Relation of ACE2 with co-morbidity factors in SARS-CoV-2 pathogenicity.

In the last 3 years of the pandemic situation, SARS-CoV-2 caused a significant number of deaths. Infection rates for symptomatic and asymptomatic patients are higher than that for death. Eventually, researchers explored that the major deaths are attributed to several comorbidity factors. The confounding factors and gender-associated infection/death rate are observed globally. This suggests that SARS-CoV-2 selects the human system recognizing the internal comorbid environment. This article explored the influences of hypertension, diabetes, cardiovascular, and renovascular disorders in COVID-19 severity and mortality. Brief mechanistic layouts have been presented here, indicating some of the comorbidity as the critical determinant in the COVID-19 pathogenesis and related mortality.

Clinicodemographic profile and predictors of poor outcome in hospitalised COVID-19 patients: a single-centre, retrospective cohort study from India.

OBJECTIVES: Primary objective was to study the clinicodemographic profile of hospitalised COVID-19 patients at a tertiary-care centre in India. Secondary objective was to identify predictors of poor outcome. SETTING: Single centre tertiary-care level. DESIGN: Retrospective cohort study. PARTICIPANTS: Consecutively hospitalised adults patients with COVID-19. PRIMARY AND SECONDARY OUTCOME MEASURES: Primary outcome variable was in-hospital mortality. Covariables were known comorbidities, clinical features, vital signs at the time of admission and on days 3-5 of admission, and initial laboratory investigations. RESULTS: Intergroup differences were tested using χ2 or Fischer's exact tests, Student's t-test or Mann-Whitney U test. Predictors of mortality were evaluated using multivariate logistic regression model. Out of 4102 SARS-CoV-2 positive patients admitted during 1-year period, 3268 (79.66%) survived to discharge and 834 (20.33%) died in the hospital. Mortality rates increased with age. Death was more common among males (OR 1.51, 95% CI 1.25 to 1.81). Out of 261 cases analysed in detail, 55.1% were in mild, 32.5% in moderate and 12.2% in severe triage category. Most common clinical presentations in the subgroup were fever (73.2%), cough/coryza (65.5%) and breathlessness (54%). Hypertension (45.2%), diabetes mellitus (41.8%) and chronic kidney disease (CKD; 6.1%) were common comorbidities. Disease severity on admission (adjusted OR 12.53, 95% CI 4.92 to 31.91, p<0.01), coagulation defect (33.21, 3.85-302.1, p<0.01), CKD (5.67, 1.08-29.64, p=0.04), high urea (11.05, 3.9-31.02, p<0.01), high prothrombin time (3.91, 1.59-9.65, p<0.01) and elevated ferritin (1.02, 1.00-1.03, p=0.02) were associated with poor outcome on multivariate regression. A strong predictor of mortality was disease progression on days 3-5 of admission (adjusted OR 13.66 95% CI 3.47 to 53.68). CONCLUSION: COVID-19 related mortality in hospitalised adult patients at our center was similar to the developed countries. Progression in disease severity on days 3-5 of admission or days 6-13 of illness onset acts as 'turning point' for timely referral or treatment intensification for optimum use of resources.

Immunoinformatic paradigm predicts macrophage and T-cells epitope responses against globally conserved spike fragments of SARS CoV-2 for universal vaccination.

BACKGROUND: Different quickly-developed vaccines are introduced against COVID-19 with inconclusive results especially against some recent variants. Eventually, somewhere COVID-19 cases decline and in some countries it revived with some new mutant-variants (i.e. D614G, Delta and Omicron). OBJECTIVES: Proposing a universal vaccination strategy by screening globally-conserved SARS-CoV-2 spike-epitopes. METHODS: Presently, several conserved (186-countries) sequences including multiple-variants (ClustalX2) epitopic-regions (SVMTriP and IEDB) and in-silico mutants of SARS-CoV-2 spike-protein-fragments (Cut1-4) were screened for their stability against proteases, antigenicity (VaxiJen V2.0 and for glycosylation effects NetOGlyc-NetNGlyc), MHCI/II reactivity (IEDB-TOOLS) and CD4+ responses by molecular-docking (Haddock2.4/PatchDock). We also examined Molecular-Dynamic-Simulation (myPresto verson-5) of MHC-II 3LQZ with 3-Cuts and T-cell 2-molecules (1KGC/4JRX) with SM3-Cut. The MD-simulation was run with 5000-cycles after 300 k-heating/1-atm pressure adjustment for the system-equilibration. Finally, 1000 fs production was run. RESULTS: The cut4-mutant (SRLFRKSNLKPFERD) showed the highest combined-score 48.23548 and Immunogenicity-Score of 92.0887. The core-sequence SRLFRKSNL showed the highest Median-Percentile-Rank (7-HLA-allele) of 19. CD4+ immunogenicity also confirms the representation of the CUT4TM2 epitope SRLFRKSNL by MHC Class II. The epitope YNYKYRLFR from CUT4 showed an IC50 of ∼30 nM with allele HLA-DRB1*11:01 and HLA-DRB5*01:01 with plenty H-bonding. Cut4 double-mutants strongly interact with the exposed T-cell surface and are facilitated by its receptors. The MD-simulation data suggest that TM2 has a maximum RMSD value of 1.7 Å, DM2 is at 1.55 Å and SM3 is at 1.5 Å. These variations correspond to structural adjustments and involve binding/unbinding chemical interactions. The RMSD plot shows that 1KGC T-cell molecule is at 2.2 Å and the 4JRX is at 1.2 Å, which increases with the simulation time. CONCLUSIONS: Screening of conserved SARS-CoV-2 spike fragments helps to find the most stable antigenic-determinant which with some mutations showed better antigenicity. Further studies are necessary to develop global vaccination strategies against COVID-19.

Global conserved RBD fraction of SARS-CoV-2 S-protein with T500S mutation in silico significantly blocks ACE2 and rejects viral spike.

BACKGROUND: SARS-CoV-2 developed global-pandemic with millions of infections/deaths. As it is urgently necessary it is assumed that some blockers/inhibitors of ACE2 could be helpful to resist the binding of viral-spike Receptor-Binding-Domain (RBD). METHODS: Here, conserved RBD from 186-countries were compared with WUHAN-Hu-1 wild-type (CLUSTAL-X2/Pymol). The RBD of ACE2-bound nCOV2 crystal-structure 6VW1 was analyzed by Haddock-PatchDock. Extensive structural study/trial to introduce point/double/triple mutations in the different locations of CUT4 (most-effective from total 4 proposed fragments; CUTs) were tested with Swiss-Model-Expacy. RESULTS: Blind-docking of mutated-CUTs in ACE2 completely rejected the nCOV2 binding to ACE2. Further, competitive-docking/binding-analyses (by PRODIGY) demonstrated few more bonding (LYS31-PHE490 and GLN42-GLN498) of CUT4 (than wild) and hindered TYR41-THR500 interaction with ACE2. Moreover, mutated-CUT4 even showed higher blocking effect against spike-ACE2 binding. CONCLUSION: In summary, CUT4-mutant rejects whole glycosylated-nCoV2 in all pre-dock, post-dock and competitive-docking conditions. The present work strategy is relevant because it could be able to block at the first level entry of the virus to the host cells. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s41231-022-00109-5.

Better binding informatics of delta variants (B.1.617.2) with ACE2 than wild, D614G or N501Y CoV-2 is fully blocked by 84 amino-acid cut of wild spike

Background and objective The B.1.617.2 known as the Delta-variant harbors diverse Spike-mutations with developed transmissibility and immune-evasion more than wild/D614G/N501Y variants. The Delta-variant claimed comparatively a large number of lives globally. In the present study, the binding-affinities of these variants’ spikes to the human lung-ACE2 were investigated. Further, a certain portion of the spike-protein with a desired mutation was tested in-silico to block the ACE2. Methods Structure of spike-variants were retrieved from PDB/GISAID and used for homology-modeling (SWISS-MODEL). A different combination of spike-ACE2 binding 1:1 or competitive blind-docking was performed using the Haddock 2.4 web-server. Eventually, two cut-segments (84 amino-acid of wild-spike, 432–516 Cut1) and its mutant T500S;Cut 2 were screened (Swiss-model Expasy-server) as blocker/inhibitor of all spike-variants (PyMOL-V2.2.2). Results It is shown that the stability and energy of the Delta binding-affinity to ACE2 is far more than others. The number H-bonding (5), their lengths (1.7 Å-2.8 Å) and energy, Van-der-Walls energy, Haddock-score were highly favorable for more stable-binding of Delta-RBD to ACE2. The Ramachandran-plot (Zlab/UMassMed Bioinfo) data supports this. We observed the best Haddock score as −120.8±2.6 for Delta with Van-der-Walls and electrostatic-energy as −62.9 and −208.7, respectively. The highest binding-affinity (ΔG) was −10.7 kcal/mol. Its THR500 and GLN506 strongly bind with the LYS353 of ACE2. The Cut1 and its mutant T500S completely blocked Delta-spike binding to ACE2 with ΔG -8.4 and −10.6 kcal/mol, respectively. But during the comparison between 2 Cuts, Cut1 showed better results. Conclusions Fractioned spike-protein from the conserved Receptor-Binding-Domain (RBD) could universally block the virus at entry-level, thus completely protecting any intercellular metabolism. Bioinformatics is an emerging field for screening of some drug/therapeutic targets from numerous options, minimizing time and expenses.

Effects of theaflavin-gallate in-silico binding with different proteins of SARS-CoV-2 and host inflammation and vasoregulations referring an experimental rat-lung injury.

Background SARS-CoV-2 claimed 5,209,104 lives, infected 260,997,910 individuals, globally. Infection is caused due to exposure or susceptibility; deaths occur due to age,comorbidity,higher-viral-load, immuno-suppression, inflammation, and multi-organ failure. Theaflavin-gallate, the major black tea component, showed previous evidence to inhibit HIV-1. Purpose As theaflavin-gallate prevents experimental rat-lung injury, the study of inhibitory effects of theaflavin-gallate was done, on SARS-CoV-2proteins and various host proteins related to some adverse effects in COVID-19 patients. Study Design Currently, some prospective phytochemical, black-tea (Camellia-sinensis) extract (BTE) was initially tested in vivo in strong oxidant-mutagen arsenic-induced model rat lung injury similar to that of COVID-19 manifestations like severe inflammation, oxidative stress, lung tissue degenerations, and apoptotic death. In silico, extensive bioinformatics and molecular docking experiments were performed on all catechin or theaflavin derivatives of C. sinensis, and finally theaflavin-3'-O-gallate (TFMG) were screened for blocking or inactivation of several proteins of SARS CoV-2 and host adversely-acting proteins or factors. Methods In vivo experiments in DNA stability (ladder, comet assay), free radicals attack (malondialdehyde; MDA, superoxide dismutase SOD, catalase gel-zymogram assay), extra cellular matrix damage (matrix metalloprotease; MMP2and9 zymogram assay) and inflammation (TNF-α, ELISA). In silico experiments- molecular docking by AutoDock-Patchdock analysis, Surface Topology Calculation by CASTp, Grid-value calculation, and Ramachandran Plot study. Results The BTE showed strong protection of lung DNA and cell-matrix by decreasing MMPs, TNF-α, and free radicals, the same factors affecting COVID-19 patients. In silico data suggest that TFMG significantly blocked the entry, exit, and amino acids at catalytic active-site of more than thirty proteins including viral (nsp1,nsp2,Mainpro,∼-9.0 kcal/mol) and host inflammatory, oxidants, apoptotic, vaso-destabilizer molecules (FAS, CD40R, BCL2, TLR2, ∼ -10 and ACE1or2 ∼ -9.0 and AT1or2∼ -7.5 kcal/mol and more). When the binding energy of TFMG ranged from -7 to -11.7 kcal/mol(average -9.0) the same for hydroxy­Chloroquine ranged (-2.5 to -7 average -4.5) and dexamethasone (-3.0 to -6.0, average -4.0 kcal/mol). Conclusions TFMG has some novel physicochemical or structural properties like (ACE values of binding to all tested proteins, -300 to -625), (for TFMG H-bond acceptor or donor: 15/10, for TFDG 20/13). Their topological-polar-surface-area (264Ų and 351Ų) and travel depth/height; 17.0/9.6 Å and 15.4/11.3 Å, respectively) were more potent than other compounds. Conclusively, the efficacy of TFMG may be further verified.

Nigellidine (Nigella sativa, black-cumin seed) docking to SARS CoV-2 nsp3 and host inflammatory proteins may inhibit viral replication/transcription and FAS-TNF death signal via TNFR 1/2 blocking.

Tissue damage occurs in COVID-19 patients due to nsp3-induced Fas-FasL interaction/TNF-related apoptosis. Presently, possible therapeutic-drug, nigellidine against was screened by bioinformatics studies COVID-19. Atomic-Contact-Energy (ACE) and binding-blocking effects were explored of nigellidine (Nigella sativa L.) in the active/catalytic sites of viral-protein nsp3 and host inflammatory/apoptotic signaling-molecules Fas/TNF receptors TNFR1/TNFR2. A control binding/inhibition of Oseltamivir to influenza-virus neuraminidase was compared here. In AutoDock, Oseltamivir binding-energy (BE) and inhibition-constant (KI) was -4.12 kcal/mol and 959.02. The ACE values (PatchDock) were -167.02/-127.61/-124.91/-122.17/-54.81/-47.07. The nigellidine BE/KI with nsp3 was -7.61 and 2.66, respectively (ACE values were -221.40/-215.62/-113.28). Nigellidine blocked FAS dimer by binding with a BE value of -7.41 kcal/mol. Its strong affinities to TNFR1 (-6.81) and TNFR2 (-5.1) are demonstrated. Our present data suggest that nigellidine may significantly block the TNF-induced inflammatory/Fas-induced apoptotic death-signaling in comparison with a positive-control drug Oseltamivir. Further studies are necessary before proposing nigellidine as medical drug.

Theaflavin-3'-O-gallate a Black-tea Constituent Blocked SARS CoV-2 RNA dependant RNA Polymerase Active-site with Better Docking Results than Remdesivir.

BACKGROUND: Replication of SARS-CoV-2 depends on viral RNA-dependent RNA-polymerase (RdRp). Remdesivir, the broad-spectrum RdRp inhibitor acts as nucleoside-analogues (NAs). Remdesivir has initially been repurposed as a promising drug against SARS-CoV-2 infection with some health hazards like liver damage, allergic reaction, low blood-pressure, and breathing-shortness, throat-swelling. In comparison, theaflavin-3'-O-gallate (TFMG), the abundant black tea component has gained importance in controlling viral infection. TFMG is a non-toxic, non-invasive, antioxidant, anticancer and antiviral molecule. RESULTS: Here, we analyzed the inhibitory effect of theaflavin-3'-O-gallate on SARS CoV-2 RdRp in comparison with remdesivir by molecular-docking study. TFMG has been shown more potent in terms of lower Atomic-Contact-Energy (ACE) and higher occupancy of surface area; -393.97 Kcal/mol and 771.90 respectively, favoured with lower desolvation-energy; -9.2: Kcal/mol. TFMG forms more rigid electrostatic and H-bond than remdesivir. TFMG showed strong affinity to RNA primer and template and RNA passage-site of RdRp. CONCLUSIONS: TFMG can block the catalytic residue, NTP entry site, cation binding site, nsp7-nsp12 junction with binding energy of -6. 72 Kcal/mol with Ki value of 11.79, and interface domain with binding energy of -7.72 and -6.16 Kcal/mol with Ki value of 2.21 and 30.71 µM. And most importantly, TFMG shows antioxidant/anti-inflammatory/antiviral effect on human studies.

In silico Nigellidine (N. sativa) bind to viral spike/active-sites of ACE1/2, AT1/2 to prevent COVID-19 induced vaso-tumult/vascular-damage/comorbidity.

COVID-19, a global-pandemic binds human-lung-ACE2. ACE2 causes vasodilatation. ACE2 works in balance with ACE1. The vaso-status maintains blood-pressure/vascular-health which is demolished in Covid-19 manifesting aldosterone/salt-deregulations/inflammations/endothelial-dysfunctions/hyper-hypo- tension, sepsis/hypovolemic-shock and vessel-thrombosis/coagulations. Here, nigellidine, an indazole-alkaloid was analyzed by molecular-docking for binding to different Angiotensin-binding-proteins (enzymes, ACE1(6en5)/ACE2(4aph)/receptors, AT1(6os1)/AT2(5xjm)) and COVID-19 spike-glycoprotein(6vsb). Nigellidine strongly binds to the spike-protein at the hinge-region/active-site-opening which may hamper proper-binding of nCoV2-ACE2 surface. Nigellidine effectively binds in the Angiotensin- II binding-site/entry-pocket (-7.54 kcal/mol, -211.76, Atomic-Contact-Energy; ACE-value) of ACE2 (Ki 8.68 and 8.3 µmol) in comparison to known-binder EGCG (-4.53) and Theaflavin-di-gallate (-2.85). Nigellidine showed strong-binding (Ki, 50.93 µmol/binding-energy -5.48 kcal/mol) to mono/multi-meric ACE1. Moreover, it binds Angiotensin-receptors, AT1/AT2 (Ki, 42.79/14.22 µmol, binding-energy, -5.96/-6.61 kcal/mol) at active-sites, respectively. This article reports the novel binding of nigellidine and subsequent blockage of angiotensin-binding proteins. The ACEs-blocking could restore Angiotensin-level, restrict vaso-turbulence in Covid patients and receptor-blocking might stop inflammatory/vascular impairment. Nigellidine may slowdown the vaso-fluctuations due to Angiotensin-deregulations in Covid patients. Angiotensin II-ACE2 binding (ACE-value -294.81) is more favorable than nigellidine-ACE2. Conversely, nigellidine-ACE1 binding-energy/Ki is lower than nigellidine-ACE2 values indicating a balanced-state between constriction-dilatation. Moreover, nigellidine binds to the viral-spike, closer-proximity to its ACE2 binding-domain. Taken together, Covid patients/elderly-patients, comorbid-patients (with hypertensive/diabetic/cardiac/renal-impairment, counting >80% of non-survivors) could be greatly benefited.

Active-site molecular docking of nigellidine with nucleocapsid-NSP2-MPro of COVID-19 and to human IL1R-IL6R and strong antioxidant role of Nigella sativa in experimental rats.

The recent outbreak of SARS CoV-2 has changed the global scenario of human lives/economy. A significant number of the non-survivors showed cardiac renal vasculature dysfunction. A 'cytokine storm' namely, interleukin IL6-IL1 receptors, i.e. IL6R-IL1R over-functioning was reported. Here, nigellidine, an indazole alkaloid and key component of Nigella sativa L. (NS) commonly known as black cumin seed was analysed for COVID-19 protein targeting and IL1R-IL6R inhibition through molecular docking study and biochemical study in experimental rat to evaluate antioxidative capacity. The NMR/X-ray crystallographic/electron microscopic structures of COVID-19 main protease (6LU7)/spike glycoprotein (6vsb)/NSP2 (QHD43415_2)/nucleocapsid (QHD43423), human IL1R (1itb)-IL6R (1pm9) from PDB were retrieved analysed for receptor-ligand interaction. Then, those structures were docked with nigellidine using AutoDock and PatchDock server. A brief comparison was made with nigellicine thymoquinone from N. sativa. Where nigellidine showed highest binding energy of -6.6 kcal/mol, ligand efficiency of -0.3 with COVID19 Nsp2 forming bonds with amino acid CYS240 present in binding pocket. Nigellidine showed strong interaction with main protease (BE: -6.38/LE: -0.29). Nigellidine showed affinity to IL1R (-6.23). The NS treated rat showed marked decline in ALP-SGPT-SGOT-malondialdehyde (MDA) than the basal levels. From the Western blot and activity analysis, it was observed that Nigellidine (sulphuryl group drug) showed no impact on phenol-catalysing ASTIV and steroid-catalysing oestrogen-sulphotransferase expressions and activities in liver tissue and thus has no influence in sulphation-mediated adverse metabolic processes. Conclusively, nigellidine has hepato-reno-protective/antioxidant-immunomodulatory/anti-inflammatory activities with inhibit potentials of COVID-19 proteins. Further validation is necessary.

Epigallocatechin gallate and theaflavin gallate interaction in SARS-CoV-2 spike-protein central channel with reference to the hydroxychloroquine interaction: Bioinformatics and molecular docking study.

SARS-CoV-2 or COVID-19 pandemic global outbreak created the most unstable situation of human health-economy. In the past two decades different parts of the word experienced smaller or bigger outbreak related to human coronaviruses. The spike glycoproteins of the COVID-19 (similar to SARS-CoV) attach to the angiotensin-converting enzyme (ACE2) and transit over a stabilized open state for the viral internalization to the host cells and propagate with great efficacy. Higher rate of mutability makes this virus unpredictable/less sensitive to the protein/nucleic acid based drugs. In this emergent situation, drug-induced destabilization of spike binding to RBD could be a good strategy. In the current study we demonstrated by bioinformatics (CASTp: computed atlas of surface topography of protein, PyMol: molecular visualization) and molecular docking (PatchDock and Autodock) experiments that tea flavonoids catechin products mainly epigallocatechin gallate or other like theaflavin gallate demonstrated higher atomic contact energy (ACE) value, binding energy, Ki value, ligand efficiency, surface area and more amino acid interactions than hydroxychloroquine (HCQ) during binding in the central channel of the spike protein. Moreover, out of three distinct binding sites (I, II and III) of spike core when HCQ binds only with site III (farthest from the nCoV-RBD of ACE2 contact), epigallocatechin gallate and theaflavin gallate bind all three sites. As sites I and II are in closer contact with open state location and viral-host contact area, these drugs might have significant effects. Taking into account the toxicity/side effects by chloroquine/HCQ, present drugs may be important. Our laboratory is working on tea flavonoids and other phytochemicals in the protection from toxicity, DNA/mitochondrial damage, inflammation and so on. The present data might be helpful for further analysis of flavonoids in this emergent pandemic situation.

Energetics and IC50 based epitope screening in SARS CoV-2 (COVID 19) spike protein by immunoinformatic analysis implicating for a suitable vaccine development.

BACKGROUND: The recent outbreak by SARS-CoV-2 has generated a chaos in global health and economy and claimed/infected a large number of lives. Closely resembling with SARS CoV, the present strain has manifested exceptionally higher degree of spreadability, virulence and stability possibly due to some unidentified mutations. The viral spike glycoprotein is very likely to interact with host Angiotensin-Converting Enzyme 2 (ACE2) and transmits its genetic materials and hijacks host machinery with extreme fidelity for self propagation. Few attempts have been made to develop a suitable vaccine or ACE2 blocker or virus-receptor inhibitor within this short period of time. METHODS: Here, attempt was taken to develop some therapeutic and vaccination strategies with a comparison of spike glycoproteins among SARS-CoV, MERS-CoV and the SARS-CoV-2. We verified their structure quality (SWISS-MODEL, Phyre2, and Pymol) topology (ProFunc), motifs (MEME Suite, GLAM2Scan), gene ontology based conserved domain (InterPro database) and screened several epitopes (SVMTrip) of SARS CoV-2 based on their energetics, IC50 and antigenicity with regard to their possible glycosylation and MHC/paratope binding (Vaxigen v2.0, HawkDock, ZDOCK Server) effects. RESULTS: We screened here few pairs of spike protein epitopic regions and selected their energetic, Inhibitory Concentration50 (IC50), MHC II reactivity and found some of those to be very good target for vaccination. A possible role of glycosylation on epitopic region showed profound effects on epitopic recognition. CONCLUSION: The present work might be helpful for the urgent development of a suitable vaccination regimen against SARS CoV-2.