GS-9191, a bis-amidate
prodrug of the
nucleotide analog 9-(2-phosphonylmethoxyethyl)-N6-cyclopropyl-2,6-diaminopurine (cPrPMEDAP), was designed as a topical agent for the treatment of papillomavirus-associated proliferative disorders, such as
genital warts. In this study, we investigated the mechanism of conversion of
GS-9191 to cPrPMEDAP. We observed that
GS-9191 is hydrolyzed in the presence of the lysosomal
carboxypeptidase cathepsin A (CatA) in vitro and is less efficiently metabolized in CatA-deficient fibroblasts than in control cells. In addition, knockdown of CatA by
small interfering RNA (
siRNA) reduced the intracellular accumulation of
GS-9191 metabolites. However, intracellular CatA levels did not correlate with the susceptibility of tested cell lines to
GS-9191, indicating that the CatA step is unlikely to be rate limiting for the activation of
GS-9191. Further analysis showed that upon the hydrolysis of the carboxylester bond in one of the
GS-9191 amidate moieties, the unmasked carboxyl group displaces
L-phenylalanine 2-methylpropyl
ester from the other amidate moiety. The cPrPMEDAP-
L-phenylalanine conjugate (cPrPMEDAP-Phe) formed is not metabolized by Hint1 (
histidine triad
nucleotide binding protein 1) phosphoramidase but undergoes spontaneous degradation to cPrPMEDAP in acidic pH that can be significantly enhanced by the addition of SiHa
cell extract. Pretreatment of SiHa cells with
bafilomycin A or
chloroquine resulted in an 8-fold increase in the intracellular concentration of cPrPMEDAP-Phe metabolite and the accumulation of
GS-9191 metabolites in the lysosomal/endosomal fraction. Together, these observations indicate that the conversion of
GS-9191 to cPrPMEDAP occurs in lysosomes via CatA-mediated
ester cleavage, followed by the release of cPrPMEDAP, most likely through the combination of
enzyme-driven and spontaneous pH-driven hydrolysis of a cPrPMEDAP-Phe intermediate.