Structural Optimization of Covalent Inhibitors for Deubiquitinase ChlaDUB1 of <i>Chlamydia trachomatis</i> as Antibiotic Agents

The cysteine protease ChlaDUB1 has proven to be a promising new target for antichlamydial therapy. This deubiquitinase manipulates protein homeostasis of the infected human host cell, concealing the chlamydial infection. In this study, we optimized a previously identified scaffold of covalently acting ChlaDUB1 inhibitors using a combination of docking, synthesis and in vitro enzymatic screening. This led to a reduction of the inhibitor size while simultaneously improving activity at ChlaDUB1 to 1 μM and enhancing the rate of target inhibition. We identified a hitherto unobserved inhibition mechanism at ChlaDUB1 and narrowed it down to a particular substitution pattern by chemical derivatization. Finally, both antichlamydial activity and cytotoxicity of the lead compounds were determined. Hereby, we present comprehensive structure–activity relationships and detailed kinetic studies that identified a small molecule lead compound for specific antichlamydial therapy, which showed drastically lowered cytotoxicity compared to previously described compounds.