Synthesis and Application of Ampicillin-derived Photoprobes in the study of resistance mechanisms of Staphylococcus aureus

MENDEZ, L.; TESTERO, S.A.*; ANTINORI, M.; LLARRULL, L.I.*; BELLUZO, B.S.

Rosario

Congreso; 4ta Reunion Internacional de Ciencias Farmaceuticas (RICiFa); 2016

The Gram-positive bacterium S. aureus is the major cause of intra- and extra-hospital infections worldwide.1 The three-component system VraSTR controls the peptidoglycan crosslinking process, and is activated by beta-lactam and glycopeptide antibiotics. VraSRT has a critical role in glycopeptide-resistance. The VraSRT system is composed of three components: VraS and VraT are membrane proteins that could quickly detect stress in the cell wall and transmit the signal to the cell cytoplasm.2 The aim of this study is to synthesize photosensitive analogs of beta-lactam antibiotics and to evaluate their interaction with membrane proteins present in S. aureus. UV irradiation of photoprobes generates highly reactive intermediates which covalently link to protein groups located nearby. Based on this property, interaction between the beta-lactam analogs and target proteins can be unequivocally revealed.We have synthesized ampicillin derivatives with different photoreactive groups bonded to the primary amino group of the side chain attached to C-6. Three photosensitive groups were incorporated: benzophenone, tetrafluorophenylazide and trifluoromethyl-phenyldiazirine. The products were characterized by 1H, 19F, 13C NMR and HPLC/MS. Spheroplasts of E. coli that expressed recombinant VraS protein were incubated with the photoprobes and irradiated with UV light at 254 nm and/or 365 nm. We detected migration shifts of the VraS-band in SDS-PAGE/Western Blot analysis which indicated formation of covalent adducts between VraS and the photoprobes. Purification of covalently modified proteins and their characterization by MALDI-TOF and protease digestion/LC/MS/MS will allow identification of the peptides bound to the photoprobe.These studies suggest the existence of a direct interaction between β-lactam antibiotics and the VraS kinase of the three-component system. Further research will let us know the site of interaction at molecular level and whether other proteins are needed for activation. VraS is an attractive target for the design of inhibitors of resistance mechanisms in S. aureus. References:1) a) Boucher HW, Talbot GH, Bradley JS, Edwards JE, Gilbert D, et al. Clin. Infect. Dis. 2009, 48, 1-12. b) Boucher HW. Clin. Infect. Dis. 2010, 50 Suppl 1, S4-9. c) Loffler CA, Macdougall C. Expert. Rev. Anti. Infect. Ther. 2007, 5, 961-981. d) Richards MJ, Edwards JR, Culver DH, Gaynes RP, Infect. Control Hosp. Epidemiol. 2000, 21, 510-515.2) a) Kuroda M, Kuwahara-Arai K, Hiramatsu K. Biochem. Biophys. Res. Commun. 2000, 269, 485-490. b) Boyle-Vavra S, Yin S, Daum RS. FEMS Microbiol. Lett. 2006, 262, 163-171. c) Yin S, Daum RS, Boyle-Vavra S. Antimicrob. Agents Chemother. 2006, 50, 336.