RNA-binding domain of SARS-CoV2 nucleocapsid: MD simulation study of the effect of the proline substitutions P67S and P80R on the structure of the protein.

The nucleocapsid component of SARS-CoV2 is involved in the viral genome packaging. GammaP.1(Brazil) and the 20 C-US(USA) variants had a high frequency of the P80R and P67S mutations respectively in the RNA-binding domain of the nucleocapsid. Since RNA-binding domain participates in the electrostatic interactions with the viral genome, the study of the effects of proline substitutions on the flexibility of the protein will be meaningful. It evinced that the trajectory of the wildtype and mutants was stable during the simulation and exhibited distinct changes in the flexibility of the protein. Moreover, the beta-hairpin loop region of the protein structures exhibited high amplitude fluctuations and dominant motions. Additionally, modulations were detected in the drug binding site. Besides, the extent of correlation and anti-correlation motions involving the protruding region, helix, and the other RNA binding sites differed between the wildtype and mutants. The secondary structure analysis disclosed the variation in the occurrence pattern of the secondary structure elements between the proteins. Protein-ssRNA interaction analysis was also done to detect the amino acid contacts with ssRNA. R44, R59, and Y61 residues of the wildtype and P80R mutant exhibited different duration contacts with the ssRNA. It was also noticed that R44, R59, and Y61 of the wildtype and P80R formed hydrogen bonds with the ssRNA. However in P67S, residues T43, R44, R45, R40, R59, and R41 displayed contacts and formed hydrogen bonds with ssRNA. Binding free energy was also calculated and was lowest for P67S than wildtype andP80R. Thus, proline substitutions influence the structure of the RNA-binding domain and may modulate viral genome packaging besides the host-immune response.Communicated by Ramaswamy H. Sarma.

Computational Grafting of Epitopes.

Epitopes are the cornerstones for the development of rational vaccine design strategies. Conventionally, epitopes are used by chemical conjugation with the carrier protein. This chapter describes our computational epitope grafting methodology to identify the preferential grafting site in a carrier protein/scaffold. We have used the mota epitope as an example, as it was already experimentally validated by an independent group. In this chapter, we have provided sufficient details to enable the wet experimentalist to employ this computational methodology in their research objective. Scripts/programs are extensively described in this chapter and freely accessible through the provided link.

Identification of conserved immunogenic peptides of SARS-CoV-2 nucleocapsid protein.

The emergence of the new SARS-CoV-2 variants has led to major concern regarding the efficacy of approved vaccines. Nucleocapsid is a conserved structural protein essential for replication of the virus. This study focuses on identifying conserved epitopes on the nucleocapsid (N) protein of SARS-CoV-2. Using 510 unique amino acid sequences of SARS-CoV-2 N protein, two peptides (193 and 215 aa) with 90% conservancy were selected for T cell epitope prediction. Three immunogenic peptides containing multiple T cell epitopes were identified which were devoid of autoimmune and allergic immune response. These peptides were also conserved (100%) in recent Omicron variants reported in Jan-August 2023. HLA analysis reveals that these peptides are predicted as binding to large number of HLA alleles and 71-90% population coverage in six continents. Identified peptides displayed good binding score with both HLA class I and HLA class II molecules in the docking study. Also, a vaccine construct docked with TLR-4 receptor displays strong interaction with 20 hydrogen bonds and molecular simulation analysis reveals that docked complex are stable. Additionally, the immunogenicity of these N protein peptides was confirmed using SARS-CoV-2 convalescent serum samples. We conclude that the identified N protein peptides contain highly conserved and antigenic epitopes which could be used as a target for the future vaccine development against SARS-CoV-2.Communicated by Ramaswamy H. Sarma.

Targeting Kaposi's sarcoma associated herpesvirus encoded protease (ORF17) by a lysophosphatidic acid molecule for treating KSHV associated diseases.

Kaposi's sarcoma associated herpesvirus (KSHV) is causative agent of Kaposi's sarcoma, Multicentric Castleman Disease and Pleural effusion lymphoma. KSHV-encoded ORF17 encodes a protease which cleaves -Ala-Ala-, -Ala-Ser- or -Ala-Thr-bonds. The protease plays an important role in assembly and maturation of new infective virions. In the present study, we investigated expression pattern of KSHV-encoded protease during physiologically allowed as well as chemically induced reactivation condition. The results showed a direct and proportionate relationship between ORF17 expression with reactivation time. We employed virtual screening on a large database of natural products to identify an inhibitor of ORF17 for its plausible targeting and restricting Kaposi's sarcoma associated herpesvirus assembly/maturation. A library of 307,814 compounds of biological origin (A total 481,799 structures) has been used as a screen library. 1-oleoyl-2-hydroxy-sn-glycero-3-phospho-(1'-myo-inositol) was highly effective against ORF17 in in-vitro experiments. The screened compound was tested for the cytotoxic effect and potential for inhibiting Kaposi's sarcoma associated herpesvirus production upon induced reactivation by hypoxia, TPA and butyric acid. Treatment of reactivated KSHV-positive cells with 1-oleoyl-2-hydroxy-sn-glycero-3-phospho-(1'-myo-inositol) resulted in significant reduction in the production of Kaposi's sarcoma associated herpesvirus. The study identified a lysophosphatidic acid molecule for alternate strategy to inhibit KSHV-encoded protease and target Kaposi's sarcoma associated herpesvirus associated malignancies.

Computational molecular interaction between SARS-CoV-2 main protease and theaflavin digallate using free energy perturbation and molecular dynamics.

Our objective was to identify the molecule which can inhibit SARS-CoV-2 main protease and can be easily procured. Natural products may provide such molecules and can supplement the current custom chemical synthesis-based drug discovery for this objective. A combination of docking approaches, scoring functions, classical molecular dynamic simulation, binding pose metadynamics, and free energy perturbation calculations have been employed in this study. Theaflavin digallate has been observed in top-scoring compounds after the three independent virtual screening simulations of 598435 compounds (unique 27256 chemical entities). The main protease-theaflavin digallate complex interacts with critical active site residues of the main protease in molecular dynamics simulation independent of the explored computational framework, simulation time, initial structure, and force field used. Theaflavin digallate forms approximately three hydrogen bonds with Glutamate166 of main protease, primarily through hydroxyl groups in the benzene ring of benzo(7)annulen-6-one, along with other critical residues. Glu166 is the most critical amino acid for main protease dimerization, which is necessary for catalytic activity. The estimated binding free energy, calculated by Amber and Schrodinger MMGBSA module, reflects a high binding free energy between theaflavin digallate and main protease. Binding pose metadynamics simulation shows the highly persistent H-bond and a stable pose for the theaflavin digallate-main protease complex. Using method control, experimental controls, and test set, alchemical transformation studies confirm high relative binding free energy of theaflavin digallate with the main protease. Computational molecular interaction suggests that theaflavin digallate can inhibit the main protease of SARS-CoV-2.

Common garlic (Allium sativum L.) has potent Anti-Bacillus anthracis activity.

ETHNOPHARMACOLOGICAL RELEVANCE: Gastrointestinal anthrax, a disease caused by Bacillus anthracis, remains an important but relatively neglected endemic disease of animals and humans in remote areas of the Indian subcontinent and some parts of Africa. Its initial symptoms include diarrhea and stomachache. In the current study, several common plants indicated for diarrhea, dysentery, stomachache or as stomachic as per traditional knowledge in the Indian subcontinent, i.e., Aegle marmelos (L.) Correa (Bael), Allium cepa L. (Onion), Allium sativum L. (Garlic), Azadirachta indica A. Juss. (Neem), Berberis asiatica Roxb. ex DC. (Daruharidra), Coriandrum sativum L. (Coriander), Curcuma longa L. (Turmeric), Cynodon dactylon (L.) Pers. (Bermuda grass), Mangifera indica L. (Mango), Morus indica L. (Black mulberry), Ocimum tenuiflorum L. (Ocimum sanctum L., Holy Basil), Ocimum gratissimum L. (Ram Tulsi), Psidium guajava L. (Guava), Zingiber officinale Roscoe (Ginger), were evaluated for their anti-Bacillus anthracis property. The usage of Azadirachta indica A. Juss. and Curcuma longa L. by Santals (India), and Allium sp. by biblical people to alleviate anthrax-like symptoms is well documented, but the usage of other plants is traditionally only indicated for different gastrointestinal disturbances/conditions. AIM OF THE STUDY: Evaluate the above listed commonly available edible plants from the Indian subcontinent that are used in the traditional medicine to treat gastrointestinal diseases including those also indicated for anthrax-like symptoms for the presence of potent anti-B. anthracis activity in a form amenable to use by the general population in the endemic areas. MATERIALS AND METHODS: Aqueous extracts made from fourteen plants indicated above were screened for their anti-B. anthracis activity using agar-well diffusion assay (AWDA) and broth microdilution methods. The Aqueous Garlic Extract (AGE) that displayed most potent anti-B. anthracis activity was assessed for its thermostability, stability under pH extremes encountered in the gastrointestinal tract, and potential antagonistic interaction with bile salts as well as the FDA-approved antibiotics used for anthrax control. The bioactive fractions from the AGE were isolated by TLC coupled bioautography followed by their characterization using GC-MS. RESULTS: Garlic (Allium sativum L.) extract was identified as the most promising candidate with bactericidal activity against B. anthracis. It consistently inhibited the growth of B. anthracis in AWDA and decreased the viable colony-forming unit counts in liquid-broth cultures by 6-logs within 6-12 h. The AGE displayed acceptable thermostability (>80% anti-B. anthracis activity retained on incubation at 50 °C for 12 h) and stability in gastric pH range (2-8). It did not antagonize the activity of FDA-approved antibiotics used for anthrax control. GC-MS analysis of the TLC separated bioactive fractions of AGE indicated the presence of previously unreported constituents such as phthalic acid derivatives, acid esters, phenyl group-containing compounds, steroids etc. CONCLUSION: The Aqueous Garlic Extract (AGE) displayed potent anti-B. anthracis activity. It was better than that displayed by Azadirachta indica A. Juss. (Neem) and Mangifera indica L., while Curcuma longa L. (Turmeric) did not show any activity under the assay conditions used. Further work should be undertaken to explore the possible application of AGE in preventing anthrax incidences in endemic areas.

Cytochrome P450 2C9 polymorphism: Effect of amino acid substitutions on protein flexibility in the presence of tamoxifen.

Tamoxifen is a prodrug and cytochrome P450 2C9 (CYP2C9) has a significant role in the formation of a therapeutically more potent metabolite (4-hydroxytamoxifen) than tamoxifen. Since CYP2C9 exhibits genetic polymorphism, it may contribute to different phenotypic drug response. Moreover, it may be misleading if the possibility of heterogeneous clinical observations of pharmacogenetic investigations is ignored. Above all, clinical investigation of all the polymorphic variants is beyond the scope of a pharmacogenetic study. Therefore, in order to understand the genotype-phenotype association, it is aimed to study the interatomic interactions of amino acid substitutions in CYP2C9 variants in the presence of tamoxifen. Computational structural biology approach was adopted to study the effect of amino acid substitutions of polymorphic variants of CYP2C9 R144C (*2), I359 L (*3), D360E (*5), R150H (*8), R335W (*11) and L90 P (*13) on the flexibility of the enzyme in the presence of tamoxifen. The mutations were selected based on previously determined associations on genotype and clinical outcome of drugs. Against the above plane, docking of tamoxifen was performed with the crystal structure representing the wild-type form of the enzyme. The docked conformation of tamoxifen was favourable for 4-hydroxylation with the site of metabolism within 5 Šof oxyferrylheme consistent with the drug metabolism pathway of tamoxifen. Further, the effect of amino acid substitutions CYP2C9 variants on the protein flexibility in the presence of tamoxifen in 4-hydroxy orientation was evaluated by molecular dynamics (MD) simulations. Distinct protein flexibility modulations between variants were observed in F/G segment constituting the substrate access/egress channels, helix B' involved with substrate specificity and helix I associated with the holding of substrates. Root Mean Square Fluctuation analysis of the trajectories of variants exhibited fluctuations in F/G segment, B' and I helix. Dominant motions in the structure were identified by performing Principal Component Analysis on trajectories and the porcupine plot depicted displaced F/G segment in variants. Thus, the interatomic interaction study of CYP2C9 variants in the presence of tamoxifen predicts the plausible effect of the investigated variants on the therapeutic outcome of tamoxifen. It is presumed that the observations of the study would be meaningful to understand tamoxifen pharmacogenetics.

Anthrax prevention through vaccine and post-exposure therapy.

INTRODUCTION: Vaccines and therapeutic antibodies are the most crucial components of anthrax prophylaxis (pre- and post-exposure) and treatment. The improvement in the availability and safety profile of vaccines and the therapeutic antibodies has helped immensely in reducing the worldwide burden of anthrax. AREAS COVERED: Current recommendations for anthrax prophylaxis and control, vaccines and therapeutic antibodies, the recent endeavors, particularly, made after 2010 toward making them safer and more efficacious along with our opinion on its future course. Primarily, PubMed and Europe PMC were searched to cover the recent developments in the above-indicated areas. EXPERT OPINION: Some key existing lacunae in our understanding of the working of biologicals-based anthrax-control measures, i.e., vaccines and therapeutic antibodies, should be addressed to improve their overall stability, safety profile, and efficacy. The identification of novel inhibitors targeting different key-molecules and vital-steps contributing to the overall anthrax pathophysiology could make a difference in anthrax control.

Studies on computational grafting of malarial epitopes in serum albumin.

BACKGROUND: Antigens (or their epitopes)-carrier protein combinations are extensively utilized for vaccine development strategies. Chemical conjugation methods are cumbersome with limited conjugation sites. Computational protein modelling methods can evaluate every position of a carrier protein for conjugation via epitope grafting in a reasonable time frame as compared to wet experimental techniques. Graftibility of positions can be estimated by the presence of native atomic contacts in resulting chimeric antigen. METHODOLOGY: Five epitopes were selected, and computational grafting at each position was performed in three templates of serum albumin. Protein modelling algorithms such as segment matching and satisfaction of spatial restraints were employed for computational grafting. Contact-based protein discriminatory function was used to evaluate the chimeric proteins having native atomic contacts. RESULTS: On the evaluation of approximately 1 million distinct protein modelling simulations, region around the 450th position of serum albumin was observed to be suitable for epitope grafting. CONCLUSION: Computational protein modelling tools may be used to design a chimeric antigen. The approach may overcome the limitations associated with chemical conjugation and furthermore harness the potential of custom gene synthesis/recombinant protein production.

Preparation and Characterization of PLGA Encapsulated Protective Antigen Domain 4 Nanoformulation.

Poly (lactic-co-glycolic acid) (PLGA) based particulate systems have been widely explored for the development of subunit based vaccines owing to its biodegradability, biocompatibility, controlled release of entrapped antigen, targeted delivery potential, and nontoxic degradation product. Here, we describe the preparation of PLGA encapsulated recombinant protective antigen domain 4 (PAD4) nanoformulation (PAD4-NP) and its characterization for antigen content, morphology, and size. We also discuss the method of immunization in mice models to evaluate such PLGA based nanoformulation vaccines.

A single-dose PLGA encapsulated protective antigen domain 4 nanoformulation protects mice against Bacillus anthracis spore challenge.

Bacillus anthracis, the etiological agent of anthrax, is a major bioterror agent. Vaccination is the most effective prophylactic measure available against anthrax. Currently available anthrax vaccines have issues of the multiple booster dose requirement, adjuvant-associated side effects and stability. Use of biocompatible and biodegradable nanoparticles to deliver the antigens to immune cells could solve the issues associated with anthrax vaccines. We hypothesized that the delivery of a stable immunogenic domain 4 of protective antigen (PAD4) of Bacillus anthracis encapsulated in a poly (lactide-co-glycolide) (PLGA)--an FDA approved biocompatible and biodegradable material, may alleviate the problems of booster dose, adjuvant toxicity and stability associated with anthrax vaccines. We made a PLGA based protective antigen domain 4 nanoparticle (PAD4-NP) formulation using water/oil/water solvent evaporation method. Nanoparticles were characterized for antigen content, morphology, size, polydispersity and zeta potential. The immune correlates and protective efficacy of the nanoparticle formulation was evaluated in Swiss Webster outbred mice. Mice were immunized with single dose of PAD4-NP or recombinant PAD4. The PAD4-NP elicited a robust IgG response with mixed IgG1 and IgG2a subtypes, whereas the control PAD4 immunized mice elicited low IgG response with predominant IgG1 subtype. The PAD4-NP generated mixed Th1/Th2 response, whereas PAD4 elicited predominantly Th2 response. When we compared the efficacy of this single-dose vaccine nanoformulation PAD4-NP with that of the recombinant PAD4 in providing protective immunity against a lethal challenge with Bacillus anthracis spores, the median survival of PAD4-NP immunized mice was 6 days as compared to 1 day for PAD4 immunized mice (p<0.001). Thus, we demonstrate, for the first time, the possibility of the development of a single-dose and adjuvant-free protective antigen based anthrax vaccine in the form of PAD4-NP. Further work in this direction may produce a better and safer candidate anthrax vaccine.