Structure–Activity Relationships of Di-2-pyridylketone, 2-Benzoylpyridine, and 2-Acetylpyridine Thiosemicarbazones for Overcoming Pgp-Mediated Drug Resistance

Abstract

Multidrug resistance (MDR) mediated by P-glycoprotein (Pgp) represents a significant impediment to successful cancer treatment. The compound, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), has been shown to induce greater cytotoxicity against resistant cells than their nonresistant counterparts. Herein, the structure− activity relationships of selected thiosemicarbazones are explored and the novel mechanism underlying their ability to overcome resistance is further elucidated. Only thiosemicarbazones with electron-withdrawing substituents at the imine carbon mediated Pgp-dependent potentiated cytotoxicity, which was reversed by Pgp inhibition. Treatment of resistant cells with these thiosemicarbazones resulted in Pgp-dependent lysosomal membrane permeabilization (LMP) that relied on copper (Cu) chelation, reactive oxygen species generation, and increased relative lipophilicity. Hence, this study is the first to demonstrate the structural requirements of these thiosemicarbazones necessary to overcome MDR. We also demonstrate the mechanism that enables the targeting of resistant tumors, whereby thiosemicarbazones "hijack" lysosomal Pgp and form redox-active Cu complexes that mediate LMP and potentiate cytotoxicity.

Tumor stressors induce two mechanisms of intracellular P-glycoprotein–mediated resistance that are overcome by lysosomal-targeted thiosemicarbazones

“…1Ci). This "half-resistant" cell model was specifically utilized throughout our experiments, instead of the "fully-resistant" KBV1 (cells grown in VBL at 1 μg/mL (14,15,17)), to enable increased Pgp expression upon exposure to stress stimuli. As shown in Fig.…”

“…1Bi) to overcome drug resistance by directly using intracellular Pgp in lysosomes against tumor cells ( Fig. 1Bii) (15)(16)(17). In fact, like DOX (Fig.…”

“…1Aii), Dp44mT is a Pgp substrate, and is effluxed out of cells, but also transported into lysosomes by utilizing Pgp on the lysosomal membrane to increase damage to this organelle ( Fig. 1Bii) (15)(16)(17).…”

“…This generation of ROS induces lysosomal membrane permeabilization (LMP), and apoptotic cell death, which effectively overcomes resistance ( Fig. 1Bii) (15)(16)(17)19). The ability of Dp44mT to "hijack" Pgp and markedly overcome resistance to standard chemotherapies (e.g., DOX) is an important therapeutic advantage, as MDR is a major impediment to successful cancer treatment.…”

“…The ability of Dp44mT to "hijack" Pgp and markedly overcome resistance to standard chemotherapies (e.g., DOX) is an important therapeutic advantage, as MDR is a major impediment to successful cancer treatment. Recently, a Dp44mT analog that has the same mechanism of action has entered multicenter clinical trials for advanced and resistant tumors (16,17,(19)(20)(21)(22)(23)(24). Hence, Dp44mT's mechanism-of-action in terms of overcoming Pgp-mediated resistance is important to elucidate.…”