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CHARACTERIZATION OF beta-D-2’-C-METHYL-2’-FLUOROCYTIDINE (PSI-6130), A NUCLEOSIDE INHIBITOR OF THE HCV RNA-DEPENDENT RNA POLYMERASE
M Powdrill, M GötteMcGill University, Montreal, Quebec
Aims: The HCV RNA-dependent RNA polymerase (NS5B) is essential for viral replication. Nucleoside analog inhibitors of this polymerase represent an important class of antiviral compounds. Such compounds that have entered clinical trials include the prodrugs of b-D-2'-deoxy-2'-C-methylcytidine (NM-107) and PSI-6130. PSI-6130 is currently in Phase 2 clinical trials. Although structurally highly related, PSI-6130 appears to be more potent, especially against virus containing the S282T mutation. Here, we attempt to determine possible underlying mechanisms for the improved potency of PSI-6130.
Methods: We considered three distinct parameters that can influence the inhibitory effects of nucleoside analogue inhibitors of HCV NS5B: rates of incorporation of the nucleoside-5'-monophosphate, rates of pyrophosphorolytic excision, and efficiency of binding of the next nucleotide. The 2'-C-methyl modification of nucleoside analogs can interfere with nucleotide binding, and cause "chain-termination".
Results: Assays with purified HCV NS5B demonstrated that PSI-6130-MP was more efficiently incorporated with IC50 values approximately 5-fold lower than with NM-107-MP. At the level of pyrophosphorolysis, PSI6130-MP was excised to a lesser extent than NM-107-MP, although combined excision/rescue reactions showed similar amounts of full-length product. These data suggest efficient re-incorporation of the PSI-6130-MP. Most importantly, excision was not inhibited by addition of the next nucleotide at high concentrations, suggesting that the nucleotide cannot bind and form a dead-end complex.
Conclusions: Differences in potency between the two 2'-C-methylated cytidine analogs correlate with different levels of incorporation. PSI-6130 retained the ability of other 2'-methylated compounds to diminish or even to prevent binding of the next nucleotide.