title: Forward genetic screening identifies a small molecule that blocks Toxoplasma gondii growth by inhibiting both host- and parasite-encoded kinases.
abstract: The simultaneous targeting of host and pathogen processes represents an untapped approach for the treatment of intracellular infections. Hypoxia-inducible factor-1 (HIF-1) is a host cell transcription factor that is activated by and required for the growth of the intracellular protozoan parasite Toxoplasma gondii at physiological oxygen levels. Parasite activation of HIF-1 is blocked by inhibiting the family of closely related Activin-Like Kinase (ALK) host cell receptors ALK4, ALK5, and ALK7, which was determined in part by use of an ALK4,5,7 inhibitor named SB505124. Besides inhibiting HIF-1 activation, SB505124 also potently blocks parasite replication under normoxic conditions. To determine whether SB505124 inhibition of parasite growth was exclusively due to inhibition of ALK4,5,7 or because the drug inhibited a second kinase, SB505124-resistant parasites were isolated by chemical mutagenesis. Whole-genome sequencing of these mutants revealed mutations in the Toxoplasma MAP kinase, TgMAPK1. Allelic replacement of mutant TgMAPK1 alleles into wild-type parasites was sufficient to confer SB505124 resistance. SB505124 independently impacts TgMAPK1 and ALK4,5,7 signaling since drug resistant parasites could not activate HIF-1 in the presence of SB505124 or grow in HIF-1 deficient cells. In addition, TgMAPK1 kinase activity is inhibited by SB505124. Finally, mice treated with SB505124 had significantly lower tissue burdens following Toxoplasma infection. These data therefore identify SB505124 as a novel small molecule inhibitor that acts by inhibiting two distinct targets, host HIF-1 and TgMAPK1.
authors.affiliation: Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America.
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Brown
authors.affiliation: Departments of Molecular Medicine & Global Health, University of South Florida, Tampa, Florida, United States of America.
authors.forename: Elena
authors.initials: E
authors.lastname: Suvorova
authors.affiliation: Department of Biology, Boston College, Chestnut Hill, Massachusetts, United States of America.
authors.forename: Andrew
authors.initials: A
authors.lastname: Farrell
authors.affiliation: Department of Microbiology and Immunology, University at Buffalo, Buffalo, New York, United States of America.
authors.forename: Aaron
authors.initials: A
authors.lastname: McLain
authors.affiliation: Department of Microbiology and Immunology, University at Buffalo, Buffalo, New York, United States of America.
authors.forename: Ashley
authors.initials: A
authors.lastname: Dittmar
authors.affiliation: Arthritis and Clinical Immunology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, United States of America.
authors.forename: Graham B
authors.initials: GB
authors.lastname: Wiley
authors.affiliation: Department of Biology, Boston College, Chestnut Hill, Massachusetts, United States of America.
authors.forename: Gabor
authors.initials: G
authors.lastname: Marth
authors.affiliation: Arthritis and Clinical Immunology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, United States of America.
authors.forename: Patrick M
authors.initials: PM
authors.lastname: Gaffney
authors.affiliation: Department of Biology, Boston College, Chestnut Hill, Massachusetts, United States of America.
authors.forename: Marc Jan
authors.initials: MJ
authors.lastname: Gubbels
authors.affiliation: Departments of Molecular Medicine & Global Health, University of South Florida, Tampa, Florida, United States of America.
authors.forename: Michael
authors.initials: M
authors.lastname: White
authors.affiliation: Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America; Department of Microbiology and Immunology, University at Buffalo, Buffalo, New York, United States of America.
authors.forename: Ira J
authors.initials: IJ
authors.lastname: Blader
mesh_terms: D030201:Activin Receptors, Type I / Q000037:antagonists & inhibitors*; D000818:Animals; D001483:Base Sequence; D052117:Benzodioxoles / Q000494:pharmacology; D020134:Catalytic Domain / Q000187:drug effects / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics; D018503:Genome, Protozoan / Q000235:genetics; D006790:Host-Parasite Interactions / Q000235:genetics; D051793:Hypoxia-Inducible Factor 1 / Q000037:antagonists & inhibitors* / Q000235:genetics; D007093:Imidazoles / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors* / Q000235:genetics; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D011725:Pyridines / Q000494:pharmacology; D017422:Sequence Analysis, DNA; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D014122:Toxoplasma / Q000235:genetics / Q000254:growth & development*
chemical_list: C519132:2-(5-benzo(1,3)dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride; D052117:Benzodioxoles; D016054:DNA, Protozoan; D051793:Hypoxia-Inducible Factor 1; D007093:Imidazoles; D015800:Protozoan Proteins; D011725:Pyridines; D019950:Mitogen-Activated Protein Kinase 1; D030201:Activin Receptors, Type I