NHC-triphosphate triammonium is an active phosphorylated intracellular metabolite of β-d-N4-Hydroxycytidine (NHC) as a triphosphate form[1]. NHC-triphosphate triammonium is a weak alternative substrate for the viral polymerase and can be incorporated into HCV replicon RNA[1][2].
In an intracellular metabolism assay, HCV replicon cells are treated with 10 μM 3H-labeled NHC, and intracellular nucleotide levels are determined after 1, 2 and 8 hours incubations. NHC is rapidly convered into the mono-, di-, and triphosphate forms, and NHC-TP reaches up to 71.12 pM after 8 hours[1].NHC-triphosphate triammonium (NHC-TP) (5-40 μM) absence leads to full-length polymerization products, it can be a weak alternative substrate. In addition, incorporation of NHC-TP instead of CTP increases the molecular weight of the polymerization product by 16 (one extra oxygen) for each event and an obvious electrophoretic shift is observed in cell-free HCV NS5B polymerization reactions[1].Huh-7 cells are incubated with (10-50 μM; 4 h) NHC or a McGuigan phosphoramidate prodrug of NHC. Intracellular levels of the parental compounds and phosphorylated metabolites are measured using LC-MS/MS. Small amounts of NHC-monophosphate (MP) and NHC-diphosphate (DP) can be observed, while NHC-triphosphate triammonium remains the most abundant metabolite[2].NHC-triphosphate triammonium (NHC-TP) metabolite may directly target the viral polymerase and behave as a nonobligate chain terminator. It plays a prominent role in inhibiting early negative-strand RNA synthesis, either through chain termination or mutagenesis, which may in turn interfere with correct replicase complex formation.
[1]. Stuyver LJ,et al. Ribonucleoside analogue that blocks replication of bovine viral diarrhea and hepatitis C viruses in culture.Antimicrob Agents Chemother. 2003 Jan;47(1):244-54. [2]. Maryam Ehteshami, et al. Characterization of β-d- N4-Hydroxycytidine as a Novel Inhibitor of Chikungunya Virus.

In an intracellular metabolism assay conducted on HCV replicon cells exposed to 10 μM 3H-labeled NHC, the transformation of NHC into its mono-, di-, and triphosphate forms is documented, exhibiting a significant increase in the NHC-triphosphate (NHC-TP) concentration up to 71.12 pM after 8 hours. The absence of NHC-TP at concentrations ranging from 5-40 μM results in complete polymerization, serving as a modest alternate substrate. Moreover, substituting NHC-TP for CTP during polymerization increases the weight of the resultant product by 16 units (attributable to an additional oxygen molecule), causing a noticeable electrophoretic shift in cell-free HCV NS5B polymerization reactions. In experiments utilizing Huh-7 cells treated with 10-50 μM NHC or a McGuigan phosphoramidate prodrug of NHC for 4 hours, analysis through LC-MS/MS reveals minor levels of NHC-mono- and diphosphates, with NHC-TP remaining the predominant metabolite. The NHC-TP metabolite is hypothesized to directly interact with the viral polymerase, acting as a potential nonobligate chain terminator and playing a crucial role in inhibiting the synthesis of early negative-strand RNA. This inhibition could lead to the disruption of proper replicase complex formation, either through chain termination or mutagenesis.