Warhead Reactivity Limits the Speed of Inhibition of the Cysteine Protease Rhodesain

Viral and parasitic pathogens rely critically on cysteine proteases for host invasion, replication, and infectivity. Their inhibition by synthetic inhibitors, such as vinyl sulfone compounds, has emerged as a promising treatment strategy. However, the individual reaction steps of protease inhibition are not fully understood. Using the trypanosomal cysteine protease rhodesain as a medically relevant target, we design photoinduced electron transfer (PET) fluorescence probes to detect kinetics of binding of reversible and irreversible vinyl sulfones directly in solution. Intriguingly, the irreversible inhibitor, apart from its unlimited residence time in the enzyme, reacts 5 times faster than the reversible one. Results show that the reactivity of the warhead, and not binding of the peptidic recognition unit, limits the rate constant of protease inhibition. The use of a reversible inhibitor decreases the risk of off-target side effects not only by allowing its release from an off-target but also by reducing the rate constant of binding.