Substrate Mimicry by Inhibitors of the Mycobacterium tuberculosis 20S Proteasome and Their Activity against Mycobacteria In Vitro and in Macrophages

The remarkable and problematic capacity of Mycobacterium tuberculosis to persist in the host is dependent on a functional 20S proteasome that is structurally and functionally analogous to its mammalian counterpart. It has been proposed that this enzyme is critical for the bacterium’s adaptive response to a variety of stressors in the host. Here, we report the structure of a rationally designed and species-selective inhibitor of the Mtb 20S proteasome in complex with the enzyme. The inhibitor was inspired by the syringolin natural products—covalent proteasome inhibitors that mimic a substrate. Structure elucidation of the Mtb 20S proteasome-inhibitor adduct via cryo-electron microscopy revealed that the inhibitor mimics a substrate yet engages the active site in a different fashion than the syringolin natural products. Corroboration of this model via the synthesis of hypothetical inhibitors led to the discovery of a more potent inhibitor, as evidenced in kinetic assays for enzyme inhibition. Both inhibitors are permeable to and active in M. tuberculosis. They sensitize the bacterium to chemically and pharmacologically induced nitrosative stress in vitro. Importantly, they suppress the persistence of live mycobacteria in murine and human macrophages.