A GST Pi-Activated Prodrug for Release of Nitric Oxide

We have used structure-based design techniques to introduce a drug named PABA/NO (O²-[2,4-dinitro-5-(N-methyl-N-4-carboxyphenylamino) phenyl] 1-N,N-dimethylamino)diazen-1-ium-1,2-diolate) that is efficiently metabolized to potentially cytolytic nitric oxide by the GSTπ, an enzyme expressed at high levels in many tumors. Mouse embryo fibroblasts (MEFs) null for GSTπ (GSTπ^-/-) show that the absence of GSTπ results in a decreased sensitivity to PABA/NO. Cytotoxicity of PABA/NO was also examined in NIH3T3 cells that were stably transfected with GSTπ and/or various combinations of γ-glutamyl cysteine synthetase (γGCS) and the ABC transporter MRP1. Overexpression of MRP1 conferred the most significant degree of resistance and in vitro transport studies confirmed that a GSTπ-activated metabolite of PABA/NO was effluxed by MRP1 in a GSH-dependent manner. Additional studies showed that in the absence of MRP1, PABA/NO activated the extracellular-regulated and stress-activated protein kinases, ERK, JNK and p38. Selective inhibitors showed that the activation of JNK and p38 were critical to the cytotoxicity of PABA/NO. In addition, PABA/NO produced antitumor effects in a human ovarian cancer model grown in SCID mice.

Glutathionylation is emerging as an important post-translational modification that influences the function and stability of a variety of proteins. Glutathionylation occurs through disulfide bond formation between glutathione (GSH) and available cysteine residues. In response to oxidative and nitrative stress, such modifications can result alter the characteristics of a spectrum of proteins. PABA-NO is novel prodrug that liberates nitric oxide (NO) following GST activation. Using two dimensional gel electrophoresis with monoclonal antibodies directed against glutathionylated cysteine residues, PABA-NO produced a time and dose dependent glutathionylation of > 50 proteins within 5 minutes of treatment. In NIH3T3 cells overexpressing MRP1, glutathionylation was significantly reduced. Similarly, in HL60 cells resistant to adriamycin (HL60-ADR), overexpression of MRP1 also reduced total protein glutathionylation in a manner consistent with collateral resistance to PABA-NO. Once NO is released, it can combine with GSH to form GSNO, a potent stimulator of protein glutathionylation and a plausible substrate for MRP1, explaining the reduced effects in MRP1 overexpressing cells. PABA-NO induced glutathionylation of JNK in vitro and this modification may be involved in drug-induced apoptosis.