New drugs are needed to treat toxoplasmosis. Toxoplasma gondii calcium-dependent protein kinases (TgCDPKs) are attractive targets because they are absent in mammals. We show that TgCDPK1 is inhibited by low nanomolar levels of bumped kinase inhibitors (BKIs), compounds designed to be inactive against mammalian kinases. Cocrystal structures of TgCDPK1 with BKIs confirm that the structural basis for selectivity is due to the unique glycine gatekeeper residue in the ATP-binding site at residue 128. We show that BKIs interfere with an early step in T. gondii infection of human cells in culture. Furthermore, we show that TgCDPK1 is the in vivo target of BKIs because T. gondii cells expressing a glycine to methionine gatekeeper mutant enzyme show significantly decreased sensitivity to this class of selective kinase inhibitors. Thus, design of selective TgCDPK1 inhibitors with low host toxicity may be achievable.The food-borne apicomplexan protozoan Toxoplasma gondii is the causative agent of toxoplasmosis and may be the most common infectious eukaryotic parasite of humans, based Correspondence should be addressed to W.C.V.V. (wesley@u.washington.edu) or E.A.M. (merritt@u.washington.edu). Accession codes. Protein Data Bank: Atomic coordinates and structure factors have been deposited with accession numbers 3I79 (apo), 3I7c (NA-PP2 complex) and 3I7b (NM-PP1 complex).Note: Supplementary information is available on the Nature Structural & Molecular Biology website. AUTHOR CONTRIBUTIONSK.K.O., K.R.K., K.K.I. and W.C.V.V. were involved in the biochemical characterization and testing of inhibitors of TgCDPK1; L.J.C., K.K.O., K.R.K., A.J.N., C.L.M.J.V., F.S.B. and W.C.V.V. selected, cloned and purified the recombinant wild-type and mutant TgCDPK1 protein; E.T.L., J.E.K., T.L.A., L.Z., W.G.J.H. and E.A.M. crystallized and solved the structure of TgCDPK1; R.M. and D.J.M. synthesized the inhibitors; A.E.D. and M.P. performed the cellular T. gondii experiments; K.K.O., E.T.L., A.E.D., D.J.M., M.P., E.A.M. and W.C.V.V. wrote the paper; all authors reviewed and edited the paper. COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests.Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/. 10 . These drugs are problematic in that they can cause rash, leucopenia and nephrotoxicity11, and sulfadiazine and pyrimethamine can result in complications during pregnancy. New therapeutics against T. gondii are needed. NIH Public AccessCalcium levels have long been associated with T. gondii's interrelated processes of invasion, gliding motility and secretion 12 . The intracellular Ca 2+ level oscillates during gliding motility and is promptly dampened upon cell invasion, preventing T. gondii from immediately gliding out of cells 13 . Calcium oscillations control many targets in the cell, and the mediation of invasion, micronemal secretion and gliding motility is thought to be largely due to T. gondii calcium-dependent protein kinases (TgCDPKs) 12,14 .Protein ...
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