Identification of inhibitors for the transmembrane Trypanosoma cruzi eIF2α kinase relevant for parasite proliferation

by Selleckchem | Nov 08 2023

The TcK2 protein kinase of Trypanosoma cruzi, the causative agent of Chagas disease, is structurally similar to the human kinase PERK, which phosphorylates the initiation factor eIF2α and, in turn, inhibits translation initiation. We have previously shown that absence of TcK2 kinase impairs parasite proliferation within mammalian cells, positioning it as a potential target for treatment of Chagas disease. To better understand its role in the parasite, here we initially confirmed the importance of TcK2 in parasite proliferation by generating CRISPR/Cas9 TcK2-null cells, albeit they more efficiently differentiate into infective forms. Proteomics indicates that the TcK2 knockout of proliferative forms expresses proteins including trans-sialidases, normally restricted to infective and nonproliferative trypomastigotes explaining decreased proliferation and better differentiation. TcK2 knockout cells lost phosphorylation of eukaryotic initiation factor 3 and cyclic AMP responsive-like element, recognized to promote growth, likely explaining both decreased proliferation and augmented differentiation. To identify specific inhibitors, a library of 379 kinase inhibitors was screened by differential scanning fluorimetry using a recombinant TcK2 encompassing the kinase domain and selected molecules were tested for kinase inhibition. Only Dasatinib and PF-477736, inhibitors of Src/Abl and ChK1 kinases, showed inhibitory activity with IC50 of 0.2 ± 0.02 mM and 0.8 ± 0.1, respectively. In infected cells Dasatinib inhibited growth of parental amastigotes (IC50 = 0.6 ± 0.2 mM) but not TcK2 of depleted parasites (IC50 > 34 mM) identifying Dasatinib as a potential lead for development of therapeutics for Chagas disease targeting TcK2.

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The provided information outlines a research study focused on understanding the role of the TcK2 protein kinase in Trypanosoma cruzi, the causative agent of Chagas disease, and its potential as a therapeutic target. Here's a summary of the key findings and implications:

1. TcK2 is important for parasite proliferation: The researchers confirmed the significance of TcK2 in parasite proliferation by generating CRISPR/Cas9 TcK2-null cells. These cells showed impaired proliferation within mammalian cells. This finding indicates that TcK2 is crucial for the parasite's ability to replicate and grow.

2. TcK2 knockout leads to better differentiation: Interestingly, although TcK2 knockout led to decreased proliferation, the knockout cells more efficiently differentiated into infective forms. This suggests that TcK2 may play a role in regulating the balance between proliferative and nonproliferative stages of the parasite's life cycle.

3. Proteomic changes in TcK2 knockout cells: The proteomic analysis of TcK2 knockout proliferative forms revealed the expression of proteins, such as trans-sialidases, which are typically restricted to infective and nonproliferative trypomastigotes. This explains the observed decreased proliferation and enhanced differentiation in TcK2 knockout cells.

4. TcK2 knockout affects phosphorylation of specific factors: The absence of TcK2 resulted in the loss of phosphorylation of eukaryotic initiation factor 3 and cyclic AMP responsive-like element, which are known to promote growth. This may explain both the decreased proliferation and augmented differentiation in TcK2 knockout cells.

5. Identification of potential inhibitors: The researchers screened a library of 379 kinase inhibitors using a recombinant TcK2 encompassing the kinase domain. Two molecules, Dasatinib and PF-477736, showed inhibitory activity against TcK2 with IC50 values of 0.2 ± 0.02 mM and 0.8 ± 0.1 mM, respectively.

6. Dasatinib as a potential lead for Chagas disease treatment: In infected cells, Dasatinib demonstrated inhibitory activity against parental amastigotes (IC50 = 0.6 ± 0.2 mM), suggesting its potential as a therapeutic agent for Chagas disease targeting TcK2. However, Dasatinib did not inhibit the growth of TcK2-depleted parasites (IC50 > 34 mM), indicating its specificity for TcK2.

Overall, the study reveals the importance of TcK2 in Trypanosoma cruzi proliferation and differentiation and identifies specific kinase inhibitors, such as Dasatinib, that could serve as potential leads for the development of therapeutics targeting TcK2 to combat Chagas disease. Further research and testing will be required to validate these findings and assess the safety and efficacy of these inhibitors for clinical use.