Identification\nof Cysteine Metabolism Regulator (CymR)-Derived\nPentapeptides as Nanomolar Inhibitors of Staphylococcus\naureus <i>O</i>‑Acetyl‑l‑serine Sulfhydrylase (CysK)

The pathway of bacterial cysteine biosynthesis is gaining\ntraction\nfor the development of antibiotic adjuvants. Bacterial cysteine biosynthesis\nis generally facilitated by two enzymes possessing <i>O</i>-acetyl-l-serine sulfhydrylases (OASS), CysK and CysM. In Staphylococcus aureus, there exists a single OASS\nhomologue, <i>Sa</i>CysK. Knockout of <i>Sa</i>CysK was found to increase sensitivity to oxidative stress, making\nit a relevant target for inhibitor development. <i>Sa</i>CysK also forms two functional complexes via interaction with the\npreceding enzyme in the pathway serine acetyltransferase (CysE) or\nthe transcriptional regulator of cysteine metabolism (CymR). These\ninteractions occur through insertion of a C-terminal peptide of CysE\nor CymR into the active site of <i>Sa</i>CysK, inhibiting\nOASS activity, and therefore represent an excellent starting point\nfor developing <i>Sa</i>CysK inhibitors. Here, we detail\nthe characterization of CysE and CymR-derived C-terminal peptides\nas inhibitors of <i>Sa</i>CysK. Using a combination of X-ray\ncrystallography, surface plasmon resonance, and enzyme inhibition\nassays, it was determined that the CymR-derived decapeptide forms\nextensive interactions with <i>Sa</i>CysK and acts as a\npotent inhibitor (<i>K</i><sub>D</sub> = 25 nM; IC<sub>50</sub> = 180 nM), making it a promising lead for the development of <i>Sa</i>CysK inhibitors. To understand the determinants of this\nhigh-affinity interaction, the structure–activity relationships\nof 16 rationally designed peptides were also investigated. This identified\nthat the C-terminal pentapeptide of CymR facilitates the high-affinity\ninteraction with <i>Sa</i>CysK and that subtle structural\nmodification of the pentapeptide is possible without impacting potency.\nUltimately, this work identified CymR pentapeptides as a promising\nscaffold for the development of antibiotic adjuvants targeting <i>Sa</i>CysK.