Intracellular metabolism of the nucleotide prodrug GS-9131, a potent anti-human immunodeficiency virus agent

Abstract

GS-9131 is a phosphonoamidate prodrug of the novel ribose-modified phosphonate nucleotide analog GS-9148 that demonstrates potent anti-human immunodeficiency virus type 1 (HIV-1) activity and an excellent resistance profile in vitro. Prodrug moieties were optimized for the efficient delivery of GS-9148 and its active diphosphate (DP) metabolite to lymphoid cells following oral administration. To understand the intracellular pharmacology of GS-9131, incubations were performed with various types of lymphoid cells in vitro. The intracellular accumulation and antiviral activity levels of GS-9148 were limited by its lack of cellular permeation, and GS-9131 increased the delivery of GS-9148-DP by 76- to 290-fold relative to that of GS-9148. GS-9131 activation was saturable at high extracellular concentrations, potentially due to a high-affinity promoiety cleavage step. Once inside the cells, GS-9148 was efficiently phosphorylated, forming similar amounts of anabolites in primary lymphoid cells. The levels of GS-9148-DP formed in peripheral blood mononuclear cells infected with HIV-1 were similar to that in uninfected PBMCs, and approximately equivalent intracellular concentrations of GS-9148-DP and tenofovir (TVF)-DP were required to inhibit viral replication by 90%. Once it was formed, GS-9148-DP was efficiently retained in activated CD4(+) cells, with a half-life of 19 h. In addition, GS-9131 showed a low potential for drug interactions with other adenine nucleoside/nucleotide reverse transcriptase inhibitors, based on the lack of competition for anabolism between suprapharmacologic concentrations of GS-9148 and TVF and the lack of activity of GS-9131 metabolites against purine nucleoside phosphorylase, an enzyme involved in the clearance of 2',3'-dideoxyinosine. Together, these observations elucidate the cellular pharmacology of GS-9131 and illustrate its efficient loading of lymphoid cells, resulting in a prolonged intracellular exposure to the active metabolite GS-9148-DP.

FIG. 1.

Structures of the ribose-modified adenosine nucleotide analog GS-9148 (Fd4AP) and its ethanolalaninyl monoamidate, monophenol prodrug GS-9131.

FIG. 2.

Enhanced intracellular levels of GS-9148-DP following the incubation of lymphoid cells with GS-9131 (open bars) relative to that of GS-9148 (filled bars). Intracellular GS-9148-DP levels are normalized based on extracellular concentration following incubation with 1 μM GS-9131 or with 100 μM GS-9148. Values represent the means ± standard deviations of three independent incubations performed in duplicate.

FIG. 3.

Intracellular metabolism of GS-9131 following incubation of CEM-CCRF cells with 1 μM GS-9131. (A) Detection of intracellular metabolites following a 24-h incubation by LC-MS-MS. GS-9148, GS-9148-MP, and GS-9148-DP were monitored in multiple-reaction-monitoring mode following the molecular ion transitions of 331.9 to 220.1, 412.1 to 220.1, and 492.0 to 220.1 m/z, respectively. Calculation of levels by comparing peak areas to authentic standard curves led to the determination of 7.15, 3.10, and 5.56 pmol/million cells of GS-9148, GS-9148-MP, and GS-9148-DP, respectively. (B) Intracellular accumulation of GS-9148 and its phosphorylated metabolites. Values represent the means ± standard deviations of three independent experiments done in duplicate.

FIG. 4.

Concentration dependence of GS-9131 metabolism following the incubation of activated PBMCs with 0.1, 1, and 10 μM extracellular GS-9131 for 24 h. Data were fitted to hyperbolic equations showing that GS-9148, GS-9148-MP, and GS-9148-DP had an apparent saturation constant (the concentration required to cause 50% saturation) of approximately 4 μM. Values represent the means ± standard deviations of two independent experiments performed in duplicate in activated PBMCs pooled from three donors.

FIG. 5.

Egress of GS-9148-DP following the incubation of activated CD4⁺ lymphocytes with 0.3 μM GS-9131 for 24 h. Following the removal of GS-9131 from culture medium, GS-9148-DP persisted in activated CD4⁺ lymphocytes, with a half-life of 19 h. Results represent the means ± standard deviations of two independent experiments done in duplicate, with cells pooled from four donors. Data were fitted to a single exponential decay curve.

FIG. 6.

Lack of an intracellular metabolic interaction between TDF and GS-9131 in activated PBMCs. (A) GS-9131 (1 μM) either alone (closed symbols and solid lines) or in combination with 1 μM TDF (open symbols and dotted lines) was metabolized to GS-9148, GS-9148-MP, and GS-9148-DP to similar extents. (B) TDF (1 μM) either alone (closed symbols and solid lines) or in combination with 1 μM GS-9131 (open symbols and dotted lines) was metabolized to TFV, TFV-MP, and TFV-DP to similar extents. Values represent the averages of duplicate samples from a representative experiment performed in activated PBMCs from a single donor.

FIG. 7.

Putative mechanism of intercellular activation of GS-9131 and GS-9148. Arrows with solid lines represent efficient metabolic steps, while arrows with dashed lines represent more inefficient metabolic processes. First, the lipophilic prodrug GS-9131 freely permeates the cell [1] and is cleaved by lysosomal protease cathepsin A and possibly by other cellular hydrolases [2]. Phenol is lost by spontaneous nucleophilic release [3], and l-alanine is then released through hydrolysis of the P-N bond yielding GS-9148, either by a previously described phosphoamidase (29) or spontaneously due to the low pH present in the lysosomal compartment [4]. The poor cell membrane permeation of the nucleotide analog GS-9148 [5] is then phosphorylated by the rate-limiting first phosphorylation step, likely catalyzed by an adenylate kinase [6], followed by the more efficient second phosphorylation step, putatively catalyzed by creatine kinase or other kinases [7], generating the active metabolite GS-9148-DP.