The role that the mitochondrial isoforms of the voltage-gated potassium channel K$_V$1.3 (mitoK$_V$1.3) and the calcium-activated potassium channel SK3 (mitoSK3) play in the regulation of apoptosis positions them at the center of cancer cell survival and makes them highly attractive anticancer targets. A distinctive advantage of these targets is that the hyperpolarized mitochondrial membrane potential (ΔΨ$_m$) of cancer cells can be exploited to selectively accumulate inhibitors conjugated to a mitochondria-targeting moiety (MTM). However, the chemical space of mitoK$_V$1.3 and mitoSK3 inhibitors has remained narrow and dominated by a few scaffolds. Among them, the best established psoralen-based conjugates are limited by poor aqueous solubility. The aim of this doctoral dissertation was to overcome these limitations by designing, synthesizing, and biologically evaluating novel mitoK$_V$1.3 and mitoSK3 inhibitors with expanded chemical diversity and improved properties.
We established a new non-psoralene class of benzamide-TPP$^+$ mitoK$_V$1.3-targeting conjugates (cis-8 and cis-9), whose mitoK$_V$1.3-dependent cytotoxicity was confirmed in K$_V$1.3-knockout cells. Systematic exploration of the linker length, stereochemical configuration, and MTM identified cis-24 (containing 4-(OMe)$_3$-TPP$^+$) as a submicromolar lead acting through mitochondrial uncoupling and permeability transition pore opening, while replacement of TPP$^+$ by 3,5-diphenylpyridinium afforded cis-7, an in vivo active lead that reduced tumor growth in a B16F10 mouse melanoma model. Using ligand-based virtual screening, we identified structurally novel K$_V$1.3 inhibitor TVS-26 and converted it into a new class of mitochondria-targeted conjugates (7a and 14d). Finally, extending this strategy to SK3, we developed the first mitochondria-targeted SK3 inhibitors, which were markedly more potent in cnacer cells than the inactive parent SK3 inhibitors.
Overall, we have demonstrated that our integrated approach, which combines medicinal chemistry, structure-activity relationship studies, ligand-based virtual screening, and pharmacological and mitochondrial profiling, can successfully expand the chemical space of mitochondria-targeted potassium channel inhibitors and overcome key challenges in this field. The doctoral dissertation has contributed to a deeper understanding of mitochondrial potassium channels as anticancer targets and has facilitated the identification of novel mitoK$_V$1.3 and mitoSK3 inhibitors, providing a solid starting point for the further development of mitochondria-directed anticancer agents.
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