Toxoplasma Calcium-Dependent Protein Kinase 1 Inhibitors: Probing Activity and Resistance Using Cellular Thermal Shift Assays.
Suzanne ScheeleJennifer A GeigerAmy E DeRocherRyan ChoiTess R SmithMatthew A HulversonRama Subba Rao VidadalaLynn K BarrettDustin J MalyEthan A MerrittKayode K OjoWesley C Van VoorhisMarilyn ParsonsPublished in: Antimicrobial agents and chemotherapy (2018)
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 50% inhibitory concentrations (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 had increased 50% effective concentrations (EC50s) 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 IC50s 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 EC50s 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.
Keyphrases
- wild type
- high throughput
- induced apoptosis
- protein kinase
- cell cycle arrest
- toxoplasma gondii
- endoplasmic reticulum stress
- single cell
- transcription factor
- oxidative stress
- stem cells
- signaling pathway
- cell proliferation
- magnetic resonance imaging
- tyrosine kinase
- cell death
- social media
- molecular dynamics simulations
- drug induced