Toxoplasma Calcium-Dependent Protein Kinase 1 Inhibitors: Probing Activity and Resistance Using Cellular Thermal Shift Assays

Abstract

In , 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 50% inhibitory concentrations (ICs) 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 had increased 50% effective concentrations (ECs) for the compound. The A and C clones had 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 RM-1-132 ICs and thermal shifts similar to those shown by wild-type CDPK1. Reduced inhibitor stabilization (and a presumed reduced interaction) was observed only 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 ECs were observed upon overexpression of the mutant proteins, as in the corresponding drug-selected parasites, although high levels of CDPK1(H201Q) only modestly increased resistance compared to that achieved with high levels of wild-type enzyme.

Bumped kinase inhibitor 1369 is effective against Cystoisospora suis in vivo and in vitro

“…S2B) as previously reported (Scheele et al, 2018). This result seems counterintuitive since calcium activates the enzymes, yet chelating Ca 2+ appears to maximize thermal stability (Scheele et al, 2018).…”

“…Further validation of the relatedness of Cs CDPK1 to Tg CDPK1 was shown by improved EGTA thermal stabilization of the enzymes relative to the control where Ca 2+ is presumably bound to the recombinant enzyme (Supplementary Fig. S2B) as previously reported (Scheele et al, 2018). This result seems counterintuitive since calcium activates the enzymes, yet chelating Ca 2+ appears to maximize thermal stability (Scheele et al, 2018).…”

In vitromaturation ofToxoplasma gondiibradyzoites in human myotubes and their metabolomic characterization

“…Furthermore, we tested the resistance against two different bumped kinase inhibitors (BKI), BKI1553 and BKI1294 (Fig. 4D), that have been shown to decrease cyst burden in brains of infected mice (Scheele et al, 2018; Winzer et al, 2015). Consistent with previous results, tachyzoites did not grow after exposure to fourfold of the IC 50 of each compound.…”

“…Up to one 1 µM of HDQ did not decrease the RS and cyst diameter but decreased the mitochondrial membrane potential estimated by the drop of intensity of Mitotracker staining intensity by 60 % (Figure SA-D). Furthermore, we tested the resistance against two different bumped kinase inhibitors (BKI), BKI1553 and BKI1294 (Figure 5D), that have been shown to decrease cyst burden in chronically infected mice 60, 61 . Consistent with previous results, tachyzoites did not grow after exposure to fourfold of the IC 50 of each compound.…”

“…Resistance of bradyzoites against pepsin digestion and temperature stress develops after 14 days post infection and increases until 28 days post infection (Fig 3C). The onset of tolerance towards inhibitors of well-established drug targets including the calcium-dependent protein kinase 1 (TgCDPK1) (Scheele et al, 2018; Winzer et al, 2015), dihydrofolate reductase-thymidylate synthase (TgDHFR-TS) (Roos, 1993) and the mitochondrial bc1-complex follows similar timing and is shared with T. gondii cysts in the brain of chronically infected mice (Bajohr et al, 2010; Saleh et al, 2007; Scheele et al, 2018; Winzer et al, 2015). Hence, our in vitro model will facilitate dissecting the molecular basis of the impressive drug tolerance and stress resistance of T. gondii tissue cysts.…”