2. Academic Validation
  3. Identification of inhibitors for the trans-membrane Trypanosoma cruzi eIF2α kinase relevant for parasite proliferation

Identification of inhibitors for the trans-membrane Trypanosoma cruzi eIF2α kinase relevant for parasite proliferation

  • J Biol Chem. 2023 May 23;104857. doi: 10.1016/j.jbc.2023.104857.
Tiago de Paula Marcelino 1,  Angela Maria Fala 2,  Matheus Monteiro da Silva 1,  Normanda Souza-Melo 1,  Amaranta Muniz Malvezzi 1,  Angélica Hollunder Klippel 3,  Martin Zoltner 4,  Norma Padilla-Mejia 5,  Samantha Kosto 5,  Mark C Field 6,  Gabriela de Assis Burle Caldas 7,  Santuza Maria Ribeiro Teixeira 7,  Rafael Miguez Couñago 2,  Katlin Brauer Massirer 8,  Sergio Schenkman 9
Affiliations

Affiliations

  • 1 Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, 04039-032, São Paulo, SP, Brazil.
  • 2 Center for Molecular Biology and Genetic Engineering - CBMEG, Center of Medicinal Chemistry - CQMED, Structural Genomics Consortium - SGC, University of Campinas - UNICAMP, 13083-886, Campinas, SP, Brazil.
  • 3 Center for Molecular Biology and Genetic Engineering - CBMEG, Center of Medicinal Chemistry - CQMED, Structural Genomics Consortium - SGC, University of Campinas - UNICAMP, 13083-886, Campinas, SP, Brazil; Departamento de Ciências Biológicas da Faculdade de Ciências Farmacêuticas da Universidade Estadual Paulista "Júlio de Mesquita Filho"-Unesp, 14800-903, Araraquara, SP, Brazil.
  • 4 Drug Discovery and Evaluation Unit, Department of Parasitology, Faculty of Science, Charles University in Prague, BIOCEV, Průmyslová 595252 50 Vestec, Czech Republic.
  • 5 School of Life Sciences, University of Dundee, Dundee, DD1 5EH, UK.
  • 6 School of Life Sciences, University of Dundee, Dundee, DD1 5EH, UK; Biology Centre, Institute of Parasitology, Czech Academy of Sciences, 37005 Ceske Budejovice, Czech Republic.
  • 7 Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, 30161-970, Belo Horizonte, MG, Brazil.
  • 8 Center for Molecular Biology and Genetic Engineering - CBMEG, Center of Medicinal Chemistry - CQMED, Structural Genomics Consortium - SGC, University of Campinas - UNICAMP, 13083-886, Campinas, SP, Brazil. Electronic address: kmassire@unicamp.br.
  • 9 Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, 04039-032, São Paulo, SP, Brazil. Electronic address: sschenkman@unifesp.br.
PMID: 37230387 DOI: 10.1016/j.jbc.2023.104857
Abstract

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 more efficiently differentiate into infective forms. Proteomics indicates that the TcK2 knockout of proliferative forms express proteins including trans-sialidases, normally restricted to infective and non-proliferative 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.

Keywords

Chagas disease; Protein kinase; T. cruzi EIF2AK2; assay eIF2α; chemical inhibitor; invasion; proteome; recombinant protein.

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