Indole-based peptidomimetic maleimides as SARS-CoV-2 Mpro inhibitor scaffolds compatible with reversible covalent inhibition: Warhead optimization and broad in vitro antiviral activity.

Although several SARS-CoV-2 main protease (Mpro) inhibitors have been clinically authorized, the continued evolution of SARS-CoV-2 and the emergence of resistance-associated mutations highlight the need for additional inhibitor scaffolds with balanced potency, chemical stability, and reversible covalent reactivity. Herein, we report the discovery and warhead optimization of indole-based peptidomimetic maleimides as Mpro inhibitor scaffolds with a binding mode compatible with reversible covalent inhibition. Initial attempts to access α-cyanoacrylate analogues afforded product-related fractions whose chromatographic and mass-spectrometric profiles changed during purification and analytical handling, preventing isolation of analytically pure samples for biological profiling. DFT calculations showed higher electrophilicity for the α-cyanoacrylate model and provided a supportive electronic rationale for the contrasting purification and handling behavior of the two warhead classes. Subsequent optimization of the maleimide series identified compound 8i as the lead inhibitor, with single-digit nanomolar inhibition of wild-type Mpro (IC50 = 6.51 nM). Jump-dilution experiments supported a reversible binding component compatible with a reversible covalent inhibition mode. In P-gp inhibitor-supplemented Vero E6 CPE assays, 8i showed broad in vitro antiviral activity against wild-type, Delta, and Omicron BA.5 SARS-CoV-2 variants, with EC50 values of 1.44, 0.49, and 1.52 μM, respectively, while 50% cytotoxicity was not reached at concentrations up to 100 μM under the applied assay conditions. Covalent docking and 100 ns molecular dynamics simulations were used to provide supportive structural interpretations of the modeled Mpro complexes and to compare compound 8i with the less active N-substituted analogue 8g. Because biochemical activity against the selected mutant proteases has not been experimentally determined, the docking results should be interpreted as hypothesis-generating structural models rather than evidence of mutation tolerance. Overall, these findings identify indole-based peptidomimetic maleimides as chemically tractable Mpro inhibitor scaffolds with a binding mode compatible with reversible covalent inhibition and support compound 8i as a lead for further antiviral optimization.