Metabolism of the Tumor Angiogenesis Inhibitor 4-(N-(S-Glutathionylacetyl)amino)phenylarsonous Acid

Abstract

4-(N-(S-glutathionylacetyl)amino) phenylarsonous acid (GSAO) is a small, synthetic mitochondrial poison that targets angiogenic endothelial cells and is currently being tested in aPhase I/IIa clinical trial. The trivalent arsenical of GSAO reacts with and perturbs adenine nucleotide translocase of the inner mitochondrial membrane of endothelial cells, which leads to proliferation arrest. Three observations indicated that the ␥-glutamyl residue of GSAO is cleaved at the endothelial cell surface by ␥-glutamyl transpeptidase (␥GT). GSAO was found to be an efficient substrate for ␥GT, endothelial cell accumulation and antiproliferative activity of GSAO was blunted by a competitive substrate and an active site inhibitor of ␥GT, and the level of cell surface ␥GT correlated strongly with the sensitivity of cells to GSAO. Using transport inhibitors, it was revealed that the resulting metabolite of GSAO cleavage by ␥GT, 4-(N-(S-cysteinylglycylacetyl)amino) phenylarsonous acid (GCAO), was transported across the plasma membrane by an organic anion transporter. Furthermore, GCAO is likely processed by dipeptidases in the cytosol to 4-(N-(S-cysteinylacetyl)amino) phenylarsonous acid (CAO), and it is this metabolite that reacts with mitochondrial adenine nucleotide translocase. Taken together, our findings indicate that ␥GT processing of GSAO at the cell surface is the rate-limiting step in its antiangiogenic activity. This information can explain the kidney toxicity at high doses of GSAO noted in preclinical studies and will aid in the anticipation of potential side effects in humans and in the design of better antimitochondrial cancer drugs. GSAO2 is a mitochondrial poison that selectively perturbs angiogenic endothelial cells in vitro and in vivo (1-3). The tripeptide trivalent arsenical inactivates the mitochondrial inner membrane transporter, adenine nucleotide translocase (ANT), by cross-linking two of the three matrix facing cysteine thiols (1, 4). Proper functioning of ANT is essential for cell viability, so targeting this protein in angiogenic endothelial cells is a powerful means of blocking angiogenesis (5). A limitation of targeting specific angiogenic proteins is that they can often be circumvented by other proteins in the angiogenic process. GSAO is currently being tested in a Phase I/IIa clinical trial in cancer patients.ANT exchanges matrix ATP for intermembrane space ADP across the inner mitochondrial membrane and is a key component of the mitochondrial permeability transition pore (6, 7). Inactivation of ANT by GSAO causes an increase in superoxide levels, proliferation arrest, ATP depletion, mitochondrial depolarization, and apoptosis in endothelial cells. The strong selectivity of GSAO for proliferating endothelial cells is a consequence of the higher mitochondrial calcium levels in proliferating cells (1). ANT is a calcium receptor that undergoes a conformational change and a change in activity upon binding of calcium ions. GSAO binds to calcium-replete ANT but binds minimally in the absence of calcium io...

The Novel Arsenical Darinaparsin Is Transported by Cystine Importing Systems

“…However, we do not exclude the possibility that a certain amount of DMAC could also arise subsequently from the dimethylarsenic being exchanged from Dar to a free cysteine (Bohle and Gu, 2013). Interestingly, another organic arsenical, GSAO [4-(N-(S-glutathionylacetyl)amino) phenylarsonous acid], also a GSH S-conjugate, is cleaved by g-GT to produce GCAO [4-(N-(S-cysteinylglycylacetyl)amino) phenylarsonous acid]) (Dilda et al, 2008). GSH is cleaved by both g-GT and a dipeptidase extracellularly and is involved in the processing of GSAO and RKO 2577 cells, and xCT levels were assessed.…”

“…Link between Darinaparsin and Cystine Import (Dilda et al, 2008). Thus, an additional step involving a dipeptidase is likely required to ultimately produce DMAC.…”

“…This assay was adapted from a previously described method (Dilda et al, 2008). Briefly, NB4 cells were incubated with or without acivicin, a g-GT inhibitor (Sigma-Aldrich) for 1 hour, then incubated for 3 hours or 24 hours in PBS containing 5 mM L-glutamic acid g-(p-nitroanilide) hydrochloride, a g-GT-specific substrate that leads to proportional color development (yellow) upon formation of p-nitroanilide.…”

“…To increase cysteine uptake, extracellular GSH can be cleaved by g-glutamyltranspeptidase (g-GT), then cleaved by a cysteinylglycine dipeptidase, both at the cell surface. Only then is cysteine/ cystine imported (Dilda et al, 2008). This is often referred to as the scavenger pathway (Zhang and Forman, 2012).…”

Necrotic platelets provide a procoagulant surface during thrombosis

“…The organic anion-transporting polypeptide (OATP) family has been implicated in transport across the plasma membrane of organic anions in the same class as GSAO. 21,22 Nine members of the OATP family have been reported in humans, 23 and platelets express OATP2B1. 24 DIDS is an inhibitor of OATP class B transporters, 25 so we tested its effect on GSAO labeling of agoniststimulated platelets.…”