Evaluation of the role of three candidate human kinases in the conversion of the hepatitis C virus inhibitor 2'-C-methyl-cytidine to its 5'-monophosphate metabolite.
Nucleoside analogs are effective inhibitors of the hepatitis C virus (HCV) in the clinical setting. One such molecule, 2'-C-methyl-cytidine (2'-MeC), entered clinical development as NM283, a valine ester prodrug form of 2'-MeC possessing improved oral bioavailability. To be active against HCV, 2'-MeC must be converted to 2'-MeC triphosphate which inhibits NS5B, the HCV RNA-dependent RNA polymerase. Conversion of 2'-MeC to 2'-MeC monophosphate is the first step in 2'-MeC triphosphate production and is thought to be the rate-limiting step. Here we investigate which of three possible enzymes, deoxycytidine kinase (dCK), uridine-cytidine kinase 1 (UCK1), or uridine-cytidine kinase 2 (UCK2), mediate this first phosphorylation step. Purified recombinant enzymes UCK2 and dCK, but not UCK1, could phosphorylate 2'-MeC in vitro. However, siRNA knockdown experiments in three human cell lines (HeLa, Huh7 and HepG2) defined UCK2 and not dCK as the key kinase for the formation of 2'-MeC monophosphate in cultured human cells. These results underscore the importance of confirming enzymatic kinase data with appropriate cell-based assays. Finally, we present data suggesting that inefficient phosphorylation by UCK2 likely limits the antiviral activity of 2'-MeC against HCV. This paves the way for the use of a nucleotide prodrug approach to overcome this limitation.
Golitsina
Nina L
NL
Idenix Pharmaceuticals, Inc., 60 Hampshire Street, Cambridge, MA 02139, USA. golistina.nina@idenix.com <golistina.nina@idenix.com>
Danehy
Francis T
FT
Fellows
Ross
R
Cretton-Scott
Erika
E
Standring
David N
DN
D000998:Antiviral Agents / Q000378:metabolism*; D001711:Biotransformation; D002460:Cell Line; D003562:Cytidine / Q000031:analogs & derivatives* / Q000378:metabolism; D003568:Cytidine Monophosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D003842:Deoxycytidine Kinase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020868:Gene Silencing; D016174:Hepacivirus / Q000187:drug effects; D006801:Humans; D011355:Prodrugs / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D014541:Uridine Kinase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D000998:Antiviral Agents; D011355:Prodrugs; D034741:RNA, Small Interfering; C513402:2'-C-methylcytidine; D003562:Cytidine; C483834:UCK2 protein, human; D014541:Uridine Kinase; D003842:Deoxycytidine Kinase; D003568:Cytidine Monophosphate