Towards a Survival-Based Cellular Assay for the Selection of Protease Inhibitors in Escherichia coli

Autor(es)

Oyadomari, William Y. ; Carvalho, Elizangela A. ; Machado, Gabriel E. ; Machado, Ana Julia O. ; Santos, Gabriel S. ; Marcondes, Marcelo ; Oliveira, Vitor

• Tipo de documento: Artigo Científico
• Fonte: BIOTECH; v. 14, n. 1, p. 10-pg., 2025-03-07.

Resumo

We describe a method tailored to the in-cell selection of protease inhibitors. In this method, a target protease is co-expressed with a selective substrate, the product of which kills host cells. Therefore, the method can be applied to identify potential inhibitors based on cell host survival when inhibition of the target protease occurs. The TEV protease was chosen for this proof-of-concept experiment. The genetically encoded selective substrate is a single polypeptide chain composed of three parts: (1) a ccdB protein, which can cause host cell death when it accumulates inside the cell; (2) a protease cleavage sequence that can be changed according to the target protease, in this case the TEV substrate ENLYFQ down arrow G (down arrow-predicted cleavage site); and (3) the ssrA sequence (AANDENYALAA), which drives the polypeptide to degradation by the ClpX/ClpP complex inside host E. coli cells. In our experiment, co-expression of the active TEV protease and this selective substrate (ccdB-ENLYFQG-ssrA) caused the death of a significant host cell population, while control assays with an inactive mutant TEV Asp81Asn did not. Details of the methodology used are given, providing the basis for the application of similar systems for other proteases of interest. (AU)

• Processo FAPESP: 23/07904-4 - Estudo dos mecanismos moleculares e celulares em transtornos mentais: estudos clínicos e modelos animais
• Beneficiário: William Yoshio Agliardi Oyadomari
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado
• Processo FAPESP: 20/09678-3 - Seleção de inibidores das proteases NS2B/NS3 dos vírus Dengue e Zika através de bibliotecas de peptídeos cíclicos e ensaios de triagem de alto desempenho (high-throughput screening)
• Beneficiário: Vitor Marcelo Silveira Bueno Brandão de Oliveira
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 21/11936-3 - Centro de Ciência Translacional e Desenvolvimento de Biofármacos
• Beneficiário: Benedito Barraviera
• Modalidade de apoio: Auxílio à Pesquisa - Centros de Ciência para o Desenvolvimento
• Processo FAPESP: 23/09167-7 - Equipando anticorpos e bactérias para uso diagnóstico e terapêutico de tumores.
• Beneficiário: Vitor Marcelo Silveira Bueno Brandão de Oliveira
• Modalidade de apoio: Auxílio à Pesquisa - Regular