Exploring Kinases of Metabolism and Signaling in the Malaria Parasite Plasmodium
Plasmodium is the causal agent of malaria, which is a parasitic disease that affects more than 215 million people annually and is endemic in 91 countries worldwide. Unfortunately, the parasites have developed resistance to all current pharmaceuticals used to treat malaria, including the front-line treatments artemisinin and artemisinin combination therapies. Due to the rapidly increasing drug resistance problem, new multi-stage inhibitors of Plasmodium are desirable. Of particular interest as multi-stage drug targets are parasite kinases since they are essential regulators in signaling, cell cycle control, and metabolism. Additionally, kinases play important roles in disease states, including cancer, heart disease, and neurodegenerative disorders. This has encouraged the work described here, which focuses on characterizing the atypical protein kinase 9 (PK9), protein kinase 5 (PK5), and shikimate kinase (SK) in Plasmodium with biochemical and chemical methods.
Specifically, target-based screening with the atypical P. falciparum PK9 revealed that benzimidazole and aminoquinoline compounds are able to bind the parasite kinase with low µM Kd(app) values. Furthermore, the top screening hit, takinib, was able to reduce parasite load in a dose-dependent manner. Takinib is the first reported binder of PfPK9 and was found to increase liver stage parasite size during later stages of infection. This unique phenotype may be the result of takinib influencing nutrient acquisition by the parasite or by modulating a cell-cycle control pathway. Takinib was also found to inhibit the human TAK1 (HsTAK1) kinase, which phosphorylates UBC13 and is involved in K63-linked ubiquitination pathways in the host. PfPK9 phosphorylates a parasite UBC13 and this work supports modulation of K63-linked ubiquitination in live parasites by takinib, suggesting similar functionalities between PfPK9 and HsTAK1.
To identify a more parasite-selective probe, 15 takinib analogs were evaluated for binding to PfPK9. HS220 was identified as an analog with the ability to bind to PfPK9, but without activity against HsTAK1. HS220 was confirmed to increase liver stage parasite size and decrease K63-linked ubiquitin on several parasite proteins, suggesting both takinib and HS220 have the same cellular target. The identification of the K63-linked ubiquitin targets will be essential to elucidating the downstream members of the PfPK9 signaling cascade. Future studies to further optimize a cellular thermal shift assay coupled with mass spectrometry may confirm on-target binding of takinib and HS220 in Plasmodium parasites. Finally, a model of PfPK9 was generated to guide hypotheses about takinib-binding and enable structural comparison with HsTAK1.
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