Functional and molecular studies of the human protein kinase Nek6 as a target candidate for drug design in Prostate Cancer

• Grant number: 18/08391-2
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date until: August 01, 2018
• End date until: April 30, 2023
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Jörg Kobarg
• Grantee: Isadora Carolina Betim Pavan
• Host Institution: Faculdade de Ciências Farmacêuticas (FCF). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil
• Associated research grant: 17/03489-1 - From functional studies to searching for new inhibitors for cancer: exploring kinases that regulate the cell cycle of the human NEK family, AP.TEM

Abstract

Members of the NEK family (NIMA related kinases) have been identified as important regulators of cell cycle checkpoints, especially in the transition to G2/M. Despite being one of the families of less studied kinases, recent studies have shown their crucial roles in mitosis, separation of centromere and signaling pathways for DNA damage. These characteristics coupled with the fact that several family members are overexpressed in Cancer or have high mutation rates make them interesting candidates for diagnosis and Cancer therapy. Overexpression of NEK6 has been reported in several types of Cancer, but recently its central role in the development of Castration-Resistant Prostate Cancer (CRPC) has recently been confirmed. Studies previously published by the group have shown that NEK6 phosphorylates NF-κB signaling pathway proteins (e.g. RelB, Prx-III and TRIP-4). This pathway is essential for cell survival and, interestingly, also appears to play an important role in the CRPC. Objective: The aim of this project is to describe the in vitro and in vivo mechanisms by which NEK6 contributes to the development of Prostate Cancer and its relation to the NF-κB signaling pathway. Investigate whether NEK6 is involved in chemotherapeutic resistance to docetaxel. Finally, identify new physiological substrates and new potential inhibitors of human NEK6. Methods: For in vitro studies, we will use castration-resistant metastatic Prostate Cancer cells (PC-3, DU-145 and 22Rv1), which will have the knockout of the NEK6 gene generated by CRISPR/Cas9 system, and thus functional studies (migration, proliferation, viability, cell cycle and apoptosis) and molecular (evaluation of the NF-κB pathway) will be performed. For in vivo studies, the PC-3 cell line with NEK6 knockout will be injected subcutaneously in immunodeficient mice nude and changes in tumor progression will be analyzed. A subsequent proteomic analysis of the tumors will be performed to verify changes in the protein expression profile. Using "Shokat approach", a NEK6 kinase which recognizes ATP analogues will be developed and, from this, new physiological substrates will be identified by mass spectrometry. To study the involvement of NEK6 in resistance to docetaxel, models of chemotherapy-resistant Prostate Cancer cells (PC-3r, DU-145r and 22Rv1r) will be generated through established protocols. We will screen compounds in vitro by the "bioassay high throughput screening" method, based on a coupled assay technology that uses luciferase to detect kinase ATP consumption, previously developed in the group. Expectations: The project will contribute to understand the mechanisms by which NEK6 is important for the development of Prostate Cancer and, with this, will be analyzed the potential of this protein kinase as a therapeutic target in order to obtain new specific inhibitors. (AU)

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

SIMABUCO, FERNANDO MOREIRA; TAMURA, RODRIGO ESAKI; BETIM PAVAN, ISADORA CAROLINA; MORALE, MIRIAN GALLIOTE; VENTURA, ARMANDO MORAIS. Molecular mechanisms and pharmacological interventions in the replication cycle of human coronaviruses. GENETICS AND MOLECULAR BIOLOGY, v. 44, n. 1, 1, 2021. (19/15619-2, 16/25139-0, 18/08391-2, 18/14818-9, 20/05346-6)

PONTE, LUIS GUSTAVO SABOIA; PAVAN, ISADORA CAROLINA BETIM; MANCINI, MARIANA CAMARGO SILVA; DA SILVA, LUIZ GUILHERME SALVINO; MORELLI, ANA PAULA; SEVERINO, MATHEUS BRANDEMARTE; BEZERRA, ROSANGELA MARIA NEVES; SIMABUCO, FERNANDO MOREIRA. The Hallmarks of Flavonoids in Cancer. Molecules, v. 26, n. 7, APR 2021. (18/08391-2, 19/00607-9, 19/25582-9, 18/14818-9)

SIMABUCO, FERNANDO MOREIRA; TAMURA, RODRIGO ESAKI; BETIM PAVAN, ISADORA CAROLINA; MORALE, MIRIAN GALLIOTE; VENTURA, ARMANDO MORAIS. Molecular mechanisms and pharmacological interventions in the replication cycle of human coronaviruses. GENETICS AND MOLECULAR BIOLOGY, v. 44, n. 1, p. 18-pg., 2021-01-01. (16/25139-0, 19/15619-2, 18/14818-9, 20/05346-6, 18/08391-2)

Academic Publications

(References retrieved automatically from State of São Paulo Research Institutions)

PAVAN, Isadora Carolina Betim. Functional and molecular study of human kinase NEK6 as a target of drugs against prostate cancer. 2023. Doctoral Thesis - Universidade Estadual de Campinas (UNICAMP). Faculdade de Ciências Farmacêuticas Campinas, SP.