title: Identification of small molecule inhibitors that block theToxoplasma gondiirhoptry kinase ROP18.
abstract: The protozoan parasiteToxoplasma gondiisecretes a family of serine-threonine protein kinases into its host cell in order to disrupt signaling and alter immune responses. One prominent secretory effector is the rhoptry protein 18 (ROP18), a serine-threonine kinase that phosphorylates immunity related GTPases (IRGs) and hence blocks interferon gamma-mediated responses in rodent cells. Previous genetic studies show that ROP18 is a major virulence component ofT. gondiistrains from North and South America. Here, we implemented a high throughput screen to identify small molecule inhibitors of ROP18in vitroand subsequently validated their specificity within infected cells. Although ROP18 was not susceptible to many kinase-directed inhibitors that affect mammalian kinases, the screen identified several sub micromolar inhibitors that belong to three chemical scaffolds: oxindoles, 6-azaquinazolines, and pyrazolopyridines. Treatment of interferon gamma-activated cells with one of these inhibitors enhanced immunity related GTPase recruitment to wild type parasites, recapitulating the defect of Δrop18mutant parasites, consistent with targeting ROP18 within infected cells. These compounds provide useful starting points for chemical biology experiments or as leads for therapeutic interventions designed to reduce parasite virulence.
authors.affiliation: Center for Integrative Chemical Biology and Drug Discovery, UNC Eshelman School of Pharmacy, University of North Carolina, 125 Mason Farm Road, CB#7363, Chapel Hill, NC 27599-7363.
authors.forename: Catherine
authors.initials: C
authors.lastname: Simpson
authors.affiliation: Department of Molecular Microbiology, Washington University Sch. Med. St. Louis MO 63110.
authors.forename: Nathaniel G
authors.initials: NG
authors.lastname: Jones
authors.affiliation: Center for Integrative Chemical Biology and Drug Discovery, UNC Eshelman School of Pharmacy, University of North Carolina, 125 Mason Farm Road, CB#7363, Chapel Hill, NC 27599-7363.
authors.forename: Emily A
authors.initials: EA
authors.lastname: Hull-Ryde
authors.affiliation: Center for Integrative Chemical Biology and Drug Discovery, UNC Eshelman School of Pharmacy, University of North Carolina, 125 Mason Farm Road, CB#7363, Chapel Hill, NC 27599-7363.
authors.forename: Dmitri
authors.initials: D
authors.lastname: Kireev
authors.affiliation: Center for Integrative Chemical Biology and Drug Discovery, UNC Eshelman School of Pharmacy, University of North Carolina, 125 Mason Farm Road, CB#7363, Chapel Hill, NC 27599-7363.
authors.forename: Michael
authors.initials: M
authors.lastname: Stashko
authors.affiliation: Department of Molecular Microbiology, Washington University Sch. Med. St. Louis MO 63110.
authors.forename: Keliang
authors.initials: K
authors.lastname: Tang
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University Sch. Med. St. Louis MO 63110.
authors.forename: Jim
authors.initials: J
authors.lastname: Janetka
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University Sch. Med. St. Louis MO 63110.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Wildman
authors.affiliation: SGC-UNC, Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, 120 Mason Farm Rd, 1070H Genetic Medicine Building, University of North Carolina, NC 27599-7363.
authors.forename: William J
authors.initials: WJ
authors.lastname: Zuercher
authors.affiliation: Structural Genomics Consortium, University of Toronto, MaRS South Tower, 101 College St, Toronto, ON, M5G 1L7, Canada; Department of Pharmacology and Toxicology, University of Toronto, 1 King's College Circle, Toronto, ON M5S 1A8, Canada.
authors.forename: Matthieu
authors.initials: M
authors.lastname: Schapira
authors.affiliation: Structural Genomics Consortium, University of Toronto, MaRS South Tower, 101 College St, Toronto, ON, M5G 1L7, Canada; Toronto General Hospital Research Institute, 200 Elizabeth St., Toronto, ON M5G 2C4, Canada.
authors.forename: Raymond
authors.initials: R
authors.lastname: Hui
authors.affiliation: Center for Integrative Chemical Biology and Drug Discovery, UNC Eshelman School of Pharmacy, University of North Carolina, 125 Mason Farm Road, CB#7363, Chapel Hill, NC 27599-7363.
authors.forename: William
authors.initials: W
authors.lastname: Janzen
authors.affiliation: Department of Molecular Microbiology, Washington University Sch. Med. St. Louis MO 63110.
authors.forename: L David
authors.initials: LD
authors.lastname: Sibley
keywords: high throughput screening; interferon; pathogen; phosphorylation; toxoplasmosis; virulence