNase is formed by swapping the N-terminal helices across the two monomeric units. A molecular-dynamics (MD) study has been performed on the protein for a simulation time of 5.5 ns to understand the factors responsible for the stability of the dimer. Essential dynamics analysis and motional correlation of the protein atoms yielded the picture of a stabilising, yet flexible, interface. We have investigated the role of intermolecular H-bonding, protein/water interaction, and protein/water networks in stabilising the dimer. The networks of interchain H-bonds involving side-chain/side-chain or side-chain/main-chain (ScHB) interactions between the two chains have also been studied. The ability of protein atoms in retaining particular H2O molecules was investigated as a function of the accessible surface area (ASA), depth, and hydration parameters, as well as their participation in protein/water networks.

Computer modeling and molecular dynamics simulations of ligand bound complexes of bovine angiogenin: dinucleotide topology at the active site of RNase a family proteins.

We have undertaken the modeling of substrate-bound structures of angiogenin. In our recent study, we modeled the dinucleotide ligand binding to human angiogenin. In the present study, the substrates CpG, UpG, and CpA were docked onto bovine angiogenin. This was achieved by overcoming the problem of an obstruction to the B1 site by the C-terminus and identifying residues that bind to the second base. The modeled complexes retain biochemically important interactions. The docked models were subjected to 1 ns of molecular dynamics, and structures from the simulation were refined by using simulated annealing. Our models explained the enzyme's specificity for both B1 and B2 bases as observed experimentally. The nature of binding of the dinucleotide substrate was compared with that of the mononucleotide product. The models of these complexes were also compared with those obtained earlier with human angiogenin. On the basis of the simulations and annealed structures, we came up with a consensus topology of dinucleotide ligands that binds to human and bovine angiogenins. This dinucleotide conformation can serve as a starting model for ligand-bound complex structures for RNase A family of proteins. We demonstrated this capability by generating the complex structure of CpA bound to eosinophil-derived neurotoxin (EDN) by fitting the consensus topology of CpA to the crystal structure of native EDN.