Abstract

The peptidomimetic drug γ-glutamyl-S-(benzyl)cysteinyl-R-(−)-phenyl glycine diethyl ester (TER199) is an analog of glutathione designed to be an isozyme-specific inhibitor of GSTP1-1 protein1–1. This compound (and the de-esterified moiety) is shown to be an effective inhibitor of multidrug resistance-associated protein1 (MRP1)-mediated drug resistance. Kinetic analyses revealed that γ-glutamyl-S-(benzyl)cysteinyl-R-(−)-phenyl glycine reversibly inhibits the transport of 2,4-dinitrophenyl-S-glutathione with aK_i of 752 μM. TER199 reversed the accumulation deficit of daunorubicin in MRP1-transfected NIH3T3 fibroblasts and maintained intracellular levels for >2 h after daunorubicin removal. Cytotoxicity assays revealed that TER199 significantly reversed the resistance of MRP1-transfected NIH3T3 cells for vincristine, doxorubicin, etoposide, and mitoxantrone. HL-60 cells made resistant to TER199 by chronic, long-term selection had increased mRNA and protein levels of multidrug resistance-associated protein, MRP1, and γ-glutamyl cysteine synthetase heavy and light subunits (the rate-limiting enzyme in GSH synthesis). In spite of increased γ-glutamyl cysteine synthetase, their glutathione content was reduced ∼35% from that of parental HL-60 cells. These cells also exhibited a drug resistance profile commensurate with the previously described MRP1 overexpressing phenotype, with resistance to Vinca alkaloids, epipodophyllotoxins, and anthracyclines; additional cross-resistance to paclitaxel (Taxol), mitoxantrone, and 5-fluorouracil was observed.