Development of Toxoplasma gondii Calcium-Dependent Protein Kinase 1 (TgCDPK1) Inhibitors with Potent Anti-Toxoplasma Activity

Abstract

Toxoplasmosis is a disease of prominent health concern that is caused by the protozoan parasite, Toxoplasma gondii. Proliferation of T. gondii is dependent on its ability to invade host cells, which is mediated, in part, by calcium-dependent protein kinase 1 (CDPK1). We have developed ATP competitive inhibitors of TgCDPK1 that block invasion of parasites into host cells, preventing their proliferation. The presence of a unique glycine gatekeeper residue in TgCDPK1 permits selective inhibition of the parasite enzyme over human kinases. These potent TgCDPK1 inhibitors do not inhibit the growth of human cell lines and represent promising candidates as toxoplasmosis therapeutics.

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

“…We show here that BKI-1294 also exhibited in vitro activity against the virulent N. caninum isolates Nc-Liv and Nc-Spain7 (IC 50 s ϭ 360 and 270 nM, respectively) and similarly against the cyst-forming T. gondii ME49 strain, while T. gondii RH tachyzoites were much more susceptible (IC 50 ϭ 20 nM). The value we obtained for T. gondii RH inhibition (20 nM) differs substantially from those reported previously (39). However, it is important to note that in this study parasite loads were quantified by real-time PCR while in earlier reports transgenic parasites expressing beta-galactosidase were used, and some degree of variation may be attributed to different culture conditions, host cells, and slight variations in protocols for parasite isolation prior to infection of HFF.…”

“…BKI-1294 was reported earlier to inhibit host cell invasion of transgenic T. gondii RH and N. caninum tachyzoites expressing beta-galactosidase, with IC 50 s of 137 and 32 nM, respectively, if the compound was added to the cultures at the time point the parasites were allowed to invade their host cell (10,39). We show here that BKI-1294 also exhibited in vitro activity against the virulent N. caninum isolates Nc-Liv and Nc-Spain7 (IC 50 s ϭ 360 and 270 nM, respectively) and similarly against the cyst-forming T. gondii ME49 strain, while T. gondii RH tachyzoites were much more susceptible (IC 50 ϭ 20 nM).…”

“…The ATP-binding site of T. gondii CDPK1, but also of other related apicomplexans, such as N. caninum and Cryptosporidium parvum, is characterized by a small glycine gatekeeper residue (10,37), while most mammalian kinases have larger residues such as methionine or phenylalanine at that position. Selective and potent inhibitors have been generated that take advantage of the enlarged space in the CDPK1 ATPbinding site (37)(38)(39). Benzoyl benzimidazole-based selective inhibitors and BKIs based on a pyrazolopyrimidine scaffold can block T. gondii CDPK1 and affect host cell invasion (40)(41)(42).…”

Novel Bumped Kinase Inhibitors Are Safe and Effective Therapeutics in the Calf Clinical Model for Cryptosporidiosis

“…The propagation of C. parvum (Iowa isolate) in calves and oocyst purification [34][35][36][37], synthesis of BKIs [22,25,27,38], in vitro CpCDPK1 enzyme assay [22,25,38], in vitro determination of C. parvum BKI sensitivity [22,25,38,39], neonatal mouse [36,40] and calf models of C. parvum infection [37,[41][42][43], and pharmacologic measurement of BKI plasma and stool levels and plasma protein binding have all been previously described [27,44]. BKI-1294 and BKI-1553 were synthesized on the pyrazolo [2,3-d] pyrimidine scaffold, while BKI-1517 was synthesized on a 5-aminopyrazole-4-carboxamide scaffold [25] (Figure 1).…”

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).…”