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

5 -O-Trityl derivatives of thymidine (dThd), (E)-5-(2-bromovinyl)-2 -deoxyuridine (BVDU), and their acyclic analogs 1-[(Z)-4triphenylmethoxy-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 M; Ki, 0.50 M; Ki/Km, 0.37) but was not inhibitory against HSV-1 TK and Dm-dNK at 100 M. 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. In mammalian cells, there are four different 2 -deoxynucleoside kinases with partially overlapping substrate specificities (Arnér and Eriksson, 1995; Johansson et al., 2001). The cytosolic thymidine (dThd) kinase (TK-1) recognizes only dThd and 2 -deoxyuridine (dUrd) as a substrate for phosphorylation. In contrast, TK-2 is located in the mitochondria and phosphorylates, besides dThd and dUrd, 2 deoxycytidine (dCyd) as a natural substrate. The cytosolic/ nuclear dCyd kinase converts dCyd, but also purine deoxynucleosides, such as 2 -deoxyguanosine (dGuo) and 2 deoxyadenosine (dAdo), to their 5 -monophosphate derivative. Finally, dGuo kinase (dGK) represents the second mitochondrial deoxynucleoside kinase phosphorylating dGuo This work was supported by a grant from the “Geconcerteerde Onderzoeksacties–Vlaanderen” (GOA) (krediet number 00/12), the “Belgische Federatie tegen Kanker”, the Spanish Comisión Interministerial de Ciencia y Tecnologica [SAF2000-0153-(02-01)], and the European Commission (QLG1-CT2001-01004). ABBREVIATIONS: dThd, 2 -deoxythymidine; TK, thymidine kinase; dUrd, 2 -deoxyuridine; dCyd, 2 -deoxycytidine; dGuo, 2 -deoxyguanosine; dAdo, 2 -deoxyadenosine kinase; dGK, 2 -deoxyguanosine kinase; dNK, 2 -deoxynucleoside kinase; Dm, Drosophila melanogaster; BVDU, (E)-5 -(2-bromovinyl)-2 -deoxyuridine; HSV, herpes simplex virus; BVU, (E)-5-(2-bromovinyl)uracil; GST, glutathione S-transferase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate; THF, tetrahydrofuran; KIN5, (E)-5-(2-bromovinyl)-1-(2 -deoxy-5 -O-triphenylmethyl)uridine; KIN6, 5 -O-triphenylmethyl-thymidine; KIN12, 1-[(Z)-4-(triphenylmethoxy)-2-butenyl]thymine; KIN39, (R,S)-1-[3-hydroxy-4-(triphenylmethoxy)butyl]thymine; KIN-52, (E)-5-(2-bromovinyl)-1-[(Z)-4-(triphenylmethoxy)-2-butenyl]uracil. 0026-895X/03/6302-263–270$7.00 MOLECULAR PHARMACOLOGY Vol. 63, No. 2 Copyright © 2003 The American Society for Pharmacology and Experimental Therapeutics 2032/1037375 Mol Pharmacol 63:263–270, 2003 Printed in U.S.A. 263 at A PE T Jornals on A uust 4, 2017 m oharm .aspeurnals.org D ow nladed from and dAdo. Both TK-2 and dGK are constitutively expressed, in contrast with TK-1, which is highly S-phase specific. However, in proliferating cells, intracellular TK-2 levels are much lower compared with TK-1, but in nonproliferating and resting cells, TK-2 is virtually the only pyrimidine deoxynucleoside kinase that is physiologically active (Wintersberger et al., 1992; Hengstschlager et al., 1994; Johansson and Karlsson, 1997). There is a high sequence homology between TK-2, dCK, and dGK, but also with herpes simplex virus type 1 (HSV-1) TK and the recently discovered multifunctional 2 -deoxynucleoside kinase (dNK) from Drosophila melanogaster (Dm-dNK) (Munch-Petersen et al., 1998; Johansson et al., 1999). The crystal structures of HSV-1 TK and Dm-dNK but not TK-2 have been resolved in complex with their natural substrate dThd and several alternative nucleoside substrates, such as (E)-5-(2-bromovinyl)-dUrd (BVDU), ganciclovir, and acyclovir (for HSV-1 TK) (Wild et al., 1997; Champness et al., 1998; Johansson et al., 2001). Whereas HSV-1 TK, like TK-2, recognizes the natural pyrimidine deoxynucleosides dThd, dUrd, and dCyd as a substrate for phosphorylation, Dm-dNK can convert all five natural purine/pyrimidine 2 -deoxynucleosides to their 5 -monophosphates (Munch-Petersen et al., 1998, 2000; Johansson et al., 1999). Only a few inhibitors of the deoxynucleoside kinases have been reported. A series of potent and selective inhibitors of HSV-1 TK consist of guanine, guanosine, or thymidine analogs that contain a lipophilic entity (i.e., halogeno/methoxysubstituted phenyl) at the 5 -position of the deoxyribose moiety (Hildebrand et al., 1990). These anti-HSV TK drugs did not recognize other TKs such as TK-1 or TK-2. Instead, 2 -alkylether or -ester derivatives of 1-D-ribofuranosylthymidine and 1-D-arabinofuranosyl-(E)-5-(2-bromovinyl)uracil were reported to be selective inhibitors of TK-2 but not to inhibit HSV-1 TK (Balzarini et al., 2001; Manfredini et al., 2001). Finally, several 5-substituted ribonucleosides [e.g., (E)-5-(2-bromovinyl)uridine] and 3 -substituted thymine riboside analogs were found to be inhibitory against TK-2 but not TK-1 (Balzarini et al., 2000). Also, Kierdaszuk et al. (1999) have reported on substrate/inhibitor properties of TK-2 toward O -alkyl sugar-modified nucleoside analogs. Their inhibitory potential against the enzyme is caused by competition of the inhibitor with thymidine as an alternative substrate, which is endowed [at least in case of (E)-5-(2bromovinyl)uridine and analogs] with a very low Vmax value. We now report on a novel class of acyclic thymine and (E)-5(2-bromovinyl)uracil (BVU) analogs that contain a bulky lipophilic (trityl) moiety linked to the pyrimidine base through an aliphatic butenyl or 2-hydroxybutyl moiety as potent inhibitors of TK-2. Interestingly, the BVU trityloxybutenyl derivative turned out to selectively inhibit TK-2 and was not measurably recognized by TK-1, HSV-1 TK, and Dm-dNK. Our enzyme kinetic studies revealed that this is the first example of a reversible non-nucleoside, nonsubstrate inhibitor of TK-2 that is competitive with respect to dThd and uncompetitive with respect to ATP. Experimental Procedures