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

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• 作者：Gustavo A. Afanador ; Stephen P. Muench ; Martin McPhillie ; Alina Fomovska ; Arne Sch?n ; Ying Zhou ; Gang Cheng ; Jozef Stec ; Joel S. Freundlich ; Hong-Ming Shieh ; John W. Anderson ; David P. Jacobus ; David A. Fidock ; Alan P. Kozikowski ; Colin W. Fishwick ; David W. Rice ; Ernesto Freire ; Rima McLeod ; Sean T. Prigge
• 刊名：Biochemistry
• 出版年：2013
• 出版时间：December 23, 2013
• 年：2013
• 卷：52
• 期：51
• 页码：9155-9166
• 全文大小：563K
• ISSN：1520-4995

文摘

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 (K_d) 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鈥揘AD⁺ complex but that they differed in their dependence on NAD⁺ concentration. Ultimately, we were able to identify compounds that bind to the TgENR鈥揘AD⁺ 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.