Based on the similarity between the active sites of the deubiquitylating and deneddylating enzyme ChlaDub1 (Cdu1) and the evolutionary related protease adenain a target-hopping approach screening on a focused set of adenain inhibitors has been pursued. The thereby identified cyano-pyrimidine based inhibitors represent the first active-site directed small molecule inhibitors for Cdu1. High-resolution crystal structures of Cdu1 in complex with two covalently bound cyano-pyrimidines as well as with its substrate ubiquitin have been obtained. These structural data were complemented by enzymatic assays and covalent docking studies to provide insight into Cdu1s substrate recognition, active site pocket flexibility and potential hotspots for ligand interaction. Combined, these data provide a strong foundation for future structure-guided medicinal chemistry optimization of this cyano-pyrimidine based scaffold towards more potent and specific Cdu1 inhibitors. [Image: see text] Covalent death sentence: Bacterial deubiquitylating enzymes (DUBs) can be utilized to enhance their pathogenicity. In our study, we have successfully harnessed the structural similarities of the active site of the chlamydial deubiquitylase 1 (Cdu1) with an evolutionarily related protease to discover the very first active-site covalent Cdu1 inhibitors. We demonstrate the molecular basis of Cdu1 ubiquitin recognition and establish criteria for the structure-informed medicinal chemistry optimization of potential Cdu1 specific inhibitors.
