Discrimination of potent inhibitors of Toxoplasma gondii enoyl-acyl carrier protein reductase by a thermal shift assay
| Autor: | Colin W. G. Fishwick, Arne Schön, David P. Jacobus, Gustavo A. Afanador, Rima McLeod, Jozef Stec, David A. Fidock, Ernesto Freire, Gang Cheng, Sean T. Prigge, Joel S. Freundlich, John W. Anderson, Alan P. Kozikowski, David W. Rice, Hong Ming Shieh, Alina Fomovska, Stephen P. Muench, Ying Zhou, Martin J. McPhillie |
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| Rok vydání: | 2013 |
| Předmět: |
Thermal shift assay
Hot Temperature Enoyl-acyl carrier protein reductase Antiprotozoal Agents Molecular Conformation Protozoan Proteins Biology Reductase Biochemistry Article chemistry.chemical_compound Inhibitory Concentration 50 Bacterial Proteins Humans Enzyme Inhibitors Fatty acid synthesis Cells Cultured Cell Proliferation Protein Unfolding chemistry.chemical_classification Fibroblasts NAD Molecular biology Enoyl-(Acyl-Carrier-Protein) Reductase (NADH) Recombinant Proteins Triclosan High-Throughput Screening Assays Dissociation constant Molecular Docking Simulation Kinetics Luminescent Proteins Enzyme chemistry Drug Design NAD+ kinase Oxidation-Reduction Toxoplasma |
| Zdroj: | Biochemistry. 52(51) |
| ISSN: | 1520-4995 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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