Expression of the kinase domains of Neks 1,4,6,8,10 e 11 for biochemical inhibitor screenings in vitro , to search for new inhibitors anti-cancer

• Grant number: 24/10662-5
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: August 01, 2024
• End date: July 31, 2028
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Jörg Kobarg
• Grantee: Bruno Henrique Ferreira Rodrigues
• Host Institution: Faculdade de Ciências Farmacêuticas (FCF). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil
• Associated research grant: 22/15126-9 - Two cell cycle regulatroy protein families: from functional studies to their use as novel diagnostic markers and therapeutic targets in cancer, AP.TEM

Abstract

Cancer is one of the most devastating diseases of man and domestic animals. Depending on the type of tissue or cell affected by mutations, a great number of cancer sub-types can de established. These sub-types are only marginally mapped and tend to be quite heterogeneous. This scenario representes one of the major frontiers of the cancer field but may lead to numerous new diagnostic and therapeutic oportunities. Protein kinases in signal transduction pathways are regulators or affect other reguladors of cell proliferation. Activating and inactivating point mutations in kinases and substrates are known to contribute to tumorigenesis. Here, we will focus on six members of the human Nek family (1,4,6, 8,10 and 11), who are known to regulate: cell cycle , mitosis, primary cillary, centrossome disjunction, DNA repair and mitochondrial homeostasis. To Discover novel candidate inibitors for these kinases we will perform high through put in vitro biochemical screenings -of up to 10.000 chemically diverse synthetic or natural compounds-, by using a coupled luciferase fluorescence reporter assay. This indirectly determinates the ATP consume by the bacterially expressed (or Baculo vírus expressed) and purified Nek kinases in the presence or abscence of selected recombinant substrate proteins o designed peptide substrates (Envision). Next, we plan to preform human cell based assays, with selected biologically active substances/inhibitor candidates. We will compare wild type and Nek knock out cells of the same linage, already generated and available , through Crispr Cas9 technology. In an ideal scenario we will expect to find diferences in importante cellular characteristics: such as proliferation, cell death, cell cycle, DNA repair. This last part of this project will allow us to see if the pre-selected inhibitors can indeed enter the cell and provoke na significant phenotype in the wild type cells but not the knock out cells, which we use to demonstrate molecular selctivity.