A mechanistic study on the tolerance of PAM distal end mismatch by SpCas9.

The therapeutic application of CRISPR-Cas9 is limited due to its off-target activity. To have a better understanding of this off-target effect, we focused on its mismatch-prone PAM distal end. The off-target activity of SpCas9 depends directly on the nature of mismatches, which in turn results in deviation of the active site of SpCas9 due to structural instability in the RNA-DNA duplex strand. In order to test the hypothesis, we designed an array of mismatched target sites at the PAM distal end and performed in vitro and cell line-based experiments, which showed a strong correlation for Cas9 activity. We found that target sites having multiple mismatches in the 18th to 15th position upstream of the PAM showed no to little activity. For further mechanistic validation, Molecular Dynamics simulations were performed, which revealed that certain mismatches showed elevated root mean square deviation values that can be attributed to conformational instability within the RNA-DNA duplex. Therefore, for successful prediction of the off-target effect of SpCas9, along with complementation-derived energy, the RNA-DNA duplex stability should be taken into account.

Repurposing of therapeutic antibodies against dengue virus envelope protein receptor binding domain.

Dengue virus (DENV) is the leading cause of numerous deaths every year due to its high infectivity. In this study we have tried to target the DENV envelope protein receptor binding domain, the region crucial for binding to host receptors which leads to membrane fusion and entry of the viral genome into the human host cell. We have taken 13 known FDA approved antiviral therapeutic antibodies from therapeutic antibody database and tried to repurpose them against the DENV envelope protein. Based on the humanness analysis, 10 antibodies were selected against the DENV envelope protein. Computational affinity maturation of the 10 selected antibodies was performed to increase their binding affinity and specificity against the DENV envelope protein which ultimately led to 8 mutant antibodies having better binding affinity than the native ones. Molecular Dynamics (MD) simulation shows that, the stability of the complexes involving both the native and mutant antibodies were found to be the same although the binding energy between the protein and the respective antibodies was seen to improve upon computational affinity maturation. Contact analyses show similar robustness of the interaction for both the mutant and native antibodies during complex formation with the DENV envelope protein. This has led to the selection of total 18 antibodies including 10 natural and 8 affinity matured mutants which have a high probability of interacting with the DENV envelope protein. Finally, based on all these analyses along with heated MD simulation, Bamlanivimab, Etesivimab and Tixagevimab with a mutation of residue 100 of the heavy chain from serine to tyrosine were selected as prospective therapeutic antibodies to combat DENV infection. This study may open a new avenue in designing therapeutics to combat Dengue viral infection.

Peptide based vaccine designing against endemic causing mammarenavirus using reverse vaccinology approach.

The rodent-borne Arenavirus in humans has led to the emergence of regional endemic situations and has deeply emerged into pandemic-causing viruses. Arenavirus have a bisegmented ambisense RNA that produces four proteins: glycoprotein, nucleocapsid, RdRp and Z protein. The peptide-based vaccine targets the glycoprotein of the virus encountered by the immune system. Screening of B-Cell and T-Cell epitopes was done based on their immunological properties like antigenicity, allergenicity, toxicity and anti-inflammatory properties were performed. Selected epitopes were then clustered and epitopes were stitched using linker sequences. The immunological and physico-chemical properties of the vaccine construct was checked and modelled structure was validated by a 2-step MD simulation. The thermostability of the vaccine was checked followed by the immune simulation to test the immunogenicity of the vaccine upon introduction into the body over the course of the next 100 days and codon optimization was performed. Finally a 443 amino acid long peptide vaccine was designed which could provide protection against several members of the mammarenavirus family in a variety of population worldwide as denoted by the epitope conservancy and population coverage analysis. This study of designing a peptide vaccine targeting the glycoprotein of mammarenavirues may help develop novel therapeutics in near future.

Computational Insights into the Conformational Dynamics of HIV-1 Vpr in a Lipid Bilayer for Ion Channel Modeling.

HIV-1 Vpr is a multifunctional accessory protein consisting of 96 amino acids that play a critical role in viral pathogenesis. Among its diverse range of activities, Vpr can create a cation-selective ion channel within the plasma membrane. However, the oligomeric state of this channel has not yet been elucidated. In this study, we investigated the conformational dynamics of Vpr helices to model the ion channel topology. First, we employed a series of multiscale simulations to investigate the specific structure of monomeric Vpr in a membrane model. During the lipid bilayer self-assembly coarse grain simulation, the C-terminal helix (residues 56-77) effectively formed the transmembrane region, while the N-terminal helix exhibited an amphipathic nature by associating horizontally with a single leaflet. All-atom molecular dynamics (MD) simulations of full-length Vpr inside a phospholipid bilayer show that the C-terminal helix remains very stable inside the bilayer core in a vertical orientation. Subsequently, using the predicted C-terminal helix orientation and conformation, various oligomeric states (ranging from tetramer to heptamer) possibly forming the Vpr ion channel were built and further evaluated. Among these models, the pentameric form exhibited consistent stability in MD simulations and displayed a compatible conformation for a water-assisted ion transport mechanism. This study provides structural insights into the ion channel activity of the Vpr protein and the foundation for developing therapeutics against HIV-1 Vpr-related conditions.

Repurposing of drugs targeting heparan sulphate binding site of dengue virus envelope protein: an in silico competitive binding study.

Dengue virus, an arbovirus, leads to millions of infections every year ultimately leading to a high rate of mortality. Highly effective and specific therapeutic option is not available till date to combat viral infection. One of the first stages in the virus lifecycle encompasses the viral entry into the host cell which is mediated by the interaction between heparan sulphate and the Dengue virus envelope protein in turn leading to the interaction between the envelope protein receptor binding domain and host cell receptors. The heparan sulphate binding site on the envelope protein was established using literature survey and the result validated using ColDock simulations. We have performed virtual screening against the heparan sulphate binding site using DrugBank database and short-listed probable inhibitors based on binding energy calculation following Molecular Dynamics (MD) simulations in this study. Two compounds (PubChem IDS 448062 and 656615) were selected for further analyses on which RAMD simulations were performed to quantitate the binding stability of both the molecules in the protein binding pocket which ultimately led to the selection of ZK-806450 molecule as the final selected compound. Competitive binding MD simulation against dengue virus envelope protein was performed for this molecule and heparan sulphate in order to ascertain the efficiency of binding of this molecule to the dengue virus envelope protein in the presence of its natural ligand molecule and found that this molecule has a higher affinity for the dengue virus envelope protein GAG binding site than heparan sulphate. This study may help in the use of this inhibitor molecule to combat dengue virus infection in foreseeable future and open a new avenue for drug repurposing methodology using competitive binding MD simulation.

Repurposing of FDA-approved drugs against oligomerization domain of dengue virus NS1 protein: a computational approach.

Dengue fever is a serious health hazard on a global scale and its primary causative agent is the dengue virus (DENV). The non-structural protein 1 (NS1) of DENV plays a pivotal role in pathogenesis. It is associated with several autoimmune events, endothelial cell apoptosis, and vascular leakage, which increase mainly during the critical phase of infection. In this study, important residues of the oligomerization domain of NS1 protein were identified by literature searches. Virtual screening has been conducted using the entire dataset of the DrugBank database and the potential small-molecule inhibitors against the NS1 protein have been chosen on the basis of binding energy values. This is succeeded by molecular dynamics (MD) simulations of the shortlisted compounds, ultimately giving rise to five compounds. These five compounds were further subjected to RAMD simulations by applying a random direction force of specific magnitude on the ligand center of mass in order to push the ligand out of the protein-binding pocket, for the quantitative estimation of their binding energy values to determine the interaction strength between protein and ligand which prevents ligand unbinding from its binding site, ultimately leading to the selection of three major compounds, DB00826 (Natamycin), DB11274 (Dihydro-alphaergocryptine), and DB11275 (Epicriptine), with the DB11274 having a role against idiopathic Parkinson's disease, and thus may have possible important roles in the prevention of dengue-associated Parkinsonism. These compounds may act as prospective drugs against dengue, by preventing the oligomerization of the NS1 protein, thereby preventing disease progression and pathogenesis.

In silico designing of an epitope-based peptide vaccine cocktail against Nipah virus: an Indian population-based epidemiological study.

Nipah virus, a zoonotic virus from the family Paramyxoviridae has led to significant loss of lives till date with the most recent outbreak in India reported in Kerala. The virus has a considerably high mortality rate along with lack of characteristic symptoms which results in the delay of the virus detection. No specific vaccine is available for the virus although monoclonal antibody treatment has been seen to be effective along with favipiravir. The high mortality and complications caused by the virus underscores the necessity to develop alternative modes of vaccination. One such method has been designed in this study using peptide cocktail consisting of the immunologically important epitopes for use as vaccine. The human leucocytic antigens that are used for the study were analyzed for their presence in various ethnic Indian populations. This study may serve as a new avenue for development of more efficient peptide cocktail vaccines in recent future based on the population genetics and ethnicity.

Repurposing of drug molecules from FDA database against Hepatitis C virus E2 protein using ensemble docking approach.

Hepatitis C virus, a member of the Flaviviridae family and genus Hepacivirus, is an enveloped, positively single stranded RNA virus. Its surface consists of a heterodimer of E1 and E2 proteins which play a crucial role in receptor binding and membrane fusion. In this study we have used in silico virtual screening by utilizing ensemble docking on the approved drugs. These drugs can bind with high efficiency to the 36 prominent conformations of the CD81 binding site clustered from a total of 3 µs MD simulation data on the E2 protein. We started with 9213 compounds from the FDA list of drugs and progressively came down to 5 compounds which have been seen to bind with very high efficiency to not only all the conformations but also the two predicted druggable pockets that encompass the CD81 binding site. MM/PBSA binding energy calculations also point to the highly stable interaction of the compounds to the E2 protein. This study may in future broaden the arsenal of therapeutics for use against HCV infection and lead to more effective care against the virus.

Identification and characterization of differentially expressed genes in the rice root following exogenous application of spermidine during salt stress.

Salinity is a major limiting factor in crop production. Exogenous spermidine (spd) effectively ameliorates salt injury, though the underlying molecular mechanism is poorly understood. We have used a suppression subtractive hybridization method to construct a cDNA library that has identified up-regulated genes from rice root under the treatment of spd and salt. Total 175 high-quality ESTs of about 100-500 bp in length with an average size of 200 bp are isolated, clustered and assembled into a collection of 62 unigenes. Gene ontology analysis using the KEGG pathway annotation database has classified the unigenes into 5 main functional categories and 13 subcategories. The transcripts abundance has been validated using Real-Time PCR. We have observed seven different types of post-translational modifications in the DEPs. 44 transmembrane helixes are predicted in 6 DEPs. This above information can be used as first-hand data for dissecting the administrative role of spd during salinity.

In silico fragment-based design and pharmacophore modelling of therapeutics against dengue virus envelope protein.

Dengue virus, an arbovirus of genus Flavivirus, is an infectious disease causing organisms in the tropical environment leading to numerous deaths every year. No therapeutic is available against the virus till date with only symptomatic relief available. Here, we have tried to design therapeutic compounds from scratch by fragment based method followed by pharmacophore based modelling to find suitable similar structure molecules and validated the same by MD simulation, followed by binding energy calculations and ADMET analysis. The receptor binding region of the dengue envelope protein was considered as the target for prevention of viral host cell entry and thus infection. This resulted in the final selection of kanamycin as a stable binding molecule against the Dengue virus envelope protein receptor binding domain. This study results in selection of a single molecule having high binding energy and prominent stable interactions as determined by post simulation analyses. This study aims to provide a direction for development of small molecule therapeutics against the dengue virus in order to control infection. This study may open a new avenue in the arena of structure based and fragment based therapeutic design to obtain novel molecules with therapeutic potential. Supplementary Information: The online version contains supplementary material available at 10.1007/s40203-024-00262-9.

In silico investigation of binding propensity of hematoxylin derivative and damnacanthal for their potential inhibitory effect on HIV-1 Vpr from different subtypes.

HIV-1, the causative agent of AIDS leads to many deaths worldwide though few options are available as therapeutics. To deal with the continuous mutation in the virus genome, requirement of new drugs is always there. Subtype variation plays a crucial role in case of HIV-1 therapeutics development. In this study, we want to investigate some pre examined molecules that can be effective for HIV-1 VPR. Inhibition of several protein-protein interactions with the small molecules will lead to identify some molecules as therapeutics other than the conventional drugs. We retrieved the sequences of different subtypes from the database and representative sequences were identified. Representative structures were modelled and validated using MD simulations. Forty molecules, showing anti Vpr activity in vitro were identified from literature survey and those were docked with each subtype representative structures. Two molecules a stable Hematoxylin Derivative (SHD) and Damnacanthal (D3), these were shown to be bind more effectively for all the subtypes. The stability of the protein and those two small molecule complexes were identified again with MD simulation followed by the binding energy calculation. Thus, these molecules can be thought as any option other than the conventional drug targeting HIV-1 Vpr.Communicated by Ramaswamy H. Sarma.

Exploring the chemical space for potential inhibitors against cell surface binding protein of Mpox virus using molecular fingerprint based screening approach.

Mpox virus is the latest member of the Poxviridae family of which small pox virus is a member. Monekypox virus has led to thousands of infections across the globe. Poxvirus gains entry into the cell making use of glycosaminoglycans like chondroitin sulphate and heparan sulphate. The interaction of the Mpox virus protein E8L also called cell surface binding protein is crucial for host cell attachment, membrane fusion and viral entry into the host cell leading to establishment of infection thus making this protein a very attractive therapeutic target. In this study we have tried to utilize the chondroitin sulphate binding groove present in the protein and identify molecules which are structurally similar to chondroitin sulphate. These molecules can thus occupy the same pocket but with a better binding affinity than chondroitin sulphate in order to outcompete the latter molecule from binding to the E8L protein and thus prevent it from performing its function. This study may pave the way for development of highly efficient therapeutics against the Mpox virus and further curb its infective potential.Communicated by Ramaswamy H. Sarma.

In silico characterization, molecular docking, and dynamic simulation of a novel fungal cell-death suppressing effector, MoRlpA as potential cathepsin B-like cysteine protease inhibitor during rice blast infection.

The blast fungus Magnaporthe oryzae is one of the most notorious pathogens affecting rice production worldwide. The cereal killer employs a special class of small secreted proteins called effectors to manipulate and perturb the host metabolism. In turn, the host plants trigger effector-triggered immunity (ETI) via localized cell death and hypersensitive response (HR). We have identified and characterized a novel secreted effector MoRlpA from M. oryzae by extensive in silico methods. The localization studies suggested that it is exclusively secreted in the host apoplasts. Interestingly, MoRlpA interacts with a protease, cathepsin B from rice with highest affinity. The 3D structural models of both the proteins were generated. Cathepsin B-like cysteine proteases are usually involved in programmed cell death (PCD) and autophagy in plants which lead to generation of HR upon infection. Our results suggest that MoRlpA interacts with rice cathepsin B-like cysteine protease and demolish the host counter-attack by suppressing cell death and HR during an active blast infection. This was further validated by molecular docking and molecular dynamic simulation analyses. The important residues involved in the rice-blast pathogen interactions were deciphered. Overall, this research highlights stable interactions between MoRlpA-OsCathB during rice blast pathogenesis and providing an insight into how this novel RlpA protease inhibitor-cum-effector modulates the host's apoplast to invade the host tissues and establish a successful infection. Thus, this research will help to develop potential fungicide to block the binding region of MoRlpA target so that the cryptic pathogen would be recognized by the host. HIGHLIGHTSFor the first time, a novel secreted effector protein, MoRlpA has been identified and characterised from M. oryzae in silicoMoRlpA contains a rare lipoprotein A-like DPBB domain which is often an enzymatic domain in other systemsMoRlpA as an apoplastic effector interacts with the rice protease OsCathB to suppress the cell death and hypersensitive response during rice blast infectionThe three-dimensional structures of both the MoRlpA and OsCathB proteins were predictedMoRlpA-OsCathB interactions were analysed by molecular docking and molecular dynamic simulation studiesCommunicated by Ramaswamy H. Sarma.

Designing of nanobodies against Dengue virus Capsid: a computational affinity maturation approach.

Dengue virus, an arbovirus, is one of the most prevalent diseases in the tropical environment and leads to huge number of casualties every year. No therapeutics are available till date against the viral disease and the only medications provide symptomatic relief. In this study, we have focused on utilizing conventional nanobodies and repurposing them for Dengue. Computationally affinity matured, best binding nanobodies tagged with constant antibody regions, could be proposed as therapeutics. These could also be applied for drug delivery purposes due to their high specificity against the viral Capsid. Another application of these nanobodies has been thought to utilize them for diagnostic purposes, to use the nanobodies for viral detection from patient samples at the earliest stage using ELISA. This study may open a new avenue for immunologic study in foreseeable future with the usage of the same molecules for multiple purposes. HighlightsNatural nanobodies against viruses were modified for use against Dengue virus Capsid conserved regions.Computational affinity maturation was performed making use of change in binding affinities upon mutating various residues in the complementary determining regions.Docking studies performed to inspect the docking groove, interface analysis and energy calculations.MM/GBSA calculations done to calculate binding free energy of the complex to determine stability of the complex.Communicated by Ramaswamy H. Sarma.

Computer-aided de novo design and optimization of novel potential inhibitors of HIV-1 Nef protein.

Nef is a small accessory protein pivotal in the HIV-1 viral replication cycle. It is a multifunctional protein and its interactions with kinases in host cells have been well characterized through many in vitro and structural studies. Nef forms a homodimer to activate the kinases and subsequently the phosphorylation pathways. The disruption of its homodimerization represents a valuable approach in the search for novel classes of antiretroviral. However, this research avenue is still underdeveloped as just a few Nef inhibitors have been reported so far, with very limited structural information about their mechanism of action. To address this issue, we have employed an in silico structure-based drug design strategy that combines de novo ligand design with molecular docking and extensive molecular dynamics simulations. Since the Nef pocket involved in homodimerization has high lipophilicity, the initial de novo-designed structures displayed poor drug-likeness and solubility. Taking information from the hydration sites within the homodimerization pocket, structural modifications in the initial lead compound have been introduced to improve the solubility and drug-likeness, without affecting the binding profile. We propose lead compounds that can be the starting point for further optimizations to deliver long-awaited, rationally designed Nef inhibitors.

Phylogenetic, structural, functional characterisation and effect of exogenous spermidine on rice (Oryza sativa) HAK transporters under salt stress.

The HAK (High-affinity K+ ) family members mediate K+ transport that confers normal plant growth and resistance against unfavourable environmental conditions. Rice (Oryza sativa L.) HAK transporters have been extensively investigated for phylogenetic analyses with other plants species with very few of them functionally characterised. But very little information is known about their evolutionary aspects, overall structural, functional characterisation, and global expression pattern of the complete HAK family members in response to salt stress. In this study, 27 rice transporters were phylogenetically clustered with different dicot and monocot family members. Subsequently, the exon-intron structural patterns, conserved motif analyses, evolutionary divergence based different substitution matrix, orthologous-paralogous relationships were studied elaborately. Structural characterisations included a comparative study of secondary and tertiary structure, post-translational modifications, correspondence analyses, normal mode analyses, K+ /Na+ binding affinities of each of the OsHAK gene members. Global expression profile under salt stress showed clade-specific expression pattern of the proteins. Additionally, five OsHAK genes were chosen for further expression analyses in root and shoot tissues of two rice varieties during short-term salinity in the presence and absence of exogenous spermidine. All the information can be used as first-hand data for dissecting the administrative role of rice HAK transporters under various abiotic stresses.

An insight into the binding mechanism of Viprinin and its morpholine and piperidine derivatives with HIV-1 Vpr: molecular dynamics simulation, principal component analysis and binding free energy calculation study.

HIV-1 Vpr is an accessory protein responsible for a plethora of functions inside the host cell to promote viral pathogenesis. One of the major functions of Vpr is the G2 cell cycle arrest. Among several small molecule inhibitors, Viprinin, a coumarin derivative, has been shown to specifically inhibit the G2 cell cycle arrest activity of Vpr thus making it an excellent choice for a lead molecule to design antiretroviral drug. But the exact mechanism of binding of the Viprinin and its two potent derivatives with Vpr is still not understood. In this study with combined molecular docking, molecular dynamics simulation, Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) method, Principal component analysis and Umbrella sampling simulation, we have explored the binding mechanism of Viprinin and its two derivatives with Vpr. MM-PBSA and Umbrella sampling calculations suggest that Viprinin and ViprininD1 have higher binding energy than ViprininD2. Molecular dynamics simulation shows that the ligands are not very stable inside the initial binding pocket and various hydrophobic interactions are responsible to hold the ligands with Vpr. Vpr backbone Principle Component Analysis (PCA) shows various unique essential motions of Vpr bound with Viprinin and its two derivatives. This study may give detailed insight of the mode of binding of the specified compounds at atomic scale and provide valuable information about the possibility of using these compounds as a potent Vpr inhibitor. Communicated by Ramaswamy H. Sarma.

Prediction of infectivity of SARS-CoV-2 virus based on Spike-hACE-2 interaction.

The COVID-19 pandemic caused by SARS-CoV-2 results almost 3 M death worldwide and till continuing in spite of having several vaccine against the virus. One of the main reasons is the mutations occur in the virus to cope with the environment. Detail study of genomics and proteomics level of each components may help to combat the situation. Spike (S) protein that covers the surface of the virus helps in entry by encountering the host receptor Human Angiotensin-Converting Enzyme-2 (hACE-2) with other different roles. In this study, we accomplish our work with the mutations in receptor binding domain (RBD) of Spike (S) protein considering different aspects like the hACE-2 variants in human populations to get an idea about the varying infectivity of different strains for different population. Several other parameters affecting the viral infectivity and in different diseased condition were also studied which may guide to a better insight in developing future therapeutics.

In silico study on miRNA regulation and NSs protein interactome characterization of the SFTS virus.

Severe fever with thrombocytopenia syndrome causing virus i.e. SFTS virus has increased in the last few years. The underlying cause and mechanism of disease progression and development of symptoms is not well known. Many viruses including Hepatitis B, Hepatitis C, HIV-1, Herpes virus, Dengue virus and many others have been seen to regulate their functions at the miRNA level. This study aimed to find out those cellular miRNAs, which can be mimicked or antagonized by the viral genome and analyze the effect of these miRNAs on various gene functions. Investigations in this study suggest a correlation between miRNA regulation with the disease symptoms and progression. By exhaustive literature survey we have tried to identify the interacting partners of the Non Structural S (NSs) protein and characterized the protein-protein interactions. The binding interface that can serve as target for therapeutic studies involving the interfacial residues was analyzed. This study would serve as an avenue to design therapeutics making use of not only protein-protein interactions but also miRNA based regulation as well.

In Silico Study of Mutational Stability of SARS-CoV-2 Proteins.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), an enveloped RNA virus transmits by droplet infection thus affects the respiratory system. Different genomes have been reported globally for SARS-CoV-2 with moderate level of mutation which makes it harder to combat the virus. Mutational profiling and the relevant evolutionary aspect of coronavirus proteins namely spike glycoprotein, membrane protein, envelope protein, nucleoprotein, ORF1ab, ORF3a, ORF6, ORF7a, ORF7b and ORF8 were studied by in silico experiments. Clustering of the protein sequences and calculation of residue relative abundance were done to get an idea about the protein conservancy as well as finding out some representative sequences for phylogenetic and ancestral reconstruction. By mutational profiling and mutation analysis, the effect of mutations on the protein stability and their functional implication were studied. This study indicates the mutational effect on the proteins and their relevance in evolution, which directs us towards a better understanding of these variations and diversification of SARS-CoV-2 for useful future therapeutic study and thus aid in designing therapeutic agents keeping the highly variable regions in mind.