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 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 ...

In Vitro and In Vivo Effects of the Bumped Kinase Inhibitor 1294 in the Related Cyst-Forming Apicomplexans Toxoplasma gondii and Neospora caninum

“…Tachyzoites of N. caninum isolates Nc-Liverpool (Nc-Liv) and Nc-Spain7, T. gondii strains ME49 and RH, the T. gondii strain CDPK1_G128M overexpressing a CDPK1 with the gatekeeper mutation G128M, and an isogenic strain, T. gondii CDPK_wt, expressing the wildtype CDPK1 (12) were all maintained by serial passages in either Vero cells or HFF in their respective media (14,15). Tachyzoites were harvested by removing infected cell layers with a rubber cell scraper followed by repeated passages through a 25-gauge needle at 4°C.…”

“…In many apicomplexan CDPK1 enzymes, including T. gondii and N. caninum, the ATP binding pocket is characterized by a glycine as the smallest possible gatekeeper residue. The T. gondii strain CDPK1_G128M, overexpressing a CDPK1 version with a muta-tion (G to M) in the ATP binding pocket, was found to be much less sensitive to BKIs (12).…”

Does protein phosphorylation govern host cell entry and egress by the Apicomplexa?

“…A Tet-inducible knockout of TgCDPK1 blocks the secretion of micronemes, which results in impaired gliding motility, invasion, and egress (Lourido et al, 2010). Several specific inhibitors were designed against TgCDPK1, including pyrazolopyrimidine derivatives for which a gatekeeper mutant was shown to restore microneme secretion in the presence of the drug (Johnson et al, 2012;Kieschnick et al, 2001;Ojo et al, 2010;Sugi et al, 2010).…”

1NM-PP1 Treatment of Mice Infected with Toxoplasma gondii

“…The in vitro susceptibility of TgCDPK1 to 1NM-PP1 (IC 50 =0.02 μM) [8] was not very different from that of susceptible mutated mouse CaMKII (IC 50 =0.03 μM) [15]. TgCDPK1 was reported to localize in the parasite cytosol and not to be secreted [9].…”

“…Using in silico prediction, we found that T. gondii calcium-dependent protein kinase 1 (TgCDPK1), which has a glycine residue at the gate site of the ATP binding pocket, was the main T. gondii analog-sensitive kinase [12]. In silico prediction, an in vitro culture assay using transgenic parasites, and a structural analysis of TgCDPK1 all showed that TgCDPK1 is a target of BKI [6,8,12]. TgCDPK1 is involved in parasite attachment, invasion, egress, and secretion in vitro [5,6].…”