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

Suzanne Scheele1, Jennifer A Geiger1, Amy E DeRocher1

  • 1Center for Infectious Disease Research, Seattle, Washington, USA.

Antimicrobial Agents and Chemotherapy
|March 21, 2018
PubMed
Summary

Cellular thermal shift assays reveal drug resistance mechanisms in Toxoplasma gondii by showing reduced on-target engagement of calcium-dependent protein kinase 1 (CDPK1) inhibitors, even when biochemical assays suggest otherwise.

Keywords:
Toxoplasma gondiibumped kinase inhibitorcellular thermal shift assayinhibitorprotein kinase

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Area of Science:

  • Parasitology
  • Molecular Biology
  • Drug Discovery

Background:

  • Toxoplasma gondii invasion relies on essential protein kinase CDPK1.
  • CDPK1 inhibitors are developed to block parasite invasion.
  • Understanding drug resistance mechanisms is crucial for effective treatment.

Purpose of the Study:

  • To investigate the mechanism of resistance to CDPK1 inhibitors in T. gondii.
  • To evaluate the utility of cellular thermal shift assays (CETSA) in assessing drug target engagement in vivo.
  • To correlate in vitro biochemical activity with cellular efficacy of CDPK1 inhibitors.

Main Methods:

  • Development of several hundred CDPK1 inhibitors.
  • Thermal shift assays (TSA) on purified CDPK1, cell lysates, and intact cells.
  • Selection of drug-resistant T. gondii clones.
  • Genetic characterization of mutations in resistant clones.
  • Comparison of inhibitor activity in biochemical and cellular assays.

Main Results:

  • Compounds inhibiting parasite growth stabilized CDPK1 in all TSA formats.
  • Poorly inhibiting compounds stabilized CDPK1 in lysates but not intact cells, suggesting cellular exclusion.
  • Drug-selected resistant clones (A and C) exhibited distinct point mutations (H201Q and L96P) in the CDPK1 kinase domain.
  • Mutant CDPK1 proteins showed similar biochemical IC50s and thermal shifts as wild-type, but reduced stabilization in cellular TSA.
  • Overexpression of mutant CDPK1 partially increased resistance, but less effectively than wild-type.

Conclusions:

  • Cellular exclusion can explain discrepancies between target inhibition and cellular activity.
  • CETSA is valuable for demonstrating reduced on-target engagement in resistant cells, even when biochemical assays are misleading.
  • Mutations conferring resistance to CDPK1 inhibitors can lead to reduced drug interaction within the cell.