20-F-20-C-Methyl Nucleosides and Nucleotides for the Treatment of Hepatitis C Virus: from Discovery to the Clinic - Accounts in Drug Discovery Case Studies in Medicinal Chemistry - page 244

Figure 11.3 PSI-6130 is anabolized to its active triphosphate by the kinases dCK,
YMPK, and NDPK. The monophosphate of PSI-6130 is also metabo-
lized to the uridine monophosphate; the latter is further phosphorylated
to the uridine triphosphate of PSI-6206, which is also an inhibitor of
HCV polymerase. However, the uridine nucleoside, PSI-6206, is not a
substrate for cellular nucleoside kinases and therefore is a dead-end
metabolite of PSI-6130.
purified recombinant HCV NS5B RdRp. In the replicase assay, PSI-6130-TP
gave a mean IC 50 of 0.34 mM. 17 The inhibition constants (K i ) were determined
in two separate studies using purified recombinant RdRp. When the 3 0 -end of
the minus strand of the HCV genome was used as template, a K i value of
0.023 mM was determined for PSI-6130-TP, and a K i value of 0.059 mM was
obtained when the minus strand of the HCV internal ribosomal entry site was
used as template. 17,18 These results confirmed that PSI-6130 was a potent
inhibitor of the HCV RdRp, and that it was a good substrate for cellular
kinases that were important in metabolizing it to the active triphosphate form
(Figure 11.3).
Since metabolism studies also revealed the presence of the uridine congener
PSI-6206 (8) and its phosphorylated derivatives in both replicon cells and
primary human hepatocytes, these compounds were further studied to under-
stand their role in HCV inhibition. 17,19 Inhibition studies with PSI-6206-TP
using the replicase assay and purified recombinant RdRp surprisingly
demonstrated that PSI-6206-TP was also a potent inhibitor of HCV RNA
synthesis. 17,19 This was the first time that a uridine analog was shown to be an
inhibitor of HCV polymerase. The mean IC 50 value for PSI-6206-TP in the
replicase assay was 1.19 mM and the K i values determined in two separate
studies were 0.141 mM and 0.42 mM, respectively. 17,19 However, our earlier SAR
work showed that the nucleoside PSI-6206 was inactive in the replicon assay.
Enzyme studies indicated that the lack of activity in the replicon assay was due
to the inability of PSI-6206 to be phosphorylated by cellular nucleoside kinases
to PSI-6206-MP. 19 Instead, the formation of PSI-6206-TP was found to involve
the deamination of PSI-6130-MP to PSI-6206-MP by deoxycytidylate deami-
nase (k cat /K m ¼ 4.1 10 4 mM 1 s 1 ). 19 Phosphorylation to PSI-6206-DP and
PSI-6206-TP was catalyzed by UMP-CMP kinase (k cat /K m ¼ 0.0091 mM 1 s 1 )
and nucleoside diphosphate kinase (k cat /K m ¼ 0.046 mM 1 s 1 ), respectively