Toxoplasma gondii calcium-dependent protein kinase 1 is a target for selective kinase inhibitors

Abstract

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 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. 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 expressing a glycine to methionine gatekeeper mutant enzyme show significantly decreased sensitivity to BKIs. Thus, design of selective TgCDPK1 inhibitors with low host toxicity may be achievable.

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Figure 1: Structure of calcium-free TgCDPK1.
Figure 2: Cellular localization of TgCDPK1 and effects of bumped kinase inhibitors on T. gondii binding to and invasion of mammalian cells.
Figure 3: TgCDPK1 gatekeeper mutant reduces sensitivity to BKIs.

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Acknowledgements

The authors would like to acknowledge the generous assistance of F. Ghomashchi and M. Gelb in delineating the calcium dependence and enzyme kinetics of TgCDPK1. This work was funded by US National Institute of Allergy and Infectious Diseases grants R01AI080625 (W.C.V.V.), R01AI50506 (M.P.) and AI067921 (C.L.M.J.V., F.S.B., W.G.J.H., E.A.M. and W.C.V.V.) and financial support from G. and K. Pigotti. K.K.I. was supported by a US National Institutes of Health grant from the Fogarty International Center 2D43 TW000924. Portions of this research were carried out at the Stanford Synchrotron Radiation Lightsource, a national user facility operated by Stanford University on behalf of the US Department of Energy, Office of Basic Energy Sciences.

Author information

K.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.C.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.

Correspondence to Ethan A Merritt or Wesley C Van Voorhis.

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Supplementary Figures 1–5, Supplementary Tables 1 and 2, Supplementary Methods (PDF 632 kb)

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Ojo, K., Larson, E., Keyloun, K. et al. Toxoplasma gondii calcium-dependent protein kinase 1 is a target for selective kinase inhibitors. Nat Struct Mol Biol 17, 602–607 (2010). https://doi.org/10.1038/nsmb.1818

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