Evaluation of thymidine triazolic derivatives as Abl kinase inhibitors
Diego de S. Paulucio (IC)a, Fernando Fumagalli (PQ)b, Flávio da Silva Emery (PQ)b, Heberth de Paulaa (PQ), Pedro A. B. Moraisa (PQ)
Federal University of Espírito Santo
a Chemistry and Physics Department, CCENS, Campus Alegre, ES, Brazil
b School of Pharmaceutical Sciences of Ribeirão Preto
*[email protected]

Introduction. Chronic myeloid leukemia (CML) is a bone marrow neoplasm that transforms the normal hematopoietic progenitor cell into malignant (1). Inhibition of the BCR-ABL gene specifically promotes energy blockade for the Abl tyrosine kinase domain. Competition with the ATP cellular receptor of the Abl tyrosine kinase domain prevents the ability of this transfer of ATP phosphate groups and phosphorylated tyrosine residues, which prevents the transduction of signals necessary for cell proliferation (2) .Aim. Considering Abl kinase is an interesting therapeutic target and the development of many additional inhibitors, here in this work is described enzymatic activity evaluation against Abl kinase with 3 novel thymidine derivatives. Methods. Enzymatic Activity. Abl kinase inhibition activity was measured via a ADP-Glo™ Kinase Assay that quantifies the kinase-dependent enzymatic production of ADP from ATP using a coupled luminescence-based reaction. ABL Tyrosine kinase enzyme (100ηg/µL) was diluted by 40 times with 1x buffer solution (8mM Tris, pH 7.5; 4mM MgCl2; 0.02mg/mL BSA; 10µM DTT). A substrate mix solution was prepared by adding: Abltide synthetic peptide substrate (YRRAAVPPSPSLSRHSSPHQ(pS)EDEEE) 50%; 4x buffer solution, 25% and ATP 100µM 25%. The reaction was made by adding 5µL of the inhibitor compound or distilled water (control) and 20µL of enzyme solution and incubated at 30°C for 10min. After, 20µL substrate mix solution was added and incubated at 30°C for 15min. All the ATP non-transformed by enzyme was depleted by adding 50 µL ADP-GLO® reagent during 40 min at room temperature. The ADP enzyme-produced reacted with 100µL ADP-GLO Detection Reagent® during 60min and the luminescence recorded (integration time 0.5-1s). The control tube was assumed as 100% enzymatic activity. All reagents were purchased from Promega, LLC.

Figura 1. Derivatives of thymidine as Abl kinase inhibitors.
Results. 3 inhibitors candidates were tested at 100µM concentration (Figure 1). Compounds inhibited less than 50% of the Abl kinase activity: T4 (34.26%), T6 (29.13%) and T11 (37.12%) (Table1).
Table 1. Abl kinase inhibition activity of thymidine triazolic derivatives
Comp. no % Abl kinase inhibition at 100µM
T4 34,26
T6 29,13
T11 37,12

Conclusion. Further investigations are ongoing in order to obtain IC50 value of these Abl kinase inhibitors. References. 1. Vendrame-Goloni CB, Carvalho-Salles AB, Ricci Júnior O, Miguel CE, Fett-Conte AC. Análise do rearranjo BCR/ABL por bandamento GTG e FISH: comparação das freqüências ao diagnóstico da LMC. Arq Cienc Saúde. 2006;13(1):7-11. 2. De Almeida Dobbin J, Gadelha MIP. Mesilato de imatinibe para tratamento da leucemia mielóide crônica. Revista brasileira de cancerologia. 2002;48(3):429-38. Keywords. Biochemsitry, Nucleosides, Medicinal Chemistry, Drug Development. Acknowledgments. This work was supported by FAPES and UFES.