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.

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.