Toxoplasma calcium-dependent protein kinase 1 inhibitors: probing activity and resistance using cellular thermal shift assays [PublishAheadOfPrint]

In Toxoplasma gondii, calcium-dependent protein kinase 1 (CDPK1) is an essential protein kinase required for invasion of host cells. We have developed several hundred CDPK1 inhibitors, many of which block invasion. Inhibitors with similar IC₅₀s were tested in thermal shift assays for their ability to stabilize CDPK1 in cell lysates, in intact cells, or in purified form. Compounds that inhibited parasite growth stabilized CDPK1 in all assays. In contrast, two compounds that showed poor growth inhibition stabilized CDPK1 in lysates but not in cells. Thus, cellular exclusion could explain exceptions in the correlation between the action on the target and cellular activity. We used thermal shift assays to examine CDPK1 in two clones that were independently selected by growth in the CDPK1 inhibitor RM-1-132 and that have increased EC₅₀s for the compound. The A and C clones have distinct point mutations in the CDPK1 kinase domain, H201Q and L96P respectively, residues that lie near one another in the inactive isoform. Purified mutant proteins showed similar RM-1-132 IC₅₀s and thermal shifts to wild type CDPK1. Reduced inhibitor stabilization (and presumed reduced interaction) was only observed in cellular thermal shift assays. This highlights the utility of cellular thermal shift assays in demonstrating that resistance involves reduced on-target engagement (even if biochemical assays suggest otherwise). Indeed, similar EC₅₀s were observed upon over-expression of the mutant proteins as in the corresponding drug-selected parasites, although high levels of CDPK1(H201Q) only modestly increased resistance as compared to high levels of wild type enzyme.

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