Structure-Based Discovery of Covalent Inhibitors Targeting the Catalytic Cysteine of Pseudomonas aeruginosa Adenosine phosphosulfate reductase

The alarming rise in antibiotic-resistant Pseudomonas aeruginosa strains underscores the urgent need to identify novel and selective therapeutic strategies. Adenosine phosphosulfate reductase (APSR) represents an attractive therapeutic target, given its central role in bacterial sulfur assimilation and its absence from host cells. [1] Key to APSR activity, the catalytic cysteine (C256) is located in the flexible C-terminal region, which has not been solved in available crystallographic structures. [2] To address this limitation, AlphaFold2 [3] was used to reconstruct the missing segment and recover the catalytic competent geometry of the active site. The resulting model enabled quantum mechanics/molecular mechanics (QM/MM) characterization of the catalytic step at the B3LYP-D3(BJ)/6-31G(d) level, yielding an activation barrier consistent with experimental kinetics. These simulations highlighted the role of the [4Fe-4S] oxidation state in active site electrostatic preorganization, with the oxidized state enabling the catalytic mechanism. [4] This model served as the structural basis to identify covalent APSR inhibitors through a multi-step workflow integrating pharmacophore modelling, covalent docking, and metadynamics rescoring. Over five million compounds across four warhead classes were screened and, eventually, nine candidates were prioritized for in vitro testing. Among them, a vinyl sulfone hit inhibited APSR at micromolar concentration and reduced bacterial proliferation without permeabilizers, indicating its ability to cross the Gram-negative double membrane. Its covalent mode of action was confirmed by mass spectrometry, demonstrating selective vinylation of C256. Genetic complementation, in which restoration of cysH rescued bacterial growth, confirmed the specificity of the APSR inhibition phenotype. Together, these findings support APSR as a promising antibacterial target and inform future lead optimization efforts.