Identification of a Salmonella PhoP/PhoQ system inhibitor from a dynamic combinatorial library

FURLAN, RLE.; CABEZUDO, I.; LOBERTTI, CA.; GARCÍA VESCOVI, E.

Salta

Congreso; LV Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB) y XIV Congreso de la Asociación Panamericana de Bioquímica y Biología Molecular (PABMB); 2019

PRESENTACIÓN ORALSalmonella is an enteropathogen that causes a wide rangeof diseases in humans and animals. PhoP/PhoQ is a two-component system (TCS)distributed amongst several Gramnegative bacteria, consisting of the histidinekinase PhoQ, and the transcriptional regulator PhoP. In S. typhimurium,the PhoP/PhoQ system regulates the adaptation to Mg2+-limitingenvironments and controls key virulence phenotypes such as the invasion andproliferation within host cells. As signal transduction in mammals does notinvolve TCS, the PhoP/PhoQ system is an attractive target to develop newantimicrobial agents. We have previously reported a methodology based on aTLC-overlay as a new strategy for the search and identification ofantimicrobial agents targeting the PhoP/PhoQ system. We applied this bioguidedstrategy using a strain carrying a PhoP-controlled reporter gene, to thescreening of a dynamic combinatorial library of hidrazones in the search forinhibitors. As a result, two libraries of hydrazones and three libraries ofthiocarbazones totalling over 370 members were screened for their inhibitory activitythrough a rapid inexpensive TLC strategy.  Satisfactorily, a complexlibrary of hydrazones that can repress the PhoP/PhoQ system was selected fromthe initial screening, to further study its members. Through iterativedeconvolution of over 100 library members we identified a potential inhibitor,A25B4. This compound could be synthesized in its pure form, characterized, andit was confirmed that it does not affect the growth of Salmonella. By quantitative β-galactosidase assayswe confirmed its inhibitory activity and it was found that the response wasdose-dependent and selective as well. Once the mechanism of action of A25B4 inthe system is known, a target protein domain of the TCS will be used totemplate a library of hydrazones, biasing the composition of the dynamiclibrary towards A25B4. This step will further confirm its affinity andmechanism of action. This strategy allow to us to establish a novel methodologyfor the discovery of PhoP/PhoQ system inhibitors to fight against Salmonella borne-disease.