Structural analysis of the kinase proteins PRPF4 and DYRK1B, from the CMGC family, and identification of small compound inhibitors

• Grant number: 19/14275-8
• Support Opportunities: Scholarships in Brazil - Post-Doctorate
• Effective date (Start): August 01, 2019
• Effective date (End): July 31, 2021
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Acordo de Cooperação: CNPq - INCTs
• Principal Investigator: Paulo Arruda
• Grantee: André da Silva Santiago
• Host Institution: Instituto de Biologia (IB). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil
• Associated research grant: 14/50897-0 - INCT 2014: Open-acess Medicinal Chemistry Centre (OpenMedChem), AP.TEM

Abstract

The human genome is composed by approximately 520 annotated kinases, which are responsible for regulating a plethora of biochemical. Kinase proteins are transferases responsible to attach the gamma phosphate from the adenosine triphosphate (ATP) onto the substrate, regulating its activity. They confer a highly dynamic hub that regulates key processes in the signalling responsive for several external stimuli. Many kinase proteins are associated with the development and progression of several types of cancer. They are commonly one of the main target of research to study and treat cancer. Hence, kinases are assured to contribute to several traits that led to dysregulation of target proteins involved in cancer development. Over the past 20 years, the FDA has approved 48 kinase inhibitors, which proves kinases as an important drug target of many diseases.Although the drugs designed for kinases correspond to approximately 6% of all FDA, most of the research focus on 25% of all kinase proteins, whereas most of them remain poorly understudied. Additionally, many of the approved drugs are multidrug kinase, such as sunitinib and cabozantinib, which means the action on the off-targets may lead to toxicities and adverse events.Among the other kinases of the CMGC group, we would like to highlight the importance of investigating PRPF4B and DYRK1B, which have attracted the interest due to their involvement in the control of spindle checkpoint and chromatids distribution and negatively regulation of the cell growth by promoting G1 phase cell cycle arrest and promoting cell resistance to chemotherapy drugs, respectivelyThe untapped kinases from the family of DYRKs and PRPF have been shown to participate of the regulation of important events during the cell cycle. DYRK1B has been received some importance due to the its role in metabolic syndrome related to abdominal fatty acid accumulation and more recently DYRK1B has been shown to participate of the regulation of cell growth by targeting CCND1, a protein required for G1/S cell cycle transition. Whereas the PRPF4 participates of the regulation of pre-mRNA splicing and spindle checkpoint. The search for inhibitors to these understudied kinases would bring some progress to promote the cells to entry in S phase and become more susceptible to chemotherapeutic compounds. In an attempt to identify small molecules able to inhibit the kinases, we propose the current project to approach some unknown questions about PRPF4 and DYRK1B, such as what are the most important residues to achieve a more efficient inhibition and what are the effects of inhibition in cancel cell lines. In an effort to speed up the inhibitor discovery for kinase, the Structural Genomics Consortium, along with medicinal chemistry groups and pharmaceutical companies, successfully developed chemical probes for some of the considered neglected kinases, such as GAK, LIMK, and AAK1. Our work will be based on the identification of inhibitor for PRPF4 and DYRK1B in collaboration with the Aché pharmaceutical company, which has provided a series of inhibitors based on a benzothiophene scaffold, previously demonstrated able to inhibit PRPF4. The new compound series will be tested regarding their capacity to preferentially inhibit PRPF4, using biochemistry and structural biology approach, instead of DYRK1B, since our previous results have identified DYRK1B as the main off-target in the kinome scan.These questions will be approached in this project using molecular biology and proteomic tools. The identification of small molecules for PRPF4 and DYRK1B will be investigated by cloning constructs designed to increase our rate of success in crystallizing it and analyzing them using kinase assays. We hope this project will also support the identification of starting points to generate specific for each of these kinases as part of a collaborative project with Aché

Scientific publications

(4)

(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)

SILVA, SUELEN FERNANDES; KLIPPEL, ANGELICA HOLLUNDER; RAMOS, PRISCILA ZONZINI; SANTIAGO, ANDREE DA SILVA; VALENTINI, SANDRO ROBERTO; BENGTSON, MARIO HENRIQUE; MASSIRER, KATLIN BRAUER; BILSLAND, ELIZABETH; COUNAGO, RAFAEL MIGUEZ; ZANELLI, CLESLEI FERNANDO. Structural features and development of an assay platform of the parasite target deoxyhypusine synthase of Brugia malayi and Leishmania major. PLoS Neglected Tropical Diseases, v. 14, n. 10, OCT 2020. (14/50897-0, 18/16672-1, 16/16970-7, 19/14275-8, 15/03553-6, 13/50724-5)

O'BYRNE, SEAN N.; SCOTT, JOHN W.; PILOTTE, JOSEPH R.; SANTIAGO, ANDRE DA S.; LANGENDORF, CHRISTOPHER G.; OAKHILL, JONATHAN S.; EDUFUL, BENJAMIN J.; COUNAGO, RAFAEL M.; WELLS, CARROW I.; ZUERCHER, WILLIAM J.; et al. In Depth Analysis of Kinase Cross Screening Data to Identify CAMKK2 Inhibitory Scaffolds. Molecules, v. 25, n. 2, JAN 2 2020. (13/50724-5, 14/50897-0, 19/14275-8)

EDUFUL, BENJAMIN J.; O'BYRNE, SEAN N.; TEMME, LOUISA; ASQUITH, CHRISTOPHER R. M.; LIANG, YI; PICADO, ALFREDO; PILOTTE, JOSEPH R.; HOSSAIN, MOHAMMAD ANWAR; WELLS, I, CARROW; ZUERCHER, WILLIAM J.; et al. Hinge Binder Scaffold Hopping Identifies Potent Calcium/Calmodulin-Dependent Protein Kinase Kinase 2 (CAMKK2) Inhibitor Chemotypes. Journal of Medicinal Chemistry, v. 64, n. 15, p. 10849-10877, AUG 12 2021. (13/50724-5, 14/50897-0, 19/14275-8)

PROFETA, GERSON S.; DOS REIS, CAIO V.; SANTIAGO, ANDRE DA S.; GODOI, PAULO H. C.; FALA, ANGELA M.; WELLS, CARROW I.; SARTORI, ROGER; SALMAZO, ANITA P. T.; RAMOS, PRISCILA Z.; MASSIRER, KATLIN B.; et al. Binding and structural analyses of potent inhibitors of the human Ca2+/calmodulin dependent protein kinase kinase 2 (CAMKK2) identified from a collection of commercially-available kinase inhibitors. SCIENTIFIC REPORTS, v. 9, NOV 11 2019. (19/14275-8, 14/50897-0, 13/50724-5)