Functional Investigation of RNA Viral Helicases as Molecular Targets for the Discovery of Novel Antiviral Agents

The recent SARS-CoV-2 pandemic highlighted the lack in pandemics preparedness for hCoVs potential future spillovers and for other (+)ssRNA viruses that still lack approved pharmacological treatments such as Flaviviruses. Small molecule development stands out as the most promising approach, as it enables possibly oral administration, stockpiling, combinatorial therapy. (+)ssRNA helicases, essential for viral replication, are among the most conserved proteins across several viral families, are a molecular class still underexplored, that lacks approved antiviral agents. In this respect, the identification of first-in-class inhibitor scaffolds is a promising achievement to pursue. The work is focused on the helicases of Coronaviruses, specifically the NSP13 of SARS-CoV-2, and of Flaviviruses, particularly the NS3 of WNV. Despite belonging to SF2 and SF1, respectively, the two proteins have strong similarities: both are non-ring forming helicases with core RecA-like domains; they share conserved motifs across the helicases and possess similar NTPase, helicase, and RTPase activities. SARS-CoV-2 NSP13 and WNV NS3helbiochemical assays and biophysical methods were established. The screening of chemically diverse compound identified first-in-class inhibitor scaffolds ranking among the strongest NSP13 and NS3 helicase inhibitors reported to date, based on IC₅₀ and EC₅₀ values and SI. Among a first set of 12 diketo acid derivatives, that inhibited both SARS-CoV-2 NSP13 enzymatic functions, three hit compounds, 5a,b,d, exhibited broad-spectrum activity against SARS-CoV-2, MERS-CoV, hCoV-OC43, and hCoV-229E. Mode of action studiesn suggested an ATP non-competitive inhibitory mechanism, likely targeting an allosteric pocket on the RecA2 domain. A rational design targeting the ATP-binding site region of SARS-CoV-2 NSP13 identified the 2-iminothiazolidin-4-one scaffold as promising core for antiviral molecules. Hit compound, 3i, potently inhibitNSP13 enzymatic functions, with a mixed-type inhibition mechanism and SARS-CoV-2 replication, with a favorable SI also in human lung epithelial cells. A specific 1:1 binding mode was confirmed by SPR (Kd = 6.20 µM). Notably, 3i was selective towards other viral and cellular targets and showed additive antiviral effect in combination with Remdesivir and Nirmatrelvir. A 3i related compound, derivative 4b, with retained NSP13 inhibitory activity and SARS-CoV-2 antiviral effect, exhibited also pan-helicases potential, showing to be active against WNV NS3 helicase. The result encouraged to target WNV NS3 helicase:4,5-dihydropyrazoles, as new WNV NS3hel inhibitors, active against viral replication, were subsequently investigated for their pan-helicases potential. Compound 21, active on WNV replication, and a dual-target inhibitor against NS3 helicase and protease functions. It was optimized by N-methylated analogue, 21a, displayed anti SARS-CoV-2 activity (EC50 6.5 μM in VeroE6-GFP and 14.5 μM in A549-ACE2), and broad-spectrum efficacy against other hCoVs with SI >45, coupled with no significant toxicity. A deep characterization of 21a confirmed a pattern coherent with the targeting of the replication step, promising potency in ex vivo systems and high genetic barrier to resistance. This hit identification with depth of biochemical, kinetic, and mechanistic characterization, represents the most extensive and detailed analysis of SARS-CoV NSP13 and WNV NS3hel inhibitors available at the best of our knowledge. The demonstration of inter-family viral helicase inhibition and broad-spectrum antiviral activity, coupled with a high barrier to resistance, supports viral RNA helicases as promising antiviral targets and provides proof-of-concept for the development of broad-spectrum inhibitors.