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Discrimination of Potent Inhibitors of Toxoplasma gondii Enoyl-Acyl Carrier Protein Reductase by a Thermal Shift Assay

DOI: 10.1021/bi400945y DOI Help

Authors: Gustavo A. Afanador (Johns Hopkins Bloomberg School of Public Health) , Stephen Muench (University of Leeds) , Martin Mcphillie (University of Sheffield) , Alina Fomovska (The University of Chicago) , Arne Schön (The Johns Hopkins University) , Ying Zhou (The University of Chicago) , Gang Cheng (University of Illinois at Chicago) , Jozef Stec (Chicago State University) , Joel S. Freundlich (Rutgers University-New Jersey Medical School) , Hong-ming Shieh (Jacobus Pharmaceutical Company) , John W. Anderson , David P. Jacobus (Jacobus Pharmaceutical Company) , David A. Fidock (Jacobus Pharmaceutical Company) , Alan P. Kozikowski (University of Illinois at Chicago) , Colin W. Fishwick (University of Leeds) , David W. Rice (University of Sheffield) , Ernesto Freire (The Johns Hopkins University) , Rima Mcleod (The University of Chicago) , Sean T. Prigge (Johns Hopkins Bloomberg School of Public Health)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Biochemistry , VOL 52 (51) , PAGES 9155 - 9166

State: Published (Approved)
Published: December 2013
Diamond Proposal Number(s): 8987

Abstract: Many microbial pathogens rely on a type II fatty acid synthesis (FASII) pathway that is distinct from the type I pathway found in humans. Enoyl-acyl carrier protein reductase (ENR) is an essential FASII pathway enzyme and the target of a number of antimicrobial drug discovery efforts. The biocide triclosan is established as a potent inhibitor of ENR and has been the starting point for medicinal chemistry studies. We evaluated a series of triclosan analogues for their ability to inhibit the growth of Toxoplasma gondii, a pervasive human pathogen, and its ENR enzyme (TgENR). Several compounds that inhibited TgENR at low nanomolar concentrations were identified but could not be further differentiated because of the limited dynamic range of the TgENR activity assay. Thus, we adapted a thermal shift assay (TSA) to directly measure the dissociation constant (Kd) of the most potent inhibitors identified in this study as well as inhibitors from previous studies. Furthermore, the TSA allowed us to determine the mode of action of these compounds in the presence of the reduced nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide (NAD+) cofactor. We found that all of the inhibitors bind to a TgENR–NAD+ complex but that they differed in their dependence on NAD+ concentration. Ultimately, we were able to identify compounds that bind to the TgENR–NAD+ complex in the low femtomolar range. This shows how TSA data combined with enzyme inhibition, parasite growth inhibition data, and ADMET predictions allow for better discrimination between potent ENR inhibitors for the future development of medicine.

Subject Areas: Biology and Bio-materials

Instruments: I02-Macromolecular Crystallography , I03-Macromolecular Crystallography , I04-1-Macromolecular Crystallography (fixed wavelength) , I04-Macromolecular Crystallography , I24-Microfocus Macromolecular Crystallography