In primary human hepatocytes, separate 50 µM exposures to PSI-7977, PSI-7976 or their mixture PSI-7851 produced similar intracellular triphosphate levels over 24 h. This finding supports cellular activation while limiting generalization of stereoisomer differences across models. [1]
Stereoselective hydrolysis of PSI-7977
Biochemical assay · Recombinant human cathepsin A (CatA) and carboxylesterase 1 (CES1); PSI-7976 and PSI-7851 comparators. [1]
CatA favored PSI-7977. CES1 converted some PSI-7977 but product formation plateaued, preventing steady-state parameter determination.
Literature-Reported Treatment Conditions
- Treatment concentration
- Varied substrate concentrations for steady-state kinetics; 100 µM in the reported CES1 product-time-course discussion.
- Exposure duration
- Time course shown to 150 min in Figure 4A,B.
- Method
- Recombinant enzyme assays at 37 °C; PSI-7976 and the PSI-7851 mixture provide stereochemical comparators.
- Reported limits
- Exact substrate concentration series, kinetic replicate count and definition of Table 5 ± are not specified in the selected methods/table.
Methods and source locations
Compound reported in the paper: PSI-7977; Compound label in the paper: PSI-7977
Activation enzyme measurements; not polymerase inhibition potency or lot-specific testing.
Materials; enzyme methods; Results Stereoselectivity of CatA and CES1; Figure 4A,B; Table 5
PSI-7977 hydrolysis by CatA: Radiolabeled substrate kinetic assay; meaning of ± and replicate count not specified in Table 5. — Table 5; Experimental Procedures, steady-state kinetics
PSI-7977 hydrolysis by CatA: Radiolabeled substrate kinetic assay; meaning of ± and replicate count not specified in Table 5. — Table 5
PSI-7977 hydrolysis by CatA: Reported table value, not recalculated. — Table 5
PSI-7977 steady-state kinetics with CES1: Product time course plateaued; do not interpret as absence of hydrolysis. — Figure 4B; Table 5 footnote a; Results, Stereoselectivity of CatA and CES1
Triphosphate formation in primary human hepatocytes
Cell-based study · Primary human hepatocytes (HHPC), with PSI-7976 and PSI-7851 comparators. [1]
At matched exposure, all three materials produced similar intracellular PSI-7409 concentrations. This metabolic endpoint does not establish equivalent clinical efficacy.
Literature-Reported Treatment Conditions
- Treatment concentration
- 50 µM of each material, tested separately.
- Exposure duration
- Up to 24 h.
- Method
- Cellular metabolism followed by HPLC analysis of intracellular metabolites.
- Reported limits
- Vehicle, number of HHPC donors and replicate/error semantics for Figure 4D are not specified in this experiment. Three donors in Figure 3 must not be assigned to Figure 4.
Methods and source locations
Compound reported in the paper: PSI-7977; Compound label in the paper: PSI-7977
Intracellular activation in primary human hepatocytes only.
Materials; Cellular Metabolism Study; HPLC; Figure 4D; Results and Discussion
Intracellular PSI-7409 formation: Cellular metabolism assay and HPLC; no exact concentration inferred from curves. — Figure 4D and caption; Results, Stereoselectivity of CatA and CES1
HCV polymerase inhibition by the active triphosphate
Biochemical assay · Purified recombinant HCV NS5BΔ21 polymerases: genotype 1b Con1, 2a JFH-1, 3a and 4a. [2]
Separately synthesized PSI-7409 inhibited all four tested polymerases. These are metabolite IC50 values, not sofosbuvir treatment concentrations.
Literature-Reported Treatment Conditions
- Treatment concentration
- Increasing concentrations of PSI-7409; assay contained 1 µM of each natural ribonucleotide.
- Exposure duration
- 30 min for GT 1b and 2a; 3 h for GT 3a and 4a.
- Method
- Cell-free RNA synthesis at 27 °C with purified NS5BΔ21; activity dose-response referenced to uninhibited activity.
- Reported limits
- Vehicle not specified for this cell-free assay. Different reaction times limit direct cross-genotype ranking.
Methods and source locations
Compound reported in the paper: PSI-7409 (active triphosphate metabolite); Compound label in the paper: PSI-7409 (active triphosphate metabolite)
Supports the polymerase-inhibition mechanism of the activated compound; cannot assign these IC50 values to the catalog prodrug.
Chemicals; Figure 1; cell-free assay methods; Figure 2; related Results
RNA polymerase activity: GT 1b, 2a, 3a and 4a, respectively: Radiolabeled nucleotide incorporation; Figure 2 reports SD from at least two independent experiments performed in duplicate. — Figure 2 graph, embedded table and caption; Results, biochemical inhibition paragraph
