title: Discrimination of potent inhibitors of Toxoplasma gondii enoyl-acyl carrier protein reductase by a thermal shift assay.
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.
authors.affiliation: Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health , Baltimore, Maryland 21205, United States.
authors.forename: Gustavo A
authors.initials: GA
authors.lastname: Afanador
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Muench
authors.forename: Martin
authors.initials: M
authors.lastname: McPhillie
authors.forename: Alina
authors.initials: A
authors.lastname: Fomovska
authors.forename: Arne
authors.initials: A
authors.lastname: Schön
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhou
authors.forename: Gang
authors.initials: G
authors.lastname: Cheng
authors.forename: Jozef
authors.initials: J
authors.lastname: Stec
authors.forename: Joel S
authors.initials: JS
authors.lastname: Freundlich
authors.forename: Hong-Ming
authors.initials: HM
authors.lastname: Shieh
authors.forename: John W
authors.initials: JW
authors.lastname: Anderson
authors.forename: David P
authors.initials: DP
authors.lastname: Jacobus
authors.forename: David A
authors.initials: DA
authors.lastname: Fidock
authors.forename: Alan P
authors.initials: AP
authors.lastname: Kozikowski
authors.forename: Colin W
authors.initials: CW
authors.lastname: Fishwick
authors.forename: David W
authors.initials: DW
authors.lastname: Rice
authors.forename: Ernesto
authors.initials: E
authors.lastname: Freire
authors.forename: Rima
authors.initials: R
authors.lastname: McLeod
authors.forename: Sean T
authors.initials: ST
authors.lastname: Prigge
mesh_terms: D000981:Antiprotozoal Agents / Q000009:adverse effects / Q000737:chemistry / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D015195:Drug Design; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH) / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000009:adverse effects / Q000737:chemistry / Q000494:pharmacology*; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000469:parasitology; D057166:High-Throughput Screening Assays; D006358:Hot Temperature; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008968:Molecular Conformation; D062105:Molecular Docking Simulation; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D058767:Protein Unfolding; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014122:Toxoplasma / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development; D014260:Triclosan / Q000009:adverse effects / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology
chemical_list: D000981:Antiprotozoal Agents; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D008164:Luminescent Proteins; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C054562:yellow fluorescent protein, Bacteria; D009243:NAD; D014260:Triclosan; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH)