4-Iminooxazolidin-2-One as a Bioisostere of Cyanohydrin Suppresses EV71 Proliferation by Targeting 3C(pro)

The fatal pathogen enterovirus 71 (EV71) is a major cause of hand-foot-and-mouth disease (HFMD), which leads to serious neurological syndromes. While there are no effective clinical agents available for EV71 treatment thus far, EV71 3C protease (3C(pro)), a cysteine protease encoded by the virus, has become a promising drug target for discovery of antiviral drugs, given that it plays a crucial role in virus proliferation and interferes with host cell function. Here, we report two inhibitors of EV71 3C(pro), FOPMC and FIOMC, that were developed from previously reported cyanohydrin derivative (R)-1 by replacing the acyl cyanohydrin group with 4-iminooxazolidin-2-one. FOPMC and FIOMC have potent antiviral activity and dramatically improved metabolic stability. These two inhibitors demonstrated broad anti-EV effects on various cell lines and five epidemic viral strains. We further illuminated the binding models between 3C(pro) and FOPMC/FIOMC through molecular docking and molecular dynamics simulations. The substitution of an acyl cyanohydrin group with 4-iminooxazolidin-2-one does make FOPMC and FIOMC potent anti-EV71 drug candidates as universal nonclassical bioisosteres with a cyanohydrin moiety. IMPORTANCE EV71 is one of the most epidemic agents of HFMD. Thus far, there are no antiviral drugs available for clinical usage. The conserved EV71 3C(pro) plays pivotal roles in virus proliferation and defense host immunity, as well as having no homology in host cells, making it a most promising antiviral target. In this work, we identified that propyl- and isopropyl-substituted 4-iminooxazolidin-2-one moieties (FOPMC and FIOMC) effectively inhibited five epidemic viral strains in rhabdomyosarcoma (RD), HEK-293T, and VeroE6 cell lines. The inhibition mechanism was also illustrated with molecular docking and molecular dynamics (MD) simulations. The successful replacement of the labile cyanohydrin greatly improved the stability and pharmacokinetic properties of (R)-1, making 4-iminooxazolidin-2-one a nonclassical bioisosteric moiety of cyanohydrin. This discovery addressed a critical issue of the primitive structural scaffold of these promising anti-EV71 inhibitors and could lead to their development as broad-spectrum anti-EV agents.