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ACS Infect Dis. 2016 Mar 11;2(3):194-206. Epub 2015 Dec 28.

Identification of small molecule inhibitors that block the Toxoplasma gondii rhoptry kinase ROP18.

Author information

1
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.
2
Department of Molecular Microbiology, Washington University Sch. Med. St. Louis MO 63110.
3
Department of Biochemistry and Molecular Biophysics, Washington University Sch. Med. St. Louis MO 63110.
4
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.
5
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.
6
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.

Abstract

The protozoan parasite Toxoplasma gondii secretes 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 of T. gondii strains from North and South America. Here, we implemented a high throughput screen to identify small molecule inhibitors of ROP18 in vitro and 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 Δrop18 mutant 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.

KEYWORDS:

high throughput screening; interferon; pathogen; phosphorylation; toxoplasmosis; virulence

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