Development of biological assays for screening thymidine analogues as potent and selective PET probes of human thymidine kinases.

Objectives The aim of this study was to develop a methodological platform for the screening of novel thymidine analogues that can be used as PET probes targeted to the measurement of thymidine kinase level in tumor proliferation.

Methods Human recombinant TK-1 and TK-2 were expressed in E. coli as fusion protein containing an N-terminal His-tag and a thrombin cleavage site, and purified by using metal affinity chromatography. Kinetic binding parameters of thymidine analogues (FMAU as a control) were determined by using a phosphotransferase assay with ³²P-ATP as a labeled substrate. The data were fitted into the Michaelis-Menten equation. The K_m, V_max, and k_cat values were calculated using subunit molecular weight of the enzymes. Inhibition assays were performed using the DE-81 filter paper with ³H-dT as a substrate. The mode of inhibition and K_i values were derived from the Dixon plots.

Results The specific activity of TK-1 and TK-2 was measured to be 2637±132 and 1872±95 nmol/min/mg, respectively. The phosphotransferase assay showed that FMAU is a substrate for both TK-1 (K_m=32.5±1.2µM) and TK-2 (K_m=72.3±3.7µM), whereas a new thymidine analogue (T07) is a good substrate of TK-2 (K_m=44.6±5.0µM) but not TK-1. As determined by the inhibition assay, FMAU inhibited the phosphorylation of ³H-dT for both TK-1 (Ki=70±4µM) and TK-2 (K_i=4.9±0.3µM). However, T07 showed excellent potency and selectivity for TK-2. T07 was determined to be an uncompetitive inhibitor of ³H-dT for TK-1 (K_i=192.2±8µM), and a competitive inhibitor of ³H-dT for TK-2 (K_i=0.017±0.001µM).

Conclusions A reliable method for the screening of thymidine analogues has been successfully developed. The hits identified from biological assays can be used as PET probes for specifically measuring thymidine kinase level in tumor proliferation.

Research Support This work was supported by the SNMMI Mitzi & William Blahd Research Grant, the Whittier Grant for Translational Research, and the USC Department of Radiology.