key: cord-0943994-ws8o94ur authors: Sarkar, Lucky; Oko, Lauren; Gupta, Soham; Bubak, Andrew N.; Das, Bishnu; Gupta, Parna; Safiriyu, Abass Alao; Singhal, Chirag; Neogi, Ujjwal; Bloom, David; Banerjee, Arup; Mahalingam, Ravi; Cohrs, Randall J.; Koval, Michael; Shindler, Kenneth S.; Pal, Debnath; Nagel, Maria; Sarma, Jayasri Das title: Azadirachta indica A. Juss bark extract and its Nimbin isomers restrict β-coronaviral infection and replication date: 2022-02-15 journal: Virology DOI: 10.1016/j.virol.2022.01.002 sha: c77181d7c544cc3848d2c4f1ec7955de4e3a3040 doc_id: 943994 cord_uid: ws8o94ur Emerging mutations in the SARS-CoV-2 genome pose a challenge for vaccine development and antiviral therapy. The antiviral efficacy of Azadirachta indica bark extract (NBE) was assessed against SARS-CoV-2 and m-CoV-RSA59 infection. Effects of in vivo intranasal or oral NBE administration on viral load, inflammatory response, and histopathological changes were assessed in m-CoV-RSA59-infection. NBE administered inhibits SARS-CoV-2 and m-CoV-RSA59 infection and replication in vitro, reducing Envelope and Nucleocapsid gene expression. NBE ameliorates neuroinflammation and hepatitis in vivo by restricting viral replication and spread. Isolated fractions of NBE enriched in Nimbin isomers shows potent inhibition of m-CoV-RSA59 infection in vitro. In silico studies revealed that NBE could target Spike and RdRp of m-CoV and SARS-CoV-2 with high affinity. NBE has a triterpenoids origin that may allow them to competitively target panoply of viral proteins to inhibit mouse and different strains of human coronavirus infections, suggesting its potential as an antiviral against pan-β-Coronaviruses. boosts host immunity and metabolism (Alzohairy, 2016) . Its antibacterial, anti-inflammatory, 183 anti-cancer, anti-allergic, anti-parasitic, and antifungal activities support repurposing of this 184 drug to combat COVID-19 (Lim, Teh, & Tan, 2021) . In silico studies suggest that Neem DBT astrocytoma cells used for titre estimation were maintained in DMEM supplemented with 237 10% FBS and 1% P/S, 10 mM HEPES, 7.5% NaHCO3 and 0.1% L-glutamine. Therapeutics Role of Azadirachta indica (Neem) and Their Active Constituents 634 in Diseases Prevention and Treatment In Vivo and In Vitro 637 Evaluation of Pharmacological Potentials of Secondary Bioactive Metabolites of Dalbergia 638 candenatensis Leaves Dysregulation in Akt/mTOR/HIF-1 signaling identified by proteo-transcriptomics of 642 SARS-CoV-2 infected cells Screening of potential 645 drug from Azadirachta Indica (Neem) extracts for SARS-CoV-2: An insight from molecular 646 docking and MD-simulation studies Gastroprotective effect of Neem (Azadirachta indica) bark extract: 650 possible involvement of H(+)-K(+)-ATPase inhibition and scavenging of hydroxyl radical Enhancement of immune responses to neem leaf extract 653 (Azadirachta indica) correlates with antineoplastic activity in BALB/c-mice SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically 706 Proven Protease Inhibitor Novel therapeutic approaches for treatment of COVID-19 Nasal priming by a murine coronavirus provides protective immunity against lethal 712 heterologous virus pneumonia Structural and functional properties CoV-2 spike protein: potential antivirus drug development for COVID-19 Neutralizing Antibodies against SARS-CoV-2 and Other Human 717 Omicron overpowers key COVID antibody treatments in early tests Protective 721 effects of neem (Azadirachta indica A. Juss.) leaf extract against cigarette smoke-and 722 lipopolysaccharide-induced pulmonary inflammation Medicinal Plants in COVID-19: Potential and Limitations Genomic characterisation and 727 epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding Neurologic Manifestations of 730 Hospitalized Patients With Coronavirus Disease Quantitation of spinal cord demyelination, 733 remyelination, atrophy, and axonal loss in a model of progressive neurologic injury Longitudinal Serological Analysis and Neutralizing Antibody Levels in Coronavirus Disease 2019 737 Convalescent Patients Juss) Capsules for Prophylaxis of COVID-19 Infection: A Pilot Topical application 742 of neem leaves prevents wrinkles formation in UVB-exposed hairless mice Azadirachta indica (Neem) Leaf Induces Apoptosis in 4T1 Breast Cancer BALB/c Mice Vaccines for SARS-CoV-2: Lessons from Other Coronavirus Strains Therapeutic effects of Nimbolide, 750 an autophagy regulator, in ameliorating pulmonary fibrosis through attenuation of TGF-beta1 751 driven epithelial-to-mesenchymal transition SARS-CoV-2/COVID-19 and advances in developing potential therapeutics and vaccines to 755 counter this emerging pandemic Cell-758 type-resolved quantitative proteomics map of interferon response against SARS-CoV-2. 759 iScience Cytoplasmic tail of coronavirus spike protein has 761 intracellular targeting signals Pharmacologic treatments for 763 coronavirus disease 2019 (COVID-19): a review Azadirachta indica A. Juss Ameliorates 765 Mouse Hepatitis Virus-Induced Neuroinflammatory Demyelination by Modulating Cell-to-Cell 766 Fusion in an Experimental Animal Model of Multiple Sclerosis Liver Disease and Coronavirus Disease 769 2019: From Pathogenesis to Clinical Care NIH Image to ImageJ: 25 years of image 772 analysis The acaricidal efficacy of aqueous 774 neem extract and ivermectin against Sarcoptes scabiei var. cuniculi in experimentally infested 775 rabbits Cell entry mechanisms of 777 SARS-CoV-2 Cell entry mechanisms of 779 SARS-CoV-2 Intracranial Inoculation Is More 781 Potent Than Intranasal Inoculation for Inducing Optic Neuritis in the Mouse Hepatitis Virus-782 Induced Model of Multiple Sclerosis A 785 proline insertion-deletion in the spike glycoprotein fusion peptide of mouse hepatitis virus 786 strongly alters neuropathology The trinity of COVID-19: immunity, 789 inflammation and intervention Natural products as home-based prophylactic 792 and symptom management agents in the setting of COVID-19 In vitro antiviral activity of neem 795 (Azardirachta indica L.) bark extract against herpes simplex virus type-1 infection Coronavirus biology and replication: 798 implications for SARS-CoV-2 Detection of SARS-CoV-2 in Different 801 Types of Clinical Specimens Drug Delivery Approaches in Addressing Clinical Pharmacology-803 Related Issues: Opportunities and Challenges NBE-treated mice showed significantly reduced inflammation 976 determined by no perivascular cuffing and less scattered Iba-1+ cells without apparent nodule 977 formation. (C) Quantification of the intensity of Iba-1 staining representing microglial 978 activation in different neuroanatomic regions of DMSO treated and NBE-treated mice brain 979 sections were plotted in the graph and showed significantly reduced inflammation in most brain 980 regions in NBE-treated mice. (D) Serial sections from liver tissues of the same DMSO treated 981 and NBE-treated mice were stained with H&E DMSO treated mice liver sections show a characteristic hepatic lesion with profuse anti-N and 983 NBE-treated mice showed comparatively much less anti-N and anti-984 The graph plotted the quantification of the intensity of anti-N staining in hepatic 985 lesions in DMSO treated and NBE-treated liver sections. Arrows indicate the presence of viral 986 antigen and Iba1+ cells in hepatic lesions. Data represent mean ± SEM and statistical 987 significance was determined by unpaired student's t-test Tukey's multiple comparison tests; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, 989 significantly different from untreated sets Figure 7: Identification of bioactive NBE components and a cartoon diagram for SARS-1062 CoV-2 proteins showing dominant binding sites for bioactive compounds.TheDCM.F1 1063 (300 µg/mL) fraction of NBE was analyzed to test the potential antiviral efficiency against m-1064 CoV-RSA59 infection by preincubating it with the virus at MOI 1, followed by infection in 1065 Effects of DCM.F1 were compared to the effects of preincubation of the virus 1066 with NBE (300 µg/mL) prior to infection. (A) Both DCM.F1 and whole NBE show reduced 1067 syncytia formation at 10 h p.i 1070 as compared with cultures infected with the untreated virus at 10 h and 18 h p.i. Viral titer assay 1071 of cell culture supernatants revealed a significant reduction in viral replication upon 1072 preincubating the virus with DCM.F1 at 18 h p.i., indicating that DCM.F1 can inhibit viral 1073 cytopathy as well as viral replication effectively. Whole NBE also reduced viral replication but 1074 was less effective than DCM.F1. Data represent mean ± SEM. Level of significance was 1075 calculated using student's t-test and RM one-way ANOVA followed by Sidak's multiple 1076 comparison test The top panel shows the total ion 1078 chromatogram obtained from the elution of DCM.F1. The extracted ions from the total ion 1079 chromatogram are shown in the middle panel. The area under the curve (AUC) for the base 1080 peak is shown in the bottom-most panel The top-ranked docking sites of the neem compounds from 1084 NBE fraction DCM.F1 are shown as large transparent spheres in red. These are anchored 1085 around residue L1024 in the Central helix for all three trimers of Spike. For ACE2, the top-1086 ranked docking sites for a majority of neem compounds are around residue F40 located close 1087 to the ACE2-Spike interaction site. A cartoon diagram of RdRp protein is drawn using 1088 coordinates from the PDB ID: 7AAP. The top-ranked docking sites of the neem compounds 1089 are shown in large green transparent spheres. Of the two sites, one is the RdRp-associated 1090 nucleotidyl transferase catalytic site and may influence the binding at the main catalytic site 1091 inhibitors from Azadirachta indica (Neem)