Promysalin is a natural product produced by Pseudomonas putida that selectively inhibits growth of the Gram-negative ESKAPE pathogen, Pseudomonas aeruginosa. Our group completed the first total synthesis, in 8 longest linear steps and an overall yield of 35%. Subsequently, we used diverted-total synthesis to access 16 synthetic analogs and carried out structure-activity relationship testing on this library. These investigations revealed that the myristic acid side chain was the most amenable to structural changes, while the salicylic acid and proline portions tolerated fewer modifications. Based on this information, we appended the minimalist probe to the amide, for use in affinity-based protein profiling. Proteomic analysis identified succinate dehydrogenase (Sdh) as the target of promysalin in P. aeruginosa. We also selected for a promysalin-resistant mutant and whole-genome sequencing revealed a mutation on Sdh, supporting our previous finding. Upon computational docking of promysalin in Sdh, we designed a new series of analogs in which the alkyl chain was modified to probe binding interactions. Synthesis and biological testing of these analogs indicated that this portion of the molecule is required for biological activity. Furthermore, computational docking revealed that analogs with a longer alkyl chain undergo a conformational change into a new binding cleft. This binding cleft contains a tryptophan residue, which has inspired a new analog series in which various aromatic groups will be appended to the alkyl chain in an attempt to induce pi-stacking and enhance binding. The synthesis of these compounds is in progress. Together this work has provided insight into the novel mechanism of action of a natural product that exhibits species-specific inhibition of P. aeruginosa.

Promysalin analogs reveal new binding cleft in succinate dehydrogenase

ACS Fall 2021

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