In Silico Drug Discovery Conference

Enteropathogenic E. coli (EPEC) is a human pathogen using type III secretion system for delivery of proteins directly into the human host. The system contains a single ATPase, EscN, which is essential for uncoupling of proteins from their complexes with chaperones before the delivery. The structure of EscN ATPase (PDB code: 2obm) was used to construct a search template to screen computationally for small molecule inhibitors blocking its active site. A total of twelve candidates out of seventy tested showed inhibitory activity against EscN ATPase and only five were non-toxic to human cells at 100 M concentration. Two lead candidates were examined but only one, Compound 54, was selected for further optimization based on the experimental data. After one stage of QSAR optimization, at least five derivatives were found to be competitive inhibitors of EscN capable of blocking virulence factor secretion as determined by Western blotting with antibodies developed to an effector. One candidate, Compound 54-6, had IC50 of 58 M but a very high (3.5 mM) inhibition constant Ki. The discrepancy was explained by proposed binding mode in the active site of enzyme. The compound was also minimally toxic to mammalian cells as determined by cell viability, apoptosis, cell cycle and mitochondrial potential interference assays. In the cell infection model of HeLa cells with EPEC, Compound 54-6 blocked effector secretion at least by 60% at 100 M concentration. The compound decreased also pedestal formation and cell entry by bacteria, when analyzed by confocal microscopy. The second best inhibitor active in blocking effector secretion and HeLa cell infection by EPEC was