부산대학교 도서관

Staphylococcus aureus is a Gram-positive human pathogen that has developed resistance to all traditionally used antibiotics. Sortase A (SrtA) is a 'house-keeping' enzyme present in a number of Gram-positive organisms including S. aureus that is responsible for the covalent anchoring of proteins to the cell wall through the recognition of a highly conserved LPXTG motif. Many of the proteins it anchors are involved in virulence and immune evasion suggesting that it is an attractive target for potential anti-infective therapies. Moreover, as SrtA is not vital for bacterial growth or survival, inhibition may not select for the development of drug-resistance, unlike conventional antibiotics. This study evaluated different assays to assay SrtA activity and includes an in vitro Fluorescence Resonance Energy Transfer (FRET) assay using purified recombinant SrtA protein and an in vivo whole cell-based assay that measured SrtA-mediated anchoring of Gaussia luciferase (GLuc) in S. aureus. A further in vivo assay was evaluated, which measured SrtA activity using fluorescence as a reporter and analysis by flow cytometry and structured illumination microscopy. In this study three novel small molecules were identified as potential inhibitors of SrtA using in silico computational docking and SAR analysis; NCC-00014270, NCC-00076932 and NCC-00032784. These compounds were shown to inhibit SrtA in vitro in a dose-dependent manner with IC50s of 140 ± 24.6 µM, 172 ± 28.1 µM and 628 ± 122 µM respectively and were shown to act as competitive inhibitors of the SrtA. With the use of an in vivo reporter, it was shown that all three compounds negatively affected SrtA-mediated anchoring in S. aureus whole cells. Moreover, the cytotoxicity of these lead compounds against eukaryotic cells was assessed. Overall, these data suggest that two of the three lead compounds were potential 'hit' molecules for further structural modification for increased inhibitory activity against SrtA.