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Author Spotlight: Identifying Compensatory Pathways in Malaria Parasites Containing Hypomorphic Allele of Essential Protein Kinases
Published on: November 22, 2024
Bumped Kinase Inhibitors Inhibit both Toxoplasma gondii MAPKL1 and CDPK1
Jemma A Montgomery1, P Holland Alday1,2, Ryan Choi3
1Division of Infectious Diseases, Portland VA Medical Center, Portland, Oregon 97239, United States.
Bumped kinase inhibitors (BKIs) target calcium-dependent protein kinase 1 (TgCDPK1) and mitogen-activated protein kinase-like 1 (TgMAPKL1) in Toxoplasma gondii. Dual targeting suggests a high genetic barrier to drug resistance, aiding therapeutic development.
Area of Science:
- Parasitology
- Drug Discovery
- Molecular Biology
Background:
- Bumped kinase inhibitors (BKIs) are optimized to target calcium-dependent protein kinase 1 (TgCDPK1) in Toxoplasma gondii and Cryptosporidium.
- BKI-1748 is a lead compound for toxoplasmosis, but its precise mechanism of action requires further investigation.
- Previous studies suggested potential off-target effects of BKIs beyond TgCDPK1.
Purpose of the Study:
- To elucidate the mechanism of action of BKI-1748.
- To identify additional targets of BKIs in Toxoplasma gondii.
- To assess the genetic basis for resistance to BKIs.
Main Methods:
- Forward genetic screen using chemical mutagenesis to generate BKI-1748 resistant clones.
- Whole genome sequencing to identify mutations in resistant clones.
- Site-directed mutagenesis to introduce specific mutations (TgMAPKL1 Leu162Gln, TgCDPK1 Gly128Met) into wild-type strains.
Main Results:
- Resistant clones exhibited single nucleotide changes in the ATP binding site of TgMAPKL1.
- Specific mutations in TgMAPKL1 and TgCDPK1 conferred 2-to-6-fold and similar resistance, respectively.
- Combined mutations in TgMAPKL1 and TgCDPK1 resulted in a significant increase in resistance (up to 157-fold).
Conclusions:
- TgMAPKL1, in addition to TgCDPK1, is identified as a target of BKIs.
- Understanding dual targeting enhances knowledge of BKI mechanism of action for therapeutic development.
- A high genetic barrier to drug resistance is suggested for this class of compounds.
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