Substrate/inhibitor properties of human deoxycytidine kinase (dCK) and thymidine kinases (TK1 and TK2) towards the sugar moiety of nucleosides, including O '-alkyl analogues

Nucleoside analogues with modified sugar moieties have been examined for th eir substrate/inhibitor specificities towards highly purified deoxycytidine kinase (dCK) and thymidine kinases (tetrameric high-affinity form of TK1, and TK2) from human leukemic spleen. In particular, the analogues included the mono- and di-O'-methyl derivatives of dC, dU and dA, syntheses of which are described. In general, purine nucleosides with modified sugar rings we re feebler substrates than the corresponding cytosine analogues. Sugar-modi fied analogues of dU were also relatively poor substrates of TK1 and TK2, b ut were reasonably good inhibitors, with generally lower Ki values vs TK2 t han TK1. An excellent discriminator between TK1 and TK2 was 3'-hexanoylamin o-2',3'-dideoxythymidine, with a Ki of similar to 600 mu M for TK1 and simi lar to 0.1 mu M for TK2. 3'-OMe-dC was a superior inhibitor of dCK to its 5 '-O-methyl congener, consistent with possible participation of the oxygen o f the (3')-OH or (3')OMe as proton acceptor in hydrogen bonding with the en zyme. Surprisingly alpha-dT was a good substrate of both TK1 and TK2, with Ki values of 120 and 30 CIM for TK1 and TK2, respectively; and a 3'-branche d alpha-L-deoxycytidine analogue proved to be as good a substrate as its al pha-D- counterpart. Several 5'-substituted analogues proved to be were good non-substrate inhibitors of dCK and, to a lesser extent, of TK2. Finally, some ribonucleosides are substrates of the foregoing enzymes; in particular C is a good substrate of dCK, and 2'-OMe-C is an even better substrate tha n dC.