LAUSR

Novel compounds developed as nitric oxide (NO) donors for cardiovascular diseases were created by linking an urea-like NO donor to the 5-carbon of the generic agent isosorbide-2-nitrate. NO also appears to have antiviral effects on SARS-CoV-2. A study of drug-protein interactions was undertaken in silico using Maestro, Schrödinger Suite software to determine if the novel compounds could be antivirals. SARS-CoV-2 papain-like protease (PL pro ) was a primary target as it mediates viral replication and inhibition of host interferon-based responses. Compared with known antiviral drugs, including the known PL pro inhibitor GRL-0617, CR-0305 had a better Glide Score (a predictor of ligand affinity) against PL pro . Molecular Mechanics / Generalized Born Surface Area (MM/GBSA) calculations and Molecular Dynamics simulations predicted CR-0305 binding to the PL pro catalytic pocket where it makes the most of key residues in the catalytic site. Subsequently, CR-0305, or (3S,3aS,6R,6aR)-6-((3-(2-hydroxyacetamido)propyl)amino) hexahydrofuro[3,2-b]furan-3-yl nitrate, was synthesized by ligating a (TBDPS) Tert-Butyldiphenylsilyl-protected side chain to the 5-carbon of 1,4:3,6-Dianhydro-L-Iditol Triflate Mononitrate followed by a de-protection reaction that removed TBDPS. Toxicity of CR-0305 was examined using MitoCheck Complex I-V activity assays in an Agilent Seahorse XFe96 analyzer in isolated mitochondria with mitochondrial stress tests in HMEC-1 human dermal endothelial cells and measurement of the effect of drug on ATP synthesis in HMEC-1 cells. No significant toxicity was identified at 10-100 μM concentrations. GRL-0617, CR-0202 and CR-0305 were tested in Vero E6 and A549-ACE2 cells exposed to SARS-CoV-2 using a drug dose response regimen from 20 μM down to 40 nM in 2-fold serial dilution. There was no evidence of cytotoxicity. GRL-0617 gave an approx. 50% reduction in SARS-CoV-2 infection compared to vehicle at the highest dose tested (20 μM), as measured by virus N protein staining with a specific antibody. CR-0305 gave a consistent but weak effect on total N protein expression in A549-ACE2 cells. In conclusion, CR-0305 binds in silico to SARS-CoV-2 PL pro and appears to inhibit viral replication in vitro , justifying further development.