Abstract 5623: Extracellular thiols and system xc− play a significant role in non-targeted in vitro activity of a folate-tubulysin B conjugate.
Authors: Parker Nikki , Pugh Michael R. , Xu Le-Cun , Vlahov Iontcho R. , Leamon Christopher P. 
Published: August 2014

Abstract
Several folate-targeted small-molecule drug conjugates (SMDCs) are under development for the treatment of folate receptor-positive (FR+) cancer. To date, all clinical SMDCs have been constructed with a disulfide-based linker strategically placed in between the targeting ligand (e.g. folate) and the therapeutically active drug. At least for SMDCs, the disulfide linker remains relatively stable in circulation, but it rapidly breaks once the SMDC enters the reductive endocytic process within the targeted cell. While this structural design has proven to be successful for folate-based SMDCs constructed with mitomycins, maytansinoids, vinca alkaloids, and epothilones, the development of a more potent folate-tubulysin B conjugate (herein called EC0531) has revealed a non-FR-specific component related to in vitro cytotoxic activity, particularly at drug concentrations exceeding that required to saturate the FR. Rationalizing that the potential instability of the disulfide linker may contribute to this effect, we explored the impact of extracellular thiols on non-FR-specific activity of EC0531. Thus, when KB cells (FR+) and A549 cells (FR−) were co-treated with high-dose EC0531 (1 μM) in the presence of cell-impermeable thiol inhibitors, such as 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) or N-(2-carboxyethyl)maleimide (NCEM), non-FR-specific cytotoxicity was inhibited along with reduced non-specific uptake of 3H-EC0531 (tritium in the tubulysin moiety). We next found that independent of cell type or FR expression status, eliminating cystine and glutathione from the incubation medium significantly attenuated the non-specific activity of EC0531; this maneuver also significantly reduced the cellular efflux of extracellular thiols, such as cysteine, which we confirmed is the predominant thiol released by the cell, and at levels reaching 11-42 μM (for KB and A549 cells, respectively) following only a 2-hour conditioning period at 37°C. System xc− is a cystine/glutamate antiporter which has been shown to be involved in regulating the extracellular redox state of the cysteine/cystine couple in vitro. Thus, using system xc− inhibitors (e.g. sulfasalazine), we observed significant reductions in: (i) extracellular thiol activity; (ii) uptake of 3H-EC0531 (i.e. 3H-tubulysin); and (iii) non-specific activity of EC0531. Finally, siRNA-mediated knockdown of xCT (the functional subunit of system xc−) also caused dramatic reduction of extracellular thiol activity as well as reduced non-FR-mediated activity of EC0531. Taken together, our data support the involvement of extracellular thiols (mainly cysteine) in the extracellular reduction of disulfide-containing SMDCs to release the active drug payload which, in turn, may allow for non-specific drug uptake and possible activity, especially when the extracellular SMDC concentration is exceedingly high.
Citation Format: Nikki Parker, Michael R. Pugh, Le-Cun Xu, Iontcho R. Vlahov, Christopher P. Leamon. Extracellular thiols and system xc− play a significant role in non-targeted in vitro activity of a folate-tubulysin B conjugate. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5623. doi:10.1158/1538-7445.AM2013-5623