20.500.12556/RUL-189109 Razvoj novih zaviralcev mitohondrijskih kanalov KV1.3 in SK3 kot protirakavih spojin Development of novel mitochondrial KV1.3 and SK3 channel inhibitors as anticancer agents Mitohondrijski izoobliki napetostno odvisnega kalijevega kanala KV1.3 (mitoKV1.3) in s kalcijem aktiviranega kanala SK3 (mitoSK3) imata pomembno vlogo pri uravnavanju apoptoze, kar je ključnega pomena za preživetje rakavih celic. Oba kanala sta zato zelo perspektivni tarči v razvoju protirakavih učinkovin. Njihova prednost je, da lahko hiperpolarizirani mitohondrijski membranski potencial (ΔΨm) rakavih celic izkoristimo za selektivno kopičenje zaviralcev mitohondrijskih tarčnih proteinov, konjugiranih na transportno skupino za dostavo v mitohondrije (MTM), v mitohondrijih. Kljub temu ostaja kemijski prostor zaviralcev mitoKV1.3 in mitoSK3 omejen na nekaj strukturnih razredov. Za najbolj uveljavljene zaviralce mitoKV1.3 veljajo psoralenski konjugati, katerih uporabo omejuje slaba vodotopnost. Namen te doktorske disertacije je bil preseči te omejitve z načrtovanjem, sintezo in biološkim vrednotenjem novih selektivnih zaviralcev mitoKV1.3 in mitoSK3 z optimiziranimi fizikalno-kemijskimi lastnostmi. Razvili smo nov razred benzamidnih zaviralcev mitoKV1.3 s trifenilfosfonijem (TPP+) kot MTM (cis-8 in cis-9) z izboljšano topnostjo, katerih selektivnost za mitoKV1.3 smo potrdili v celicah z utišanim genom za ionske kanale KV1.3. S sistematičnim spreminjanjem dolžine distančnika, stereokemijske konfiguracije in MTM smo razvili cis-24 (s 4-(OMe)3-TPP+) kot spojino vodnico s submikromolarno jakostjo delovanja, ki deluje preko razklopa celičnega dihanja in sinteze ATP v mitohondrijih in odpiranja mitohondrijske prepustne prehodne pore. Zamenjava TPP+ s 3,5-difenilpiridinijem kot MTM pa je vodila do in vivo učinkovite spojine cis-7, ki je zmanjšala rast tumorja v mišjem modelu melanoma B16F10. Z virtualnim rešetanjem na osnovi liganda smo odkrili strukturno nov zaviralec KV1.3 TVS-26 in ga pretvorili v nov razred v mitohondrije usmerjenih konjugatov (7a, 14d). Nazadnje smo strategijo razširili še na SK3 in razvili prve v mitohondrije usmerjene zaviralce mitoSK3, ki so imeli bistveno močnejše citotoksično delovanje od izhodiščnega zaviralca SK3. Z integriranim pristopom, ki združuje racionalno načrtovanje, raziskave odnosa med strukturo in delovanjem, virtualno rešetanje na osnovi liganda ter farmakološko vrednotenje s poudarkom na učinkih na mitohondrije, smo uspešno razširili kemijski prostor mitohondrijsko usmerjenih zaviralcev kalijevih kanalov in premagali ključne izzive na tem področju. Doktorska disertacija tako prispeva k boljšemu razumevanju mitohondrijskih kalijevih kanalov kot tarč za razvoj protirakavih učinkovin ter omogoča identifikacijo novih zaviralcev mitoKV1.3 in mitoSK3. Naši rezultati predstavljajo novo izhodišče za nadaljnji razvoj v mitohondrije usmerjenih protirakavih učinkovin. The role that the mitochondrial isoforms of the voltage-gated potassium channel KV1.3 (mitoKV1.3) 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 (ΔΨm) of cancer cells can be exploited to selectively accumulate inhibitors conjugated to a mitochondria-targeting moiety (MTM). However, the chemical space of mitoKV1.3 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 mitoKV1.3 and mitoSK3 inhibitors with expanded chemical diversity and improved properties. We established a new non-psoralene class of benzamide-TPP+ mitoKV1.3-targeting conjugates (cis-8 and cis-9), whose mitoKV1.3-dependent cytotoxicity was confirmed in KV1.3-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 KV1.3 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 cancer 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 mitoKV1.3 and mitoSK3 inhibitors, providing a solid starting point for the further development of mitochondria-directed anticancer agents. apoptoza benzamid kalijevi kanali mitoKV1.3 mitoSK3 usmerjanje v mitohondrij protirakavo delovanje sulfonamid TPP+ virtualno rešetanje na osnovi liganda apoptosis benzamide potassium channels mitoKV1.3 mitoSK3 mitochondria-targeting anticancer sulfonamide TPP+ ligand-based virtual screening true false false [M. Fois] Slovenski jezik Angleški jezik Doktorsko delo/naloga 2026-10-02 07:15:14 2026-10-02 07:15:54 2026-10-12 13:55:38 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 2027-09-24 576.36(043.3) 41825 294439683 10854.pdf 10854.pdf 1 7BEE21B3315E9957CF86E98DAC8A33F9 16b6e3df27da5fc3af6557cf934cdba80310260c724f7a1c641c52cf3c7b55d1 3d2bc8b6-be20-11f1-8bc5-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=249573 Fakulteta za farmacijo 0 0 0