Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. Montoya, J.G., Boothroyd, J.C. & Kovacs, J.A. Toxoplasma gondii. in Principles and Practice of Infectious Diseases 7th ed. (eds. Mandell, G.L., Bennett, J.E., & Dolin, R.) 3485–3526 (Churchill Livingstone Elsevier, Philadelphia, 2010).

  2. Mead, P.S. et al. Food-related illness and death in the United States. Emerg. Infect. Dis. 5, 607–625 (1999).

    Article  CAS  Google Scholar 

  3. Baril, L. et al. Risk factors for Toxoplasma infection in pregnancy: a case-control study in France. Scand. J. Infect. Dis. 31, 305–309 (1999).

    Article  CAS  Google Scholar 

  4. Jones, J.L. et al. Toxoplasma gondii infection in the United States: seroprevalence and risk factors. Am. J. Epidemiol. 154, 357–365 (2001).

    Article  CAS  Google Scholar 

  5. Wallace, M.R., Rossetti, R.J. & Olson, P.E. Cats and toxoplasmosis risk in HIV-infected adults. J. Am. Med. Assoc. 269, 76–77 (1993).

    Article  CAS  Google Scholar 

  6. Vastava, P.B. et al. MRI features of Toxoplasma encephalitis in the immunocompetent host: a report of two cases. Neuroradiology 44, 834–838 (2002).

    Article  CAS  Google Scholar 

  7. Hermentin, K. et al. Comparison of different serotests for specific Toxoplasma Igm-antibodies (Isaga, Spiha, Ifat) and detection of circulating antigen in 2 cases of laboratory acquired Toxoplasma infection. Zentralbl. Bakteriol. Mikrobiol. Hyg. [A] 270, 534–541 (1989).

    CAS  Google Scholar 

  8. Bach, M.C. & Armstrong, R.M. Acute toxoplasmic encephalitis in a normal adult. Arch. Neurol. 40, 596–597 (1983).

    Article  CAS  Google Scholar 

  9. Pelphrey, P.M. et al. Highly efficient ligands for dihydrofolate reductase from Cryptosporidium hominis and Toxoplasma gondii inspired by structural analysis. J. Med. Chem. 50, 940–950 (2007).

    Article  CAS  Google Scholar 

  10. Dannemann, B. et al. Treatment of toxoplasmic encephalitis in patients with AIDS-a randomized trial comparing pyrimethamine plus clindamycin to pyrimethamine plus sulfadiazine. Ann. Intern. Med. 116, 33–43 (1992).

    Article  CAS  Google Scholar 

  11. Jacobson, J.M. et al. Pyrimethamine pharmacokinetics in human immunodeficiency virus-positive patients seropositive for Toxoplasma gondii. Antimicrob. Agents Chemother. 40, 1360–1365 (1996).

    Article  CAS  Google Scholar 

  12. Nagamune, K. & Sibley, L.D. Comparative genomic and phylogenetic analyses of calcium ATPases and calcium-regulated proteins in the apicomplexa. Mol. Biol. Evol. 23, 1613–1627 (2006).

    Article  CAS  Google Scholar 

  13. Lovett, J.L. & Sibley, L.D. Intracellular calcium stores in Toxoplasma gondii govern invasion of host cells. J. Cell Sci. 116, 3009–3016 (2003).

    Article  CAS  Google Scholar 

  14. Kieschnick, H. et al. Toxoplasma gondii attachment to host cells is regulated by a calmodulin-like domain protein kinase. J. Biol. Chem. 276, 12369–12377 (2001).

    Article  CAS  Google Scholar 

  15. Canduri, F. et al. Protein kinases as targets for antiparasitic chemotherapy drugs. Curr. Drug Targets 8, 389–398 (2007).

    Article  CAS  Google Scholar 

  16. Doerig, C. et al. Protein kinases as targets for antimalarial intervention: kinomics, structure-based design, transmission-blockade, and targeting host cell enzymes. Biochim. Biophys. Acta 1754, 132–150 (2005).

    Article  CAS  Google Scholar 

  17. Harper, J.F. & Harmon, A. Plants, symbiosis and parasites: a calcium signalling connection. Nat. Rev. Mol. Cell Biol. 6, 555–566 (2005).

    Article  CAS  Google Scholar 

  18. Raichaudhuri, A. et al. Domain analysis of a groundnut calcium-dependent protein kinase: nuclear localization sequence in the junction domain is coupled with nonconsensus calcium binding domains. J. Biol. Chem. 281, 10399–10409 (2006).

    Article  CAS  Google Scholar 

  19. Wernimont et al. Structures of apicomplexan calcium-dependent protein kinases reveal mechanism of activation by calcium. Nat. Struct. Mol. Biol. advance online publication, doi:10.1038/nsmb.1796 (02 May 2010).

  20. Noble, M.E., Endicott, J.A. & Johnson, L.N. Protein kinase inhibitors: insights into drug design from structure. Science 303, 1800–1805 (2004).

    Article  CAS  Google Scholar 

  21. Knight, Z.A. & Shokat, K.M. Features of selective kinase inhibitors. Chem. Biol. 12, 621–637 (2005).

    Article  CAS  Google Scholar 

  22. Cohen, M.S. et al. Structural bioinformatics-based design of selective, irreversible kinase inhibitors. Science 308, 1318–1321 (2005).

    Article  CAS  Google Scholar 

  23. Liao, J.J. Molecular recognition of protein kinase binding pockets for design of potent and selective kinase inhibitors. J. Med. Chem. 50, 409–424 (2007).

    Article  CAS  Google Scholar 

  24. Bishop, A.C. et al. A chemical switch for inhibitor-sensitive alleles of any protein kinase. Nature 407, 395–401 (2000).

    Article  CAS  Google Scholar 

  25. Bishop, A.C. et al. Design of allele-specific inhibitors to probe protein kinase signaling. Curr. Biol. 8, 257–266 (1998).

    Article  CAS  Google Scholar 

  26. Bishop, A.C. et al. Generation of monospecific nanomolar tyrosine kinase inhibitors via a chemical genetic approach. J. Am. Chem. Soc. 121, 627–631 (1999).

    Article  CAS  Google Scholar 

  27. Zhang, C. et al. A second-site suppressor strategy for chemical genetic analysis of diverse protein kinases. Nat. Methods 2, 435–441 (2005).

    Article  CAS  Google Scholar 

  28. Bishop, A.C., Buzko, O. & Shokat, K.M. Magic bullets for protein kinases. Trends Cell Biol. 11, 167–172 (2001).

    Article  CAS  Google Scholar 

  29. Johnson, A.W. et al. The brain-derived neurotrophic factor receptor TrkB is critical for the acquisition but not expression of conditioned incentive value. Eur. J. Neurosci. 28, 997–1002 (2008).

    Article  Google Scholar 

  30. Morgan, D.J. et al. Tissue-specific PKA inhibition using a chemical genetic approach and its application to studies on sperm capacitation. Proc. Natl. Acad. Sci. USA 105, 20740–20745 (2008).

    Article  CAS  Google Scholar 

  31. Chen, X. et al. A chemical-genetic approach to studying neurotrophin signaling. Neuron 46, 13–21 (2005).

    Article  Google Scholar 

  32. Kafsack, B.F., Beckers, C. & Carruthers, V.B. Synchronous invasion of host cells by Toxoplasma gondii. Mol. Biochem. Parasitol. 136, 309–311 (2004).

    Article  CAS  Google Scholar 

  33. Alexandrov, A. et al. A facile method for high-throughput co-expression of protein pairs. Mol. Cell. Proteomics 3, 934–938 (2004).

    Article  CAS  Google Scholar 

  34. Ojo, K.K. et al. Glycogen synthase kinase 3 is a potential drug target for African trypanosomiasis therapy. Antimicrob. Agents Chemother. 52, 3710–3717 (2008).

    Article  CAS  Google Scholar 

  35. Hendrickson, W.A., Horton, J.R. & Lemaster, D.M. Selenomethionyl proteins produced for analysis by multiwavelength anomalous diffraction (Mad)-a vehicle for direct determination of 3-dimensional structure. EMBO J. 9, 1665–1672 (1990).

    Article  CAS  Google Scholar 

  36. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    Article  CAS  Google Scholar 

  37. Terwilliger, T. SOLVE and RESOLVE: automated structure solution, density modification, and model building. J. Synchrotron Radiat. 11, 49–52 (2004).

    Article  CAS  Google Scholar 

  38. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  Google Scholar 

  39. Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997).

    Article  CAS  Google Scholar 

  40. McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  Google Scholar 

  41. Cohen, S.X. et al. ARP/wARP and molecular replacement: the next generation. Acta Crystallogr. D Biol. Crystallogr. 64, 49–60 (2008).

    Article  CAS  Google Scholar 

  42. Schuttelkopf, A.W. & van Aalten, D.M.F. PRODRG: a tool for high-throughput crystallography of protein-ligand complexes. Acta Crystallogr. D Biol. Crystallogr. 60, 1355–1363 (2004).

    Article  Google Scholar 

  43. Lovell, S.C. et al. Structure validation by C α geometry: φ,ψ and C β deviation. Proteins 50, 437–450 (2003).

    Article  CAS  Google Scholar 

  44. Matrajt, M. et al. Amino-terminal control of transgenic protein expression levels in Toxoplasma gondii. Mol. Biochem. Parasitol. 120, 285–289 (2002).

    Article  CAS  Google Scholar 

  45. Striepen, B. et al. Expression, selection, and organellar targeting of the green fluorescent protein in Toxoplasma gondii. Mol. Biochem. Parasitol. 92, 325–338 (1998).

    Article  CAS  Google Scholar 

  46. DeRocher, A. et al. Analysis of targeting sequences demonstrates that trafficking to the Toxoplasma gondii plastid branches off the secretory system. J. Cell Sci. 113, 3969–3977 (2000).

    CAS  PubMed  Google Scholar 

  47. Karnataki, A. et al. Cell cycle-regulated vesicular trafficking of Toxoplasma APT1, a protein localized to multiple apicoplast membranes. Mol. Microbiol. 63, 1653–1668 (2007).

    Article  CAS  Google Scholar 

  48. Fruth, I.A. & Arrizabalaga, G. Toxoplasma gondii: induction of egress by the potassium ionophore nigericin. Int. J. Parasitol. 37, 1559–1567 (2007).

    Article  CAS  Google Scholar 

  49. Seeber, F. & Boothroyd, J.C. Escherichia coli β-galactosidase as an in vitro and in vivo reporter enzyme and stable transfection marker in the intracellular protozoan parasite Toxoplasma gondii. Gene 169, 39–45 (1996).

    Article  CAS  Google Scholar 

Download references

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

Authors and Affiliations

Authors

Contributions

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.

Corresponding authors

Correspondence to Ethan A Merritt or Wesley C Van Voorhis.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

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

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.1818

This article is cited by

Search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research