Načrtovanje in sinteza novih zaviralcev napetostno odvisnih kalijevih kanalov KV 1.3 in KV 10.1 s protitumornim delovanjem : doktorska disertacija

Gubič, Špela (author), Peterlin-Mašič, Lucija (mentor), Tomašič, Tihomir (co-mentor)

Voltage-gated potassium channels are proteins found in cell membranes. They include channels KV1.3 and KV10.1 (hEAG1), which are significantly involved in cancer development. High expression of KV10.1 (hEAG1) has been found in more than 70% of human cancers. KV1.3 is directly involved in promoting cancer cell proliferation, and inhibition of the channel in mitochondria can selectively induce cancer cell apoptosis. As part of our work, we have developed new benzamide-thiophene-based KV1.3 inhibitors. Since the cryo-electronic (cryo-EM) structure of the channel was not known at the time of development, new inhibitors were designed based on the structure of the known benzamide KV1.3 inhibitor. Two new benzamide hit compounds were identified by ligand-based virtual screening. New thiophene-based inhibitors were developed by structural optimization of the hit compounds and by preparation of a large number of new analogues, which were also patented. The most selective among them was the tetrahydropyran-type compound, which exhibited KV1.3 inhibitory activity in the mid nanomolar range. Cyclohexane-type compounds with two chiral centres and with hydroxyl or carbamate groups showed the most potent KV1.3 inhibition in the low nanomolar range in T cells, affecting their activation, intracellular Ca2+ levels, and colony formation. KV1.3 is involved in proliferation of healthy and cancer cells. The new benzamide compounds either inhibited the growth of Panc-1 cells or were cytotoxic to them. Certain benzamide compounds also had a cytotoxic effect on Colo357 cells. Only one compound showed specific anticancer activity without effects on non-cancer hTERT-RPE1 cells. KV1.3 is also found in the inner mitochondrial membrane (mitoKV1.3). Since a crucial step for selectively inducing apoptosis in cancer cells is for the compounds to enter mitochondria in large numbers, we developed new benzamide-based mitoKV1.3 inhibitors. The latter were prepared by attaching the triphenylphosphonium+ (TPP+) group to benzamide KV1.3 inhibitors with two chiral centers. The mitochondrial benzamide inhibitors greatly reduced the number of live Colo357 cells in the 2D and 3D cell models. The two compounds induced apoptosis at 5 μM in the 2D and 3D cell models. Both compounds also significantly decreased the number of live B16F10 mouse melanoma cells expressing mitoKV1.3. On the other hand, the two compounds had a significantly less potent effect on B16F10 mouse melanoma cells not expressing mitoKV1.3. Like KV1.3, the hEAG1 channel is also significantly involved in cancer cell proliferation. Because it is found in healthy tissues only in the central nervous system, it is an almost ideal IV target for the development of new anticancer drugs. We developed new diphenylamine hEAG1 inhibitors by structurally optimising previously published diphenylamine inhibitors. Our new inhibitors showed improved potency compared to previsouly published compounds. The best compound had a better ratio of IC50 values for hEAG1 and hERG than known compounds of the same structural type. The compounds inhibited the growth of breast cancer cells MCF-7, which highly express hEAG1, and Panc-1 cells, which have high hERG expression. As part of the doctoral work, we have developed new structural types of KV1.3 and KV10.1 inhibitors, which represent an important contribution to the development of new drugs and immunosuppressants. The KV1.3 and KV10.1 inhibitors have a new mechanism of action, as there are no drugs with such a mechanism of action on the market so far. The PhD thesis represents an original contribution to the field of research on the involvement of ion channels in cancer processes.

• Language: English
• Work type: Dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: FFA - Faculty of Pharmacy
• Place of publishing: Ljubljana
• Publisher: [Š. Gubič]
• Year: 2022
• Number of pages: IX, 384 str.
• PID: 20.500.12556/RUL-143394
• UDC: 615.4:54:616.006(043.3)
• COBISS.SI-ID: 133516291
• Publication date in RUL: 19.12.2022

Secondary language

• Language: Slovenian
• Title: Design and synthesis of novel voltage-gated potassium channel KV 1.3 and KV 10.1 inhibitors with anticancer activity

Abstract

Napetostno odvisni kalijevi kanali so beljakovine, ki se nahajajo v celičnih membranah. Med njih sodita kanala KV1.3 in KV10.1 (hEAG1), ki sta pomembno vpletena v nastanek rakavih obolenj. Visoko izražanje hEAG1 so odkrili v več kot 70 odstotkih rakavih celic pri ljudeh. Kanal KV1.3 spodbuja proliferacijo rakavih celic in zaviranje kanala v mitohondrijih lahko selektivno povzroči apoptozo rakavih celic. V sklopu doktorske disertacije smo razvili nove benzamidne tiofenske zaviralce KV1.3. Ker krio-elektronska (krio-EM) struktura kanala v času načrtovanja še ni bila znana, smo nove zaviralce načrtovali na podlagi strukture znanega benzamidnega zaviralca. Z izvedbo virtualnega rešetanja na osnovi liganda smo prišli do dveh novih benzamidnih spojin zadetkov. Novi strukturni tip tiofenskih zaviralcev smo razvili s kemijsko optimizacijo spojin zadetkov in pripravo zelo obsežnega števila novih analogov, ki smo jih tudi patentno zaščitili. Med njimi je bila najbolj selektivna spojina tetrahidropiranskega tipa, ki je imela jakost zaviranja KV1.3 v nanomolarnem območju. Spojine cikloheksanskega tipa z dvema kiralnima centroma in s hidroksilnimi ali karbamatnimi skupinami so izkazovale najmočnejšo jakost zaviranja KV1.3 v nizkem nanomolarnem območju na T-celicah in vplivale na njihovo aktivacijo, znotrajcelični nivo Ca2+ in tvorbo kolonij. Za kanal KV1.3 so ugotovili, da sodeluje pri procesu proliferacije zdravih in rakavih celic. Novi tiofenski zaviralci so zavirali rast rakavih celic Panc-1 ali pa so bili za njih citotoksični. Določene spojine so imele citotoksičen učinek tudi na celicah Colo357. Ena spojina je izkazovala tudi specifično protirakavo delovanje brez citotoksičnih učinkov na nerakavi humani celični liniji hTERT-RPE1. Kanal KV1.3 se poleg v celičnih membranah nahaja tudi v notranji mitohondrijski membrani (mitoKV1.3). Ker je za selektivno sprožitev apoptoze rakavih celic ključen korak, da se spojine v velikem številu dostavljajo v notranjost mitohondrijev, smo razvili nove benzamidne zaviralce mitoKV1.3. Slednje smo pripravili z vezavo trifenilfosfonijeve+ (TPP+) skupine na benzamidne zaviralce KV1.3 z dvema kiralnima centroma. Mitohondrijski zaviralci so izrazito zmanjšali število živih rakavih celic Colo357 v 2D in 3D celičnih modelih. Dve najboljši spojini sta sprožili apoptozo celic Colo357 že pri koncentraciji 5 μM in sta pomembno zmanjšali število živih celic mišjega melanoma B16F10, ki izraža mitoKV1.3. Po drugi strani sta imeli spojini bistveno manjši učinek na celicah mišjega melanoma B16F10, ki niso izražale mitoKV1.3. II Tudi kanal hEAG1 podobno kot KV1.3 vpliva na proliferacijo rakavih celic. Ker se v zdravih tkivih nahaja zgolj v osrednjem živčnem sistemu, je primerna tarča za razvoj novih protirakavih učinkovin. V doktorskem delu smo s kemijsko optimizacijo znanih spojin pridobili nove difenilaminske zaviralce hEAG1, ki so izkazovali močnejšo jakost zaviranja hEAG1 kot znane spojine. Najboljša spojina je imela izboljšano razmerje med vrednostima IC50 za hEAG1 in hERG kot znani spojini istega strukturnega tipa. Obe spojini sta zavirali rast celic raka dojke MCF-7, ki izraža kanale hEAG1 v visokem številu in tudi rast celic Panc-1, ki v povečanem številu izražajo hERG. V okviru doktorskega dela smo razvili nove strukturne tipe zaviralcev napetostno odvisnih ionskih kanalov KV1.3 in KV10.1, ki predstavljajo pomemben prispevek k razvoju novih protirakavih in imunosupresivnih učinkovin. Novi zaviralci KV1.3 in KV10.1 imajo nov mehanizem delovanja, saj zdravil s takim mehanizmom delovanja še ni na tržišču. Doktorsko delo predstavlja izviren prispevek na področju raziskav vključenosti ionskih kanalov v rakave procese.

• Keywords: kalijevi kanali KV1.3, zaviralci KV1.3, benzamidne spojine, zaviralci hEAG1, izoforma 1 človeškega kanala eag, imatinib mesilat