TABLE 3.

Intracellular metabolism and NS5B-catalyzed incorporation rates for nucleoside triphosphate analogs

NTP substrate	Intracellular NTP (pmol/106 cells)	Intracellular NTP (μM)	keff, NS5Bc (s−1)	FDd
ATP	8,700.0a	4,400.0	1.3
2′-C-Methyl-DAPN-TP	96.0b	48.0	0.03	43
GTP	1,600.0a	790.0	4.1
2′-C-Methyl-GTP	47.0b	24.0	0.5	8.0

^aIntracellular ATP and GTP concentrations were determined in Huh-7 cell lines as described in Materials and Methods. Primary human hepatocytes were estimated to contain amounts of natural NTPs comparable to those of Huh-7 cells. Nucleotide levels measured by LC-MS/MS were converted to micromolar concentrations, estimating a cell volume of 2 pl/cell (33).

^bIntracellular 2′-C-methyl-DAPN-TP and 2′-C-methyl-GTP levels measured in primary human hepatocytes after treatment with 50 μM DAPN-PD1 for 24 h.

^cRate constant for incorporation was determined with the equation K_{eff, NS5B} (per second) = (k_pol × [NTP])/(K_d,app + [NTP]). k_pol and K_d,app values were determined experimentally as reported in Table 2.

^dFold difference (FD) was calculated by dividing the K_{eff, NS5B} of natural NTP substrate by that of the corresponding rNAI-TP.