Chemical Evolution of Antivirals Against Enterovirus D68 through Protein-Templated Knoevenagel Reactions

The generation of bioactive molecules from inactive precursors is a crucial step in the chemical evolution of life, however, mechanistic insights into this aspect of abiogenesis are scarce. Here, we investigate the protein-catalyzed formation of antivirals by the 3C-protease of enterovirus D68. The enzyme induces aldol condensations yielding inhibitors with antiviral activity in cells. Kinetic and thermodynamic analyses reveal that the bioactivity emerges from a dynamic reaction system including inhibitor formation, alkylation of the protein target by the inhibitors, and competitive addition of non-protein nucleophiles to the inhibitors. The most active antivirals are slowly reversible inhibitors with elongated target residence times. The study reveals first examples for the chemical evolution of bio-actives through protein-catalyzed, non-enzymatic C-C couplings. The discovered mechanism works under physiological conditions and might constitute a native process of drug development.