20.500.12556/RUL-185740 Razvoj novih zaviralcev mitohondrijskih kanalov K$_V$1.3 in SK3 kot protirakavih spojin raziskovalni podatki, obravnavani v doktorskem delu Development of novel mitochondrial K$_V$1.3 and SK3 channel inhibitors as anticancer agents research data underlying the doctoral dissertation Mitohondrijski izoobliki napetostno odvisnega kalijevega kanala K$_V$1.3 (mitoK$_V$1.3) in s kalcijem aktiviranega kanala SK3 (mitoSK3) imata pomembno vlogo pri uravnavanju apoptoze, kar je ključnega pomena za preživetje rakavih celic. Oba kanala sta zato zelo perspektivni tarči v razvoju protirakavih učinkovin. Njihova prednost je, da lahko hiperpolarizirani mitohondrijski membranski potencial (ΔΨ$_m$) rakavih celic izkoristimo za selektivno kopičenje zaviralcev mitohondrijskih tarčnih proteinov, konjugiranih na transportno skupino za dostavo v mitohondrije (MTM), v mitohondrijih. Kljub temu ostaja kemijski prostor zaviralcev mitoK$_V$1.3 in mitoSK3 omejen na nekaj strukturnih razredov. Za najbolj uveljavljene zaviralce mitoK$_V$1.3 veljajo psoralenski konjugati, katerih uporabo omejuje slaba vodotopnost. Namen te doktorske disertacije je bil preseči te omejitve z načrtovanjem, sintezo in biološkim vrednotenjem novih selektivnih zaviralcev mitoK$_V$1.3 in mitoSK3 z optimiziranimi fizikalno-kemijskimi lastnostmi. Razvili smo nov razred benzamidnih zaviralcev mitoK$_V$1.3 s trifenilfosfonijem (TPP$^+$) kot MTM (cis-8 in cis-9) z izboljšano topnostjo, katerih selektivnost za mitoK$_V$1.3 smo potrdili v celicah z utišanim genom za ionske kanale K$_V$1.3. S sistematičnim spreminjanjem dolžine distančnika, stereokemijske konfiguracije in MTM smo razvili cis-24 (s 4-(OMe)$_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 K$_V$1.3 TVS-26 in ga pretvorili v nov razred v mitohondrije usmerjenih konjugatov (7a, 14d). Nazadnje smo strategijo razširili še na SK3 in razvili prve v mitohondrije usmerjene zaviralce mitoSK3, ki so imeli bistveno močnejše citotoksično delovanje od izhodiščnega zaviralca SK3. Z integriranim pristopom, ki združuje racionalno načrtovanje, raziskave odnosa med strukturo in delovanjem, virtualno rešetanje na osnovi liganda ter farmakološko vrednotenje s poudarkom na učinkih na mitohondrije, smo uspešno razširili kemijski prostor mitohondrijsko usmerjenih zaviralcev kalijevih kanalov in premagali ključne izzive na tem področju. Doktorska disertacija tako prispeva k boljšemu razumevanju mitohondrijskih kalijevih kanalov kot tarč za razvoj protirakavih učinkovin ter omogoča identifikacijo novih zaviralcev mitoK$_V$1.3 in mitoSK3. Naši rezultati predstavljajo novo izhodišče za nadaljnji razvoj v mitohondrije usmerjenih protirakavih učinkovin. The role that the mitochondrial isoforms of the voltage-gated potassium channel K$_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. 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 Slovenski jezik Angleški jezik Neznano 2026-08-19 14:16:15 2026-08-20 08:43:53 2026-08-21 06:50:44 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 2027-09-25 README.docx README.docx README 1 A72247AEA4CCB71EA2C34635A8758A21 9e85330031fe4af1cb0cc7a93ab70f9dcded2944b8cc71cd8d14fa54f82cd8ba 23ab288a-9c9b-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=244788 1_article_1.zip 1_article_1.zip Data for the first article 1 1 B4E66639490AC854BD439A074B0BE9C4 77f94aae8fc953ae27f5728016e245660a0112e7693121a6b627a4e3200a1aea f75f1c5b-9bdb-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=244607 1_article_2.zip 1_article_2.zip Data for the first article 2 1 DA66A0A2DB90853693734B11621B7EF4 1de85d2346fbcda6bc8d11296dd7382138cc743a77e057c480849e670c9e8556 b84669b0-9bdc-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=244608 1_article_3.zip 1_article_3.zip Data for the first article 3 1 1B4B98EEC302B7305A1544C1E3C7A9A0 0a37d5cf3cad7f98d293975a2a4bf4020ca5b1dc70c0b5503ab5cb48d37c46ab cb1ae851-9bdd-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=244609 2_article_1.zip 2_article_1.zip Data for the second article 1 1 4D58CF4375BF4DEA637DA8E420F39F58 f6a45d7ae7e628182320203820b9ee929e3e6104dbf445a2b67deb0a78795acc 98687c46-9be0-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=244610 Fakulteta za farmacijo Meritve in testi 0 0 0