Metabolic Activation of the Anti-Hepatitis C Virus Nucleotide Prodrug PSI-352938

Abstract

c PSI-352938 is a novel cyclic phosphate prodrug of ␤-D-2=-deoxy-2=-␣-fluoro-2=-␤-C-methylguanosine-5=-monophosphate with potent anti-HCV activity. In order to inhibit the NS5B RNA-dependent RNA polymerase, PSI-352938 must be metabolized to the active triphosphate form, PSI-352666. During in vitro incubations with PSI-352938, significantly larger amounts of PSI-352666 were formed in primary hepatocytes than in clone A hepatitis C virus (HCV) replicon cells. Metabolism and biochemical assays were performed to define the molecular mechanism of PSI-352938 activation. The first step, removal of the isopropyl group on the 3=,5=-cyclic phosphate moiety, was found to be cytochrome P450 (CYP) 3A4 dependent, with other CYP isoforms unable to catalyze the reaction. The second step, opening of the cyclic phosphate ring, was catalyzed by phosphodiesterases (PDEs) 2A1, 5A, 9A, and 11A4, all known to be expressed in the liver. The role of these enzymes in the activation of PSI-352938 was confirmed in primary human hepatocytes, where prodrug activation was reduced by inhibitors of CYP3A4 and PDEs. The third step, removal of the O 6 -ethyl group on the nucleobase, was shown to be catalyzed by adenosine deaminase-like protein 1. The resulting monophosphate was consecutively phosphorylated to the diphosphate and to the triphosphate PSI-352666 by guanylate kinase 1 and nucleoside diphosphate kinase, respectively. In addition, formation of nucleoside metabolites was observed in primary hepatocytes, and ecto-5=-nucleotidase was able to dephosphorylate the monophosphate metabolites. Since CYP3A4 is highly expressed in the liver, the CYP3A4-dependent metabolism of PSI-352938 makes it an effective liver-targeted prodrug, in part accounting for the potent antiviral activity observed clinically.

Activation of Peripheral Blood Mononuclear Cells by Dengue Virus Infection Depotentiates Balapiravir

“…Nucleoside analogs are prone to cell type-specific activity (18,19). This is reflected by the EC 50 variation when R1479 was tested in PBMCs and various immortalized cell lines; the EC 50 s were estimated to be 0.249, 3.57, 0.626, 14.25, and 22.85 M in DENVinfected PBMCs, THP-1, KU812, A549, and Huh-7 cells, respectively (Table 1 and data not shown).…”

Sofosbuvir: The Discovery of a Curative Therapy for the Treatment of Hepatitis C Virus

“…Each of these enzymes was a liver-associated enzyme, thus supporting the liver targeting theory for this prodrug. The prodrug cleavage intermediate di-acid PSI-352707 was also isolated and characterised as the first confirmed example of this proposed phosphoramidate cleavage intermediate thus solidifying the prodrug degradation mechanism [35,36].…”

“…In support of future clinical development efforts, the metabolism of PSI-7851 was extensively profiled (Figure 8.7) [35,36]. These studies included both in vitro and in vivo radiolabelled whole-cell studies and in vivo metabolite profiling.…”

Nucleosides and Nucleotides for the Treatment of Viral Diseases

“…A significant effort was focused on purine nucleotides because of early work that showed them to be exceptionally potent in the whole cell replicon assay. 51 PSI-352938 demonstrated modest replicon activity (EC 90 ¼ 1.37 μM) but was shown to be active against the NS5B S282T mutation known to confer resistance to 2 0 -F-2 0 -C-methyl pyrimidine nucleosides; three other mutations (S15G, C223H, and V321I) were required to confer a high level of resistance. PSI-352938 (44) was the first in a class of cyclic phosphate prodrugs and also employed a double prodrug strategy.…”

“…To investigate the potential for C-nucleosides as inhibitors of HCV replication, several reports have investigated both purine and pyrimidine C-nucleoside derivatives (49)(50)(51)(52)(53)(54)(55). To investigate the potential for C-nucleosides as inhibitors of HCV replication, several reports have investigated both purine and pyrimidine C-nucleoside derivatives (49)(50)(51)(52)(53)(54)(55).…”