A Pharmacophore Approach for Novel Inhibitors of Pseudomonas Aeruginosa Exotoxin A

Abstract

Pseudomonas aeruginosa (PA) is an opportunistic pathogen that causes lung infections in cystic fibrosis and other immune-compromised individuals. The most toxic factor secreted by PA is a 66 kDa protein, exotoxin A (ExoA), which belongs to a larger (mART) family of enzymes that catalyze the transfer of the ADP-ribosyl moiety from NAD+ to a protein target. COMPUTATIONAL APPROACH: Here, we used the high resolution crystal structure of cholix as the structural model system for ExoA. We considered structures in complex with several ligands (NAD+ and various inhibitors: NAP, V30, etc.) in order to address the mode of ligand binding. All the analyses were performed with the Molecular Operative Environment suite (MOE.2011). For each residue in the pocket, the interaction energy was evaluated along with various other descriptors. Binding free energy was calculated according to the GBVI/WSA function. Based upon the consensus features of the cholix:ligand complexes, a pharmacophore model was developed and was used to dock these molecules as standards for the purpose of training, followed by the docking of a new inhibitor library (M-series) obtained from an in silico screening against another mART toxin, Iota toxin from C. perfringens. EXPERIMENTAL APPROACH: A cell based assay was used for a coarse screen of the potency of the M-series inhibitors based on the viability of C38 cystic fibrosis human lung cells treated with ExoA. Compound M19 arose as the best inhibitor for protection of the cystic fibrosis human lung cell line with an EC50 of 2 μM. Future research will involve improvement of the M19 scaffold based on the information from computational approaches. This work was funded by a Cystic Fibrosis Canada (CFC) summer studentship to AP, a CFC Canada-Kin postdoctoral fellowship to MRL, and CFC operating grant to ARM.