Optimization and biological evaluation of l-DOPA derivatives as potent influenza PAN endonuclease inhibitors with multi-site binding characteristics

[Display omitted] •Two sets of l-DOPA derivatives as novel PAN inhibitors were obtained based on the multi-site binding and SBDD strategies.•The optimized compound T-31 showed broad-spectrum anti-influenza activity in vitro.•Compound T-31 proved to be a potent PAN inhibitor with multi-site binding characteristics.•T-31 could effectively inhibit replication of influenza viruses by targeting HA2 and PAN. Emerging and potential influenza pandemics still are an enormous worldwide public health challenge. The PAN endonuclease has been proved to be a promising target for anti-influenza drug design. Here, we report the discovery and optimization of potent Y-shaped PAN inhibitors featuring multi-site binding characteristics with l-DOPA as a starting point. We systematically modified the hit 1 bearing two-binding characteristics based on structure-based rational design combined with multisite binding and conformational constraint strategies, generating four families of l-DOPA derivatives for SARs analysis. Among these substances, N, 3-di-substituted 1, 2, 3, 4-tetrahydroisoquinoline derivative T-31 displayed superior properties as a lead PAN endonuclease inhibitor and antiviral agent. The lead T-31 inhibited PAN endonuclease activity with an IC50 value of 0.15 μM and showed broad and submicromolar anti-influenza potency in cell-based assays. More importantly, T-31 could simultaneously target both influenza HA and the RdRp complex, thus interfering with virus entry into host cells and viral replication. This study offers a set of novel PAN endonuclease inhibitors with multi-site binding characteristics starting from the l-DOPA skeleton.