Vol. 63, Issue 2, 263-270, February 2003

ACCELERATED COMMUNICATION
Non-Nucleoside Inhibitors of Mitochondrial Thymidine Kinase (TK-2) Differentially Inhibit the Closely Related Herpes Simplex Virus Type 1 TK and Drosophila melanogaster Multifunctional Deoxynucleoside Kinase

Jan Balzarini, Ana-Isabel Hernández, Philippe Roche, Robert Esnouf, Anna Karlsson, Maria-José Camarasa, and Maria-Jesus Pérez-Pérez

Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium (J.B.); Instituto de Química Médica, Consejo Superior de Investigaciones Cientificas, Madrid, Spain (A.-I.H., M.-J.C., M.-J.P.-P.); the Wellcome Trust Centre for Human Genetics, Oxford, United Kingdom (P.R., R.E.); and the Karolinska Institute, Huddinge University Hospital, Huddinge, Sweden (A.K.)

5'-O-Trityl derivatives of thymidine (dThd), (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), and their acyclic analogs 1-[(Z)-4-triphenylmethoxy-2-butenyl]thymine (KIN-12) and (E)-5-(2-bromovinyl)-1-[(Z)-4-triphenylmethoxy-2-butenyl]uracil (KIN-52) have been synthesized and evaluated for their inhibitory activity against the amino acid sequence related mitochondrial dThd kinase (TK-2), herpes simplex virus type 1 (HSV-1) TK, and Drosophila melanogaster multifunctional 2'-deoxynucleoside kinase (Dm-dNK). Several compounds proved markedly inhibitory to these enzymes and represent a new generation of nucleoside kinase inhibitors. KIN-52 was the most potent and selective inhibitor of TK-2 (IC₅₀, 1.3 µM; K_i, 0.50 µM; K_i/K_m, 0.37) but was not inhibitory against HSV-1 TK and Dm-dNK at 100 µM. As found for the alternative substrate BVDU, the tritylated compounds competitively inhibited the three enzymes with respect to dThd. However, whereas BVDU behaved as a noncompetitive inhibitor (alternative substrate) of TK-2 and HSV-1 TK with respect to ATP as the varying substrate, the novel tritylated enzyme inhibitors emerged as reversible purely uncompetitive inhibitors of these enzymes. Computer-assisted modeling studies are in agreement with these findings. The tritylated compounds do not act as alternative substrates and they showed a type of kinetics against the nucleoside kinases different from that of BVDU. KIN-12, and particularly KIN-52, are the very first non-nucleoside specific inhibitors of TK-2 reported and may be useful for studying the physiological role of the mitochondrial TK-2 enzyme.