TABLE 6.

Generation of 2′-C-methyl-GTP from DAPN-PD1 and BMS-986094 in Huh-7 cells

Prodrug	2′-C-Methyl-GTPa (pmol/106 cells)	2′-C-Methyl-GTPb (μM)	keff, POLRMTc (s−1)	Selectivityd
DAPN-PD1	7.8 ± 0.2	3.9	0.0007
BMS-986094	680 ± 40	340	0.04	57.0

^arNAI prodrug (50 μM) was incubated with Huh-7 cells for 4 h at 37°C. Intracellular 2′-C-methyl-GTP levels generated from each prodrug were determined using LC-MS/MS. Each value is the average from two separate replicates (± standard deviations).

^bAmount of nucleoside 5′-triphosphate metabolites was converted from picomoles per million cells to molarity using an estimated cellular volume of 2 pl per cell (33).

^cRate constant for incorporation was determined with the equation K_{eff, POLRMT} (per second) = (k_pol × [NTP])/(K_d,app + [NTP]). K_{eff, POLRMT} was calculated based on reported kinetic properties of 2′-C-methyl-GTP incorporation by POLRMT enzyme (33).

^dSelectivity was determined as (K_{eff, POLRMT})_{2′-C-methyl-GTP from DAPN-PD1}/(K_{eff, POLRMT}) _{2′-Cmethyl-GTP from BMS-986094}.