Optimizacija in vrednotenje 4,6-substituiranih-1,3,5-triazin-2(1H)-onov kot katalitičnih zaviralcev človeške DNA topoizomeraze IIα

Korošec, Aljoša (avtor), Sosič, Izidor (mentor), Perdih, Andrej (komentor)

Rak je oznaka za številna bolezenska stanja in je eden najpogostejših vzrokov smrti. Razvoj protirakavih učinkovin poskuša odkriti nove zdravilne učinkovine, ki bi ta obolenja učinkovito pozdravile in imele hkrati čim manj neželenih učinkov za bolnika. Uveljavljena tarča za razvoj protirakavih učinkovin je encim DNA topoizomeraza IIα (topo IIα). Gre za kompleksen protein, ki ima ključno vlogo pri uspešnem podvajanju celic, saj omogoča topološke spremembe molekule DNA. Z zaviranjem tega encima ustavimo proces celične delitve, kar pripelje do celične smrti. Trenutno je v klinični uporabi več zaviralcev topo IIα, kot sta na primer zdravilni učinkovin mitoksantron in etopozid. Težava pri teh učinkovinah je, da delujejo kot topoizomerazni strupi in povzročajo vrsto resnih stranskih učinkov, prav tako pa se lahko pojavi tudi rezistenca rakavih celic na tak tip terapije. Trenuten razvoj topoizomeraznih zaviralcev je zato usmerjen k odkrivanju učinkovin, ki delujejo kot katalitični zaviralci. Med njimi je tudi kemijski razred 4,6-substituiranih-1,3,5-triazin-2(1H)onov, ki deluje preko vezave na ATPazno domeno enicma topo IIα. Na podlagi predhodnih raziskav tega kemijskega razreda, v katerih smo optimizirali zaviralno aktivnost in odkrili, da so sintetizirane spojine slabo topne, smo v sklopu magistrske naloge z uporabo strukturno podprtega načrtovanja sintetizirali novo serijo 4,6-substituiranih-1,3,5-triazin-2(1H)onov, pri kateri smo želeli ohraniti biološko aktivnost in izboljšati topnost. Najprej smo sintetizirali potrebne tarčne tiosečninske derivate in nato z intermolekularno kondenzacijo pripravili 1,3,5-triazin-2(1H)-onske obroče, na katere smo pripeli 3-fluoro-5-(triflourometil)benzilni fragment. Sintetizirali smo štiri končne spojine, ki smo jih ovrednotili s spektroskopskimi metodami ter določili zaviralno aktivnost na encimu topo IIα. Vse pripravljene spojine 5, 6, 13 in 14 so izkazovale primerljivo zaviralno aktivnost topo IIα z znanimi zaviralci tega razreda in bile hkrati tudi bolj aktivne kot referenčna zdravilna učinkovina etopozid. Zaradi boljše topnosti smo spojini 13 in 14 izbrali za nadaljnje vrednotenje s testom aktivnosti razpletanja ter testom spodbujanja cepitve. S testom spodbujanja cepitve smo potrdili, da spojini ne delujeta kot topoizomerazna strupa ampak kot katalitična zaviralca encima topo IIα. S testom aktivnosti razpletanja smo vizualno pokazali, da spojini uspešno zavirata encim tudi pri bistveno nižjih koncentracijah kot referenčni topo IIα zaviralec etopozid. Za te spojini smo z molekulskim sidranjem postavili tudi vezavni model v vezavnem mestu za ATP, ki se nahaja na N-terminalni domeni encima. Rezultati študije predstavljajo pomemben korak naprej pri razvoju tega kemijskega razreda do spojin z učinkovitim delovanjem na to protirakavo tarčo in ustreznimi fizikalno-kemijskimi lastnosti.

• Jezik: Slovenski jezik
• Ključne besede: rak, topoizomeraza IIα, katalitični zaviralci, 4, 6-substituirani-1, 3, 5-triazin-2(1H)-oni
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2019
• PID: 20.500.12556/RUL-111809
• Datum objave v RUL: 14.10.2019

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Optimization and evaluation of 4,6-substitued-1,3,5-triazin-2(1H)-ones as catalytic inhibitors of the human DNA topoisomerase IIα

Izvleček

Cancer is a term describing multiple disease states and is one of the most common causes of death. A significant part of the development of anti-cancer drugs is devoted to the research of new molecules, which could effectively and without side-effects treat patients. One of the studied and important targets for the development of new anti-cancer drugs is the human DNA topoisomerase IIα (topo IIα). This is a complex molecular motor, which plays a crucial part in successful cell division, because it enables topological changes of the DNA molecule. By inhibiting this enzyme, the process of cell replication is halted, leading ultimately to cell death. Multiple inhibitors of topo IIα are used in the clinic, such as mitoxantrone and etoposide. The problem with these drugs is that they act as topoisomerase poisons and cause multiple serious side–effects. In addition, cancer cells can eventually develop resistance to the treatment with these drugs. Recent developments of topoisomerase inhibitors focuses on finding substances that work as catalytic inhibitors. One of such classes of compounds are the 4,6-substituted-1,3,5-triazine-2(1H)ones, which bind to the ATPase domain of the topo IIα enzyme. Based on previous research, which was focused on the optimization of the inhibitory activity (which, unfortunately, led to poorly soluble compounds) we here focused on the synthesis of a new series of 4,6-substituted-1,3,5-triazine-2(1H)ones with improved solubility and retained biological activity. First, we synthesized the corresponding thiourea derivatives, followed by the construction of the 1,3,5-triazin-2(1H)-one scaffold with the use of intramolecular condensation. On this ring, we then attached the 3-fluoro-5-(trifluoromethyl)benzyl fragment. We synthesized four compounds and characterized them with spectroscopic methods and determined their inhibitory activity. All prepared compounds 5, 6, 13, and 14 showed comparable inhibition of topo IIα as previously published compounds, with inhibitory values superior than the reference drug topo II poison etoposide. Because of better solublity, additional assays with compounds 13 and 14 were performed. With the cleaveage assay we showed that compounds act as catalytic inhibiotrs. Moreover, with the decantenation assay we proved that 13 and 14 inhibit the enzyme at lower concentrations than the reference drug etoposide. We used molecular docking to establish the binding modes in the ATP binding site for these two compounds, which is located on the N-terminal domain of the enzyme. The results of our study represent an important step for further development of this chemical class of compounds with effective inhibiton of the target and appropriate physico-chemical properties.

• Ključne besede: cancer, topoisomerase IIα, catalytic inhibitors, 4, 6-substituted-1, 3, 5-triazine-2(1H)-one