Načrtovanje in optimizacija katalitičnih zaviralcev človeške DNA topoizomeraze ▫$II\alpha$▫ kot potencialnih protirakavih učinkovin : doktorska disertacija

Bergant Loboda, Kaja (avtor), Perdih, Andrej (mentor), Sosič, Izidor (komentor), Minovski, Nikola (član komisije za zagovor)

DNA topoisomerases are a diverse family of enzymes, that work as molecular motors and enable topological changes of the DNA molecule. While there are several subgroups of topoisomerases, the subject of our research was the human DNA topoisomerase II[alpha], which is a validated and established anticancer target, as its concentration is higher in rapidly dividing cells compared to normal cells. Type II topoisomerases act via a complex catalytic cycle that offers multiple points for its modulation and consequently drug design. Clinically used inhibitors of this enzyme, topoisomerase II poisons, act by stabilizing a short-lived complex between the DNA and the enzyme turning it into a cellular toxin. However, they at the same time also damage the DNA molecule, and this can lead to serious side effects, such as cardiotoxicity and the onset of secondary forms of cancer. Therefore, in recent years, research has focused on the development of catalytic topo II inhibitors that do not cause DNA damage. In our research outlined by four research hypotheses, we investigated the identification, optimization, and evaluation of new catalytic inhibitors of human topoisomerase II[alpha] that bind to its ATP binding site. In our first study substituted 4,5%-bitiazoles, known inhibitors of the bacterial topoisomerase type II DNA gyrase were used as a design starting point. By comparing the ATP binding sites of both enzymes, we pinpointed their key differences and similarities that were used in a virtual screening of a targeted chemical library to identify inhibitors that would better bind to the topoisomerase II[alpha] ATP binding site. For selected compounds, we demonstrated at the in vitro level that they act as catalytic ATP-competitive inhibitors of topoisomerase II[alpha] and bind to its isolated ATPase domain. The compounds were also effective at the cellular level, as some of them exhibited cytotoxicity on the HepG2 and MFC-7 cell lines in the same range as clinically used anticancer drug etoposide. The substituted 4,5%-bitiazoles stopped the cell cycle in the G1 phase, affected the cell proliferation, and did not cause the occurrence of DNA double-strand breaks, a further evidence that these compounds exhibit a different mode of action at the cellular level compared to topoisomerase II poisons. Our second study dealt with the optimization of position 4 of the 4,6-disubstituted 1,3,5 triazin-2(1H)-ones, for which previous studies have shown they act as catalytic topoisomerase II[alpha] inhibitors and bind to the ATPase domain. Using a developed docking binding model, we screened a focused virtual library of possible analogues with different substituents at the position 4 of the 1,3,5-triazine ring that could form additional interactions with the ATP binding site. After screening, selected in silico optimized analogues were synthesized and were shown to be more potent catalytic inhibitors than the parent compounds. 4 Moreover, two of them also exhibited promising cytotoxicity on the HepG2 cancer cell line. In addition, this chemical class did not induce the formation of DNA double-strand breaks at the cellular level. In our final study, we started from the inactive compounds from the class of 3,5-disubstituted 1,2,4-oxadiazoles, which were originally designed as DNA gyrase inhibitors. We added more rigid moieties to the core structure along with functional groups, which could enable stronger interactions with the ATP binding pocket. In addition to the synthesized compounds, several commercially available substituted oxadiazoles were included in the evaluation of topo II% inhibition. The assays identified several 3,5-disubstituted 1,2,4-oxadiazoles with introduced rigid structures which possessed human topoisomerase II[alpha] inhibition activity. These compounds acted via a catalytic inhibition mechanism and were able to bind to the isolated ATPase domain. They further displayed a cytotoxic potential on the MCF-7 cancer cell line and did not lead to DNA double-strand breaks. During our research we were able to confirm all four research hypotheses. In addition, the discovered lead compounds could via proper optimization lead to preclinical candidates with comparable efficacy as clinically used topoisomerase II poisons, and decreased incidence of serious side effects associated with this group of anticancer agents.

• Jezik: Angleški jezik
• Vrsta gradiva: Doktorska disertacija
• Tipologija: 2.08 - Doktorska disertacija
• Organizacija: FFA - Fakulteta za farmacijo
• Kraj izida: Ljubljana
• Založnik: [K. Bergant Loboda]
• Leto izida: 2021
• Št. strani: 279 str.
• PID: 20.500.12556/RUL-137085
• UDK: 615.28:616-006-085(043.3)
• COBISS.SI-ID: 79768579
• Datum objave v RUL: 01.06.2022

Sekundarni jezik

• Jezik: Slovenski jezik
• Naslov: Design and optimization of catalytic inhibitors of the human DNA topoisomerase IIα as potential anticancer agents

Izvleček

DNA topoizomeraze so družina encimov, ki delujejo kot molekulski motorji in omogočajo topološke spremembe v molekuli DNA ter s tem mnoge procese v celici. V naših raziskavah smo se osredotočili na človeško DNA topoizomerazo II[alfa], ki je validirana in uveljavljena protirakava tarča, saj je njena koncentracija višja v hitro delečih se celicah v primerjavi z normalnimi. Topoizomeraze tipa II delujejo preko kompleksnega katalitičnega cikla, ki ponuja več prijemališč za načrtovanje zdravilnih učinkovin. Klasični zaviralci tega encima, topoizomerazni II strupi, stabilizirajo kovalentni kompleks med DNA in encimom ter ga spremenijo v celični strup. Pri tem tudi poškodujejo DNA molekulo in lahko povzročajo resne neželene učinke, kot sta kardiotoksičnost in nastanek sekundarnih oblik raka. Zato so se v zadnjih letih raziskave usmerile v razvoj katalitičnih zaviralcev, ki ne povzročijo poškodb DNA. V okviru doktorske disertacije smo želeli preko štirih raziskovalnih hipotez identificirati in ovrednotiti nove katalitične zaviralce topoizomeraze II[alfa] ter tudi optimizirati obstoječe katalitične zaviralce, ki se vežejo v vezavno mesto za ATP. V prvi raziskavi smo kot izhodiščno točko načrtovanja uporabili poznane zaviralce bakterijske topoizomeraze tipa II, substituirane 4,5%-bitiazole. S primerjavo vezavnih mest za ATP bakterijske DNA giraze in človeške topoizomeraze II[alfa] smo določili ključne razlike in podobnosti v vezavnih mestih, ki so omogočile virtualno rešetanje usmerjene kemijske knjižnice za identifikacijo zaviralcev, ki bi se bolje vezali na človeško topoizomerazo II[alfa]. Spojine tega razreda delujejo kot katalitični ATP kompetitivni zaviralci človeške DNA topoizomeraze II[alfa] in se vežejo na izolirano ATPazno domeno. Spojine so bile učinkovite tudi na celičnem nivoju, saj so nekatere izmed njih izkazovale citotoksičnost na HepG2 in MFC-7 celični linijah v enakem obsegu kot uveljavljena protirakava zdravilna učinkovina etopozid. Za izbrano spojino smo ugotovili, da ustavi celični cikel v G1 fazi, vpliva na proliferacijo celic ter dokazali, da ne povzroča dvojnih prelomov DNA, kar je dodatni dokaz, da spojine tega razreda tudi na celičnem nivoju delujejo drugače kot topoizomerazni II strupi. V drugi raziskavi smo se osredotočili na optimizacijo mesta substitucije 4 kemijskega razreda 4,6-disubstituiranih 1,3,5-triazin-2(1H)-onov, za katere so v prejšnjih študijah pokazali, da so katalitični zaviralci človeške topoizomeraze II[alfa] in se vežejo na ATPazno domeno, kjer se nahaja ATP vezavno mesto. S pomočjo in silico vezavnega modela smo zgradili in rešetali usmerjeno kemijsko knjižnico možnih analogov z različnimi substituenti na mestu 4 1,3,5-triazinskega obroča, da bi tako spojini omogočili dodatne interakcije z vezavnim mestom za ATP. Po načrtovanju so bili sintetizirani novi analogi, ki so se izkazali kot boljši katalitični zaviralci človeške topoizomeraze II[alfa] kot izhodne spojine. 2 Dve izmed njih sta izkazovala tudi obetavno citotoksičnost na HepG2 rakavi celični liniji. Na celičnem nivoju tudi ta kemijski razred ne povzroča nastanka dvojnih prelomov DNA molekule. V tretji raziskavi smo se lotili optimizacije neaktivnih spojin iz razreda 3,5-disubstituiranih 1,2,4-oksadiazolov, prvotno načrtovanih kot zaviralcev DNA giraze. V molekule smo uvedli rigidnejše fragmente, da bi s tem omogočili stabilnejše interakcije v fosfatnem delu ATP žepa. Poleg sintetiziranih spojin smo ovrednotili tudi nekaj komercialno dostopnih substituiranih oksadiazolov. Identificirali smo več aktivnih spojin, ki nakazujejo, da rigidnejše strukture omogočajo zaviralno aktivnost človeške topoizomeraze II[alfa] preko katalitičnega mehanizma zaviranja ter vezave na ATPazno domeno. Spojine so bile citotoksične na MCF-7 rakavi celični liniji ter niso vodile do nastanka dvojnih prelomov DNA. Tekom raziskav doktorske disertacije smo uspeli potrditi vse štiri izhodiščne raziskovalne hipoteze. Odkrite spojine vodnice so primerne za nadaljnjo optimizacijo do predkliničnih kandidatov, ki bi izkazovali primerljivo učinkovitost kot klinično uporabni topoizomerazni II strupi ter imeli sočasno manjšo pojavnost neželenih učinkov kot ta skupina protirakavih učinkovin.

• Ključne besede: proti rakave učinkovine, DNA topoizomeraza II[alfa], sintetizirane spojine

Projekti

• Financer: ARRS - Agencija za raziskovalno dejavnost Republike Slovenije
• Številka projekta: P1-0012
• Naslov: Molekulske simulacije, bioinformatika in načrtovanje zdravilnih učinkovin