Engineering PD-1-targeted small protein variants for in vitro diagnostics and in vivo PET imaging.

BACKGROUND: Programmed cell death 1 (PD-1) belongs to immune checkpoint proteins ensuring negative regulation of the immune response. In non-small cell lung cancer (NSCLC), the sensitivity to treatment with anti-PD-1 therapeutics, and its efficacy, mostly correlated with the increase of tumor infiltrating PD-1+ lymphocytes. Due to solid tumor heterogeneity of PD-1+ populations, novel low molecular weight anti-PD-1 high-affinity diagnostic probes can increase the reliability of expression profiling of PD-1+ tumor infiltrating lymphocytes (TILs) in tumor tissue biopsies and in vivo mapping efficiency using immune-PET imaging. METHODS: We designed a 13 kDa ß-sheet Myomedin scaffold combinatorial library by randomization of 12 mutable residues, and in combination with ribosome display, we identified anti-PD-1 Myomedin variants (MBA ligands) that specifically bound to human and murine PD-1-transfected HEK293T cells and human SUP-T1 cells spontaneously overexpressing cell surface PD-1. RESULTS: Binding affinity to cell-surface expressed human and murine PD-1 on transfected HEK293T cells was measured by fluorescence with LigandTracer and resulted in the selection of most promising variants MBA066 (hPD-1 KD = 6.9 nM; mPD-1 KD = 40.5 nM), MBA197 (hPD-1 KD = 29.7 nM; mPD-1 KD = 21.4 nM) and MBA414 (hPD-1 KD = 8.6 nM; mPD-1 KD = 2.4 nM). The potential of MBA proteins for imaging of PD-1+ populations in vivo was demonstrated using deferoxamine-conjugated MBA labeled with 68Galium isotope. Radiochemical purity of 68Ga-MBA proteins reached values 94.7-99.3% and in vitro stability in human serum after 120 min was in the range 94.6-98.2%. The distribution of 68Ga-MBA proteins in mice was monitored using whole-body positron emission tomography combined with computerized tomography (PET/CT) imaging up to 90 min post-injection and post mortem examined in 12 mouse organs. The specificity of MBA proteins was proven by co-staining frozen sections of human tonsils and NSCLC tissue biopsies with anti-PD-1 antibody, and demonstrated their potential for mapping PD-1+ populations in solid tumors. CONCLUSIONS: Using directed evolution, we developed a unique set of small binding proteins that can improve PD-1 diagnostics in vitro as well as in vivo using PET/CT imaging.

Current understanding on TREM-2 molecular biology and physiopathological functions.

Triggering receptor expressed on myeloid cells 2 (TREM-2), a glycosylated receptor belonging to the immunoglobin superfamily and especially expressed in the myeloid cell lineage, is frequently explained as a reminiscent receptor for both adaptive and innate immunity regulation. TREM-2 is also acknowledged to influence NK cell differentiation via the PI3K and PLCγ signaling pathways, as well as the partial activation or direct inhibition of T cells. Additionally, TREM-2 overexpression is substantially linked to cell-specific functions, such as enhanced phagocytosis, reduced toll-like receptor (TLR)-mediated inflammatory cytokine production, increased transcription of anti-inflammatory cytokines, and reshaped T cell function. Whereas TREM-2-deficient cells exhibit diminished phagocytic function and enhanced proinflammatory cytokines production, proceeding to inflammatory injuries and an immunosuppressive environment for disease progression. Despite the growing literature supporting TREM-2+ cells in various diseases, such as neurodegenerative disorders and cancer, substantial facets of TREM-2-mediated signaling remain inadequately understood relevant to pathophysiology conditions. In this direction, herein, we have summarized the current knowledge on TREM-2 biology and cell-specific TREM-2 expression, particularly in the modulation of pivotal TREM-2-dependent functions under physiopathological conditions. Furthermore, molecular regulation and generic biological relevance of TREM-2 are also discussed, which might provide an alternative approach for preventing or reducing TREM-2-associated deformities. At last, we discussed the TREM-2 function in supporting an immunosuppressive cancer environment and as a potential drug target for cancer immunotherapy. Hence, summarized knowledge of TREM-2 might provide a window to overcome challenges in clinically effective therapies for TREM-2-induced diseases in humans.

Targeting neuroblastoma by small-molecule inhibitors of human ALYREF protein: mechanistic insights using molecular dynamics simulations.

Neuroblastoma is a tumour of the sympathetic nervous system mainly prevalent in children. Many strategies have been employed to target several drug-targetable proteins for the clinical management of neuroblastoma. However, the heterogeneous nature of neuroblastoma presents serious challenges in drug development for its treatment. Albeit numerous medications have been developed to target various signalling pathways in neuroblastoma, the redundant nature of the tumour pathways makes its suppression unsuccessful. Recently, the quest for neuroblastoma therapy resulted in the identification of human ALYREF, a nuclear protein that plays an essential role in tumour growth and progression. Therefore, this study used the structure-based drug discovery method to identify the putative inhibitors targeting ALYREF for the Neuroblastoma treatment. Herein, a library of 119 blood-brain barrier crossing small molecules from the ChEMBL database was downloaded and docked against the predicted binding pocket of the human ALYREF protein. Based on docking scores, the top four compounds were considered for intermolecular interactions and molecular dynamics simulation analysis, which revealed CHEMBL3752986 and CHEMBL3753744 with substantial affinity and stability with the ALYREF. These results were further supported by binding free energies and essential dynamics analysis of the respective complexes. Hence, this study advocates the sorted compounds targeting ALYREF for further in vitro and in vivo assessment to develop a drug against neuroblastoma.Communicated by Ramaswamy H. Sarma.

Current therapeutic targets and multifaceted physiological impacts of caffeine.

Caffeine, which shares consubstantial structural similarity with purine adenosine, has been demonstrated as a nonselective adenosine receptor antagonist for eliciting most of the biological functions at physiologically relevant dosages. Accumulating evidence supports caffeine's beneficial effects against different disorders, such as total cardiovascular diseases and type 2 diabetes. Conversely, paradoxical effects are also linked to caffeine ingestion in humans including hypertension-hypotension and tachycardia-bradycardia. These observations suggest the association of caffeine action with its ingested concentration and/or concurrent interaction with preferential molecular targets to direct explicit events in the human body. Thus, a coherent analysis of the functional targets of caffeine, relevant to normal physiology, and disease pathophysiology, is required to understand the pharmacology of caffeine. This review provides a broad overview of the experimentally validated targets of caffeine, particularly those of therapeutic interest, and the impacts of caffeine on organ-specific physiology and pathophysiology. Overall, the available empirical and epidemiological evidence supports the dose-dependent functional activities of caffeine and advocates for further studies to get insights into the caffeine-induced changes under specific conditions, such as asthma, DNA repair, and cancer, in view of its therapeutic applications.

Understanding the self-assembly dynamics of A/T absent 'four-way DNA junctions with sticky ends' at altered physiological conditions through molecular dynamics simulations.

Elucidation of structure and dynamics of alternative higher-order structures of DNA such as in branched form could be targeted for therapeutics designing. Herein, we are reporting the intrinsically dynamic and folds transitions of an unusual DNA junction with sequence d(CGGCGGCCGC)4 which self-assembles into a four-way DNA junction form with sticky ends using long interval molecular simulations under various artificial physiological conditions. The original crystal structure coordinates (PDB ID: 3Q5C) for the selected DNA junction was considered for a total of 1.1 µs molecular dynamics simulation interval, including different temperature and pH, under OPLS-2005 force field using DESMOND suite. Following, post-dynamics structure parameters for the DNA junction were calculated and analyzed by comparison to the crystal structure. We show here that the self-assembly dynamics of DNA junction is mitigated by the temperature and pH sensitivities, and discloses peculiar structural properties as function of time. From this study it can be concluded on account of temperature sensitive and pH dependent behaviours, DNA junction periodic arrangements can willingly be synthesized and redeveloped for multiple uses like genetic biomarkers, DNA biosensor, DNA nanotechnology, DNA Zipper, etc. Furthermore, the pH dis-regulation behaviour may be used to trigger the functionality of DNA made drug-releasing nanomachines.

Exploration of Potent Antiviral Phytomedicines from Lauraceae Family Plants against SARS-CoV-2 Main Protease.

A new Coronaviridae strain, Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), emerged from Wuhan city of China and caused one of the substantial global health calamities in December 2019. Even though several vaccines and drugs have been developed worldwide since COVID-19, a cost-effective drug with the least side effects is still unavailable. Currently, plant-derived compounds are mostly preferred to develop antiviral therapeutics due to its less toxicity, easy access, and cost-effective characteristics. Therefore, in this study, 124 phytochemical compounds from plants of Lauraceae family with medicinal properties were virtually screened against SARS-CoV-2 Mpro. Identification of four phytomolecules, i.e., cassameridine, laetanine, litseferine and cassythicine, with docking scores -9.3, -8.8, -8.6, and -8.6 kcal/mol, respectively, were undertaken by virtual screening, and molecular docking. Furthermore, the molecular dynamic simulation and essential dynamics analysis have contributed in understanding the stability and inhibitory effect of these selected compounds. These phytomolecules can be considered for further in vitro and in vivo experimental study to develop anti-SARS-CoV-2 therapeutics targeting the main protease (Mpro).

Myomedin replicas of gp120 V3 loop glycan epitopes recognized by PGT121 and PGT126 antibodies as non-cognate antigens for stimulation of HIV-1 broadly neutralizing antibodies.

Introduction: Imprinting broadly neutralizing antibody (bNAb) paratopes by shape complementary protein mimotopes represents a potential alternative for developing vaccine immunogens. This approach, designated as a Non-Cognate Ligand Strategy (NCLS), has recently been used for the identification of protein variants mimicking CD4 binding region epitope or membrane proximal external region (MPER) epitope of HIV-1 envelope (Env) glycoprotein. However, the potential of small binding proteins to mimic viral glycan-containing epitopes has not yet been verified. Methods: In this work, we employed a highly complex combinatorial Myomedin scaffold library to identify variants recognizing paratopes of super candidate bNAbs, PGT121 and PGT126, specific for HIV-1 V3 loop epitopes. Results: In the collection of Myomedins called MLD variants targeted to PGT121, three candidates competed with gp120 for binding to this bNAb in ELISA, thus suggesting an overlapping binding site and epitope-mimicking potential. Myomedins targeted to PGT126 designated MLB also provided variants that competed with gp120. Immunization of mice with MLB or MLD binders resulted in the production of anti-gp120 and -Env serum antibodies. Mouse hyper-immune sera elicited with MLB036, MLB041, MLB049, and MLD108 moderately neutralized 8-to-10 of 22 tested HIV-1-pseudotyped viruses of A, B, and C clades in vitro. Discussion: Our data demonstrate that Myomedin-derived variants can mimic particular V3 glycan epitopes of prominent anti-HIV-1 bNAbs, ascertain the potential of particular glycans controlling neutralizing sensitivity of individual HIV-1 pseudoviruses, and represent promising prophylactic candidates for HIV-1 vaccine development.

Viral informatics: bioinformatics-based solution for managing viral infections.

Several new viral infections have emerged in the human population and establishing as global pandemics. With advancements in translation research, the scientific community has developed potential therapeutics to eradicate or control certain viral infections, such as smallpox and polio, responsible for billions of disabilities and deaths in the past. Unfortunately, some viral infections, such as dengue virus (DENV) and human immunodeficiency virus-1 (HIV-1), are still prevailing due to a lack of specific therapeutics, while new pathogenic viral strains or variants are emerging because of high genetic recombination or cross-species transmission. Consequently, to combat the emerging viral infections, bioinformatics-based potential strategies have been developed for viral characterization and developing new effective therapeutics for their eradication or management. This review attempts to provide a single platform for the available wide range of bioinformatics-based approaches, including bioinformatics methods for the identification and management of emerging or evolved viral strains, genome analysis concerning the pathogenicity and epidemiological analysis, computational methods for designing the viral therapeutics, and consolidated information in the form of databases against the known pathogenic viruses. This enriched review of the generally applicable viral informatics approaches aims to provide an overview of available resources capable of carrying out the desired task and may be utilized to expand additional strategies to improve the quality of translation viral informatics research.

Computational Investigations on the Natural Small Molecule as an Inhibitor of Programmed Death Ligand 1 for Cancer Immunotherapy.

Several therapeutic monoclonal antibodies approved by the FDA are available against the PD-1/PD-L1 (programmed death 1/programmed death ligand 1) immune checkpoint axis, which has been an unprecedented success in cancer treatment. However, existing therapeutics against PD-L1, including small molecule inhibitors, have certain drawbacks such as high cost and drug resistance that challenge the currently available anti-PD-L1 therapy. Therefore, this study presents the screening of 32,552 compounds from the Natural Product Atlas database against PD-L1, including three steps of structure-based virtual screening followed by binding free energy to refine the ideal conformation of potent PD-L1 inhibitors. Subsequently, five natural compounds, i.e., Neoenactin B1, Actinofuranone I, Cosmosporin, Ganocapenoid A, and 3-[3-hydroxy-4-(3-methylbut-2-enyl)phenyl]-5-(4-hydroxybenzyl)-4-methyldihydrofuran-2(3H)-one, were collected based on the ADMET (absorption, distribution, metabolism, excretion, and toxicity) profiling and binding free energy (>−60 kcal/mol) for further computational investigation in comparison to co-crystallized ligand, i.e., JQT inhibitor. Based on interaction mapping, explicit 100 ns molecular dynamics simulation, and end-point binding free energy calculations, the selected natural compounds were marked for substantial stability with PD-L1 via intermolecular interactions (hydrogen and hydrophobic) with essential residues in comparison to the JQT inhibitor. Collectively, the calculated results advocate the selected natural compounds as the putative potent inhibitors of PD-L1 and, therefore, can be considered for further development of PD-L1 immune checkpoint inhibitors in cancer immunotherapy.

Discovery of Bispecific Lead Compounds from Azadirachta indica against ZIKA NS2B-NS3 Protease and NS5 RNA Dependent RNA Polymerase Using Molecular Simulations.

Zika virus (ZIKV) has been characterized as one of many potential pathogens and placed under future epidemic outbreaks by the WHO. However, a lack of potential therapeutics can result in an uncontrolled pandemic as with other human pandemic viruses. Therefore, prioritized effective therapeutics development has been recommended against ZIKV. In this context, the present study adopted a strategy to explore the lead compounds from Azadirachta indica against ZIKV via concurrent inhibition of the NS2B-NS3 protease (ZIKVpro) and NS5 RNA dependent RNA polymerase (ZIKVRdRp) proteins using molecular simulations. Initially, structure-based virtual screening of 44 bioflavonoids reported in Azadirachta indica against the crystal structures of targeted ZIKV proteins resulted in the identification of the top four common bioflavonoids, viz. Rutin, Nicotiflorin, Isoquercitrin, and Hyperoside. These compounds showed substantial docking energy (-7.9 to -11.01 kcal/mol) and intermolecular interactions with essential residues of ZIKVpro (B:His51, B:Asp75, and B:Ser135) and ZIKVRdRp (Asp540, Ile799, and Asp665) by comparison to the reference compounds, O7N inhibitor (ZIKVpro) and Sofosbuvir inhibitor (ZIKVRdRp). Besides, long interval molecular dynamics simulation (500 ns) on the selected docked poses reveals stability of the respective docked poses contributed by intermolecular hydrogen bonds and hydrophobic interactions. The predicted complex stability was further supported by calculated end-point binding free energy using molecular mechanics generalized born surface area (MM/GBSA) method. Consequently, the identified common bioflavonoids are recommended as promising therapeutic inhibitors of ZIKVpro and ZIKVRdRp against ZIKV for further experimental assessment.

Understanding on the possible routes for SARS CoV-2 invasion via ACE2 in the host linked with multiple organs damage.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), accountable for causing the coronavirus diseases 2019 (COVID-19), is already declared as a pandemic disease globally. Like previously reported SARS-CoV strain, the novel SARS-CoV-2 also initiates the viral pathogenesis via docking viral spike-protein with the membranal angiotensin-converting enzyme 2 (ACE2) - a receptor on variety of cells in the human body. Therefore, COVID-19 is broadly characterized as a disease that targets multiple organs, particularly causing acute complications via organ-specific pathogenesis accompanied by destruction of ACE2+ cells, including alveolus, cardiac microvasculature, endothelium, and glomerulus. Under such circumstances, the high expression of ACE2 in predisposing individuals associated with anomalous production of the renin-angiotensin system (RAS) may promote enhanced viral load in COVID-19, which comparatively triggers excessive apoptosis. Furthermore, multi-organ injuries were found linked to altered ACE2 expression and inequality between the ACE2/angiotensin-(1-7)/mitochondrial Ang system (MAS) and renin-angiotensin-system (RAS) in COVID-19 patients. However, the exact pathogenesis of multi-organ damage in COVID-19 is still obscure, but several perspectives have been postulated, involving altered ACE2 expression linked with direct/indirect damages by the virus-induced immune responses, such as cytokinin storm. Thus, insights into the invasion of a virus with respect to ACE2 expression site can be helpful to simulate or understand the possible complications in the targeted organ during viral infection. Hence, this review summarizes the multiple organs invasion by SARS CoV-2 linked with ACE2 expression and their consequences, which can be helpful in the management of the COVID-19 pathogenesis under life-threatening conditions.

SARS-CoV-2 Mpro inhibitors: identification of anti-SARS-CoV-2 Mpro compounds from FDA approved drugs.

Recent outbreak of COVID-19 pandemic caused by severe acute respiratory syndrome-Coronavirus-2 (SARS-CoV-2) has raised serious global concern for public health. The viral main 3-chymotrypsin-like cysteine protease (Mpro), known to control coronavirus replication and essential for viral life cycle, has been established as an essential drug discovery target for SARS-CoV-2. Herein, we employed computationally screening of Druglib database containing FDA approved drugs against active pocket of SARS-CoV-2 Mpro using MTiopen screen web server, yields a total of 1051 FDA approved drugs with docking energy >-7 kcal/mol. The top 10 screened potential compounds against SARS-CoV-2 Mpro were then studied by re-docking, binding affinity, intermolecular interaction, and complex stability via 100 ns all atoms molecular dynamics (MD) simulation followed by post-simulation analysis, including end point binding free energy, essential dynamics, and residual correlation analysis against native crystal structure ligand N3 inhibitor. Based on comparative molecular simulation and interaction profiling of the screened drugs with SARS-CoV-2 Mpro revealed R428 (-10.5 kcal/mol), Teniposide (-9.8 kcal/mol), VS-5584 (-9.4 kcal/mol), and Setileuton (-8.5 kcal/mol) with stronger stability and affinity than other drugs and N3 inhibitor; and hence, these drugs are advocated for further validation using in vitro enzyme inhibition and in vivo studies against SARS-CoV-2 infection.Communicated by Ramaswamy H. Sarma.

Determination of tyrosinase-cyanidin-3-O-glucoside and (-/+)-catechin binding modes reveal mechanistic differences in tyrosinase inhibition.

Tyrosinase, exquisitely catalyzes the phenolic compounds into brown or black pigment, inhibition is used as a treatment for dermatological or neurodegenerative disorders. Natural products, such as cyanidin-3-O-glucoside and (-/+)-catechin, are considered safe and non-toxic food additives in tyrosinase inhibition but their ambiguous inhibitory mechanism against tyrosinase is still elusive. Thus, we presented the mechanistic insights into tyrosinase with cyanidin-3-O-glucoside and (-/+)-catechin using computational simulations and in vitro assessment. Initial molecular docking results predicted ideal docked poses (- 9.346 to - 5.795 kcal/mol) for tyrosinase with selected flavonoids. Furthermore, 100 ns molecular dynamics simulations and post-simulation analysis of docked poses established their stability and oxidation of flavonoids as substrate by tyrosinase. Particularly, metal chelation via catechol group linked with the free 3-OH group on the unconjugated dihydropyran heterocycle chain was elucidated to contribute to tyrosinase inhibition by (-/+)-catechin against cyanidin-3-O-glucoside. Also, predicted binding free energy using molecular mechanics/generalized Born surface area for each docked pose was consistent with in vitro enzyme inhibition for both mushroom and murine tyrosinases. Conclusively, (-/+)-catechin was observed for substantial tyrosinase inhibition and advocated for further investigation for drug development against tyrosinase-associated diseases.

Insights into cyclooxygenase-2 inhibition by isolated bioactive compounds 3-caffeoyl-4-dihydrocaffeoyl quinic acid and isorhamnetin 3-O-ß-D-glucopyranoside from Salicornia herbacea.

BACKGROUND: Cyclooxygenase-2 (COX-2) is an important enzyme with numerous biological functions. Overexpression of COX-2 has been associated with various inflammatory-related diseases and therefore, projected as an important pharmacological target. PURPOSE: We aimed to investigate the inhibitory potential of isolated bioactive compounds, 3-caffeoyl-4-dihydrocaffeoyl quinic acid (CDQ) and isorhamnetin 3-O-ß-d-glucopyranoside (IDG), from Salicornia herbacea against COX-2 using both computational and in vitro approaches. METHODS: Computational analysis, including molecular docking, molecular dynamics (MD) simulations, and post-simulations analysis, were employed to estimate the binding affinity and stability of CDQ and IDG in the catalytic pocket of COX-2 against Celecoxib as positive control. These predictions were further evaluated using in vitro enzyme inhibition as well as gene expression mediation in macrophages cells. RESULTS: Molecular docking analysis revealed substantial binding energy of CDQ (-6.1 kcal/mol) and IDG (-5.9 kcal/mol) with COX-2, which are lower than Celecoxib (-8.1 kcal/mol). MD simulations (100 ns) and post simulation analysis exhibited the substantial stability and binding affinity of docked CDQ and IDG compounds with COX-2. In vitro assays indicated significant COX-2 inhibition by CDQ (IC50 = 76.91 ± 2.33 µM) and IDG (IC50 = 126.06 ± 9.44 µM). This result supported the inhibitory potential of isolated bioactive compounds against COX-2. Also, a cellular level study revealed a downregulation of COX-2 expression in tumor necrosis factor-alpha stimulated RAW 264.7 macrophages treated with CDQ and IDG. CONCLUSION: Computational and experimental analysis of CDQ and IDG from S. herbacea established their potential in the inhibition and mediation of COX-2. Hence, CDQ and IDG can be considered for therapeutic development against COX-2 linked disorders, such as inflammation and cancer. Furthermore, CDQ and IDG structures can be served as a lead compound for the development of advanced novel anti-inflammatory drugs.

Drug repurposing for ligand-induced rearrangement of Sirt2 active site-based inhibitors via molecular modeling and quantum mechanics calculations.

Sirtuin 2 (Sirt2) nicotinamide adenine dinucleotide-dependent deacetylase enzyme has been reported to alter diverse biological functions in the cells and onset of diseases, including cancer, aging, and neurodegenerative diseases, which implicate the regulation of Sirt2 function as a potential drug target. Available Sirt2 inhibitors or modulators exhibit insufficient specificity and potency, and even partially contradictory Sirt2 effects were described for the available inhibitors. Herein, we applied computational screening and evaluation of FDA-approved drugs for highly selective modulation of Sirt2 activity via a unique inhibitory mechanism as reported earlier for SirReal2 inhibitor. Application of stringent molecular docking results in the identification of 48 FDA-approved drugs as selective putative inhibitors of Sirt2, but only top 10 drugs with docking scores > - 11 kcal/mol were considered in reference to SirReal2 inhibitor for computational analysis. The molecular dynamics simulations and post-simulation analysis of Sirt2-drug complexes revealed substantial stability for Fluphenazine and Nintedanib with Sirt2. Additionally, developed 3D-QSAR-models also support the inhibitory potential of drugs, which exclusively revealed highest activities for Nintedanib (pIC50 ≥ 5.90 µM). Conclusively, screened FDA-approved drugs were advocated as promising agents for Sirt2 inhibition and required in vitro investigation for Sirt2 targeted drug development.

Structure-Based Identification of Natural Products as SARS-CoV-2 Mpro Antagonist from Echinacea angustifolia Using Computational Approaches.

Coronavirus disease-19 (COVID-19) pandemic, caused by the novel SARS-CoV-2 virus, continues to be a global threat. The number of cases and deaths will remain escalating due to the lack of effective therapeutic agents. Several studies have established the importance of the viral main protease (Mpro) in the replication of SARS-CoV-2 which makes it an attractive target for antiviral drug development, including pharmaceutical repurposing and other medicinal chemistry approaches. Identification of natural products with considerable inhibitory potential against SARS-CoV-2 could be beneficial as a rapid and potent alternative with drug-likeness by comparison to de novo antiviral drug discovery approaches. Thereof, we carried out the structure-based screening of natural products from Echinacea-angustifolia, commonly used to prevent cold and other microbial respiratory infections, targeting SARS-CoV-2 Mpro. Four natural products namely, Echinacoside, Quercetagetin 7-glucoside, Levan N, Inulin from chicory, and 1,3-Dicaffeoylquinic acid, revealed significant docking energy (>-10 kcal/mol) in the SARS-CoV-2 Mpro catalytic pocket via substantial intermolecular contacts formation against co-crystallized ligand (<-4 kcal/mol). Furthermore, the docked poses of SARS-CoV-2 Mpro with selected natural products showed conformational stability through molecular dynamics. Exploring the end-point net binding energy exhibited substantial contribution of Coulomb and van der Waals interactions to the stability of respective docked conformations. These results advocated the natural products from Echinacea angustifolia for further experimental studies with an elevated probability to discover the potent SARS-CoV-2 Mpro antagonist with higher affinity and drug-likeness.

Exploration of natural compounds with anti-SARS-CoV-2 activity via inhibition of SARS-CoV-2 Mpro.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a dreaded pandemic in lack of specific therapeutic agent. SARS-CoV-2 Mpro, an essential factor in viral pathogenesis, is recognized as a prospective therapeutic target in drug discovery against SARS-CoV-2. To tackle this pandemic, Food and Drug Administration-approved drugs are being screened against SARS-CoV-2 Mpro via in silico and in vitro methods to detect the best conceivable drug candidates. However, identification of natural compounds with anti-SARS-CoV-2 Mpro potential have been recommended as rapid and effective alternative for anti-SARS-CoV-2 therapeutic development. Thereof, a total of 653 natural compounds were identified against SARS-CoV-2 Mpro from NP-lib database at MTi-OpenScreen webserver using virtual screening approach. Subsequently, top four potential compounds, i.e. 2,3-Dihydroamentoflavone (ZINC000043552589), Podocarpusflavon-B (ZINC000003594862), Rutin (ZINC000003947429) and Quercimeritrin 6"-O-L-arabinopyranoside (ZINC000070691536), and co-crystallized N3 inhibitor as reference ligand were considered for stringent molecular docking after geometry optimization by DFT method. Each compound exhibited substantial docking energy >-12 kcal/mol and molecular contacts with essential residues, including catalytic dyad (His41 and Cys145) and substrate binding residues, in the active pocket of SARS-CoV-2 Mpro against N3 inhibitor. The screened compounds were further scrutinized via absorption, distribution, metabolism, and excretion - toxicity (ADMET), quantum chemical calculations, combinatorial molecular simulations and hybrid QM/MM approaches. Convincingly, collected results support the potent compounds for druglikeness and strong binding affinity with the catalytic pocket of SARS-CoV-2 Mpro. Hence, selected compounds are advocated as potential inhibitors of SARS-CoV-2 Mpro and can be utilized in drug development against SARS-CoV-2 infection.

Anti-dengue infectivity evaluation of bioflavonoid from Azadirachta indica by dengue virus serine protease inhibition.

Dengue virus (DENV) serine protease enzyme, i.e. NS2B-NS3pro (non-structural protein 2B-non-structural protein 3) has been approved as prime drug target for the drug discovery against dengue infection, because of its essential role in viral replication. This study demonstrates the potential of bioflavonoids from Azadirachta indica against dengue infection using computational and experimental approach. Initially, 49 bioflavonoids reported in Azadirachta indica were collected and virtually screened on the catalytic triad of DENV protease, results in the identification of kaempferol-3-O-rutinoside (-9.555 kcal/mol), rutin (-9.324 kcal/mol), hyperoside (-7.879 kcal/mol), and epicatechin (-7.622 kcal/mol) as potent viral protease inhibitors against reference compound quercetin (-6.94 kcal/mol). Subsequently, these docked complexes were analyzed for the stability via molecular dynamics simulations and free binding energy calculations, suggested the considerable stability of selected bioflavonoids with viral protease. Additionally, density functional theory and ADMET (Absorption, Distribution, Metabolism, Excretion and Toxicity) analysis indicated the least chemical reactivity and considerable medicinal properties, respectively for the screened bioflavonoids by comparison to quercetin. Accordingly, kaempferol 3-O-ß-rutinoside and epicatechin were evaluated at various concentrations for cell viability (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay) and in vitro antiviral activity (focus forming unit assay) against DENV-2 strain. The antiviral assay showed dose dependent inhibition of DENV-2 infectivity by the selected compounds while maximum 77.7% and 66.2% viral inhibition were recorded for 100 µM kaempferol 3-O-ß-rutinoside and 1000 µM epicatechin, respectively without significant cell toxicity. These results suggested the potential of bioflavonoids from Azadirachta indica in the development of effective drug against dengue infection.Communicated by Ramaswamy H. Sarma.

Structure-based screening and validation of bioactive compounds as Zika virus methyltransferase (MTase) inhibitors through first-principle density functional theory, classical molecular simulation and QM/MM affinity estimation.

Recent Zika virus (ZIKV) outbreak and association with human diseases such as neurological disorders have raised global health concerns. However, in the absence of an approved anti-ZIKV drug has generated urgency for the drug development against ZIKV infection. Here, structure-based virtual screening of 8589 bioactive compounds, screened at the substrate-binding site of ZIKV nonstructural 5 (NS5)-based structure N-terminal methyltransferase (MTase) domain followed by ADMET (absorption, distribution, metabolism, excretion and toxicity) profiling concluded the four potential lead inhibitors, i.e. (4-acetylamino-benzenesulfonylamino)-acetic acid (F3342-0450), 3-(5-methylfuran-2-yl)-N-(4-sulfamoylphenyl)propanamide (F1736-0142), 8-(2-hydroxy-ethylamino)-1,3-dimethyl-7-(3-methyl-benzyl)-3,7-dihydro-purine-2,6-dione (F0886-0080) and N-[4-(aminosulfonyl)phenyl]-2,3-dihydro-1,4-benzodioxine-2-carboxamide (F0451-2187). Collectively, extra precision docking and Density Functional Theory(DFT) calculations studies identified the F3342-0450 molecule, having strong interactions on the active site of MTase, further supported by molecular dynamics simulation, binding affinity and hybrid QM/MM calculations, suggest a new drug molecule for the antiviral drug development against ZIKV infection. Communicated by Ramaswamy H. Sarma.