Eksperimentalno in računsko ovrednotenje vpliva povezovalnih heteroatomov v 4,6-substituiranih 1,3,5-triazin-2(1H)-onih na zaviranje človeške DNA topoizomeraze IIα

Tomšič, Gašper (author), Sosič, Izidor (mentor), Perdih, Andrej (comentor)

Rak je stalni spremljevalec večceličnega življenja in pogost vzrok smrti, zaradi katerega umre približno 10 milijonov ljudi letno. Razred 4,6-substituiranih 1,3,5-triazin-2(1H)-onov je bil razvit kot nova možnost zaviranja človeške DNA topoizomeraze IIα (topo IIα), ki je uveljavljena tarča kemoterapije zaradi vloge pri replikaciji DNA in posledično delitvi celic. V magistrski nalogi smo žveplova atoma, ki povezujeta substituenta na mestih 4 in 6 s triazinonskim obročem, zamenjali za kisikova, da bi izboljšali fizikalno-kemijske lastnosti spojin ter ovrednotili vpliv povezovalnih atomov na topo IIα zaviralno aktivnost. Sintezo tarčnih spojin smo izvedli po novem postopku s tvorbo etrov na trikloro-1,3,5-triazinu ter sledečo pretvorbo klorotriazina v triazinon. Sintetizirali smo pet O-triazinonov 8-12, med katerimi sta dva neposredna analoga že predhodno ovrednotenih S-triazinonov. Istovetnost spojin smo preverili z NMR in HRMS-ESI spektrometričnimi metodami. Najboljšo topo IIα zaviralno aktivnost sta izkazali spojini 9 in 10. Ker je spojina 9 neposredni analog S-triazinona 13, smo ugotovili, da zamenjava povezovalnih atomov nakazuje manjše znižanje topo IIα zaviralne aktivnosti ter izboljšanje fizikalno kemijskih lastnosti. Za par analognih spojin 9 in 13 smo izvedli še simulacijo molekulske dinamike v dolžini 2 µs. Analize trajektorij z dinamičnimi farmakofori in izračuni proste energije vezave so pokazale, da je O-triazinon 9 v aktivnem mestu za ATP (adenozin trifosfat) bolj gibljiv kot njegov S-analog 13 ter večino časa zavzema tudi drugačno vezavno pozo, ki preko interakcije s Tyr151 lahko rigidizira segmenta tarčnega proteina. Ugotovili smo tudi pomembnost molekul vode pri vezavi O-triazinona ter v ta namen razvili novo metodo za izračun dinoforonih modelov, ki omogoča bolj celosten uvid v medmolekulsko prepoznavanje kot dosedanje metode. Razvita metodologija bo uporabna tudi za analizo dinofornih modelov pri drugih sistemih protein-ligand. Z raziskavami smo razširili poznavanje odnosa med strukturo in delovanjem tega razreda spojin in pripomogli k racionalnemu načrtovanju nadaljnjih korakov optimizacije teh obetavnih katalitičnih zaviralcev topo IIα.

• Language: Slovenian
• Keywords: 4, 6-substituirani 1, 3, 5-triazin-2(1H)-oni, katalitični zaviralci, protirakave učinkovine, molekulska dinamika, načrtovanje učinkovin
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2023
• PID: 20.500.12556/RUL-150106
• Publication date in RUL: 14.09.2023

Secondary language

• Language: English
• Title: Experimental and computational investigation of the linker heteroatom influence in 4,6-substituted 1,3,5-triazin-2(1H)-ones on inhibitory activity against human DNA topoisomerase IIα

Abstract

Cancer has been a constant companion to multicellular life since its emergence and is a common cause of death among humans, attributed to approximately 10 million deaths each year. The 4,6-disubstituted 1,3,5-triazin-2(1H)-one structural class has shown promising inhibitory effects on human topoisomerase IIα (topo IIα), which is an established chemotherapy target, owing to its role in DNA and cell replication. In this master’s thesis, two sulfur atoms, connecting the triazinone scaffold to the substituents at positions 4 and 6, were substituted for oxygen atoms with the aim to improve the physico-chemical properties of the compounds and to investigate the effects of linker atoms on topo IIα inhibition. The target compound synthesis was carried out by initial ether formation on the trichloro-1,3,5-triazine and subsequent conversion of the chlorotriazine scaffold into a triazinone. Five O-triazinones 8-12 were synthesized, among which two were direct analogues of previously assayed S-triazinones. The identity of compounds was confirmed by NMR and HRMS-ESI spectrometric methods. Compounds 9 and 10 exhibited the strongest topo IIα inhibition. As compound 9 is a direct analogue of the S-triazinone 13, we concluded that the change in linker atoms results in marginally reduced topo IIα inhibitory activity and improved physico-chemical properties. Molecular dynamics simulations for compounds 9 and 13, each in the length of 2 µs, were performed. The trajectory analysis through dynamic pharmacophores and binding free energy calculations revealed, that O-triazinone 9 is more flexible in the ATP binding site than its S-analogue 13, as well as assuming a different binding pose, in which, through interactions with Tyr151, it is able to rigidize a segment of the target protein. Additionally, we showed the importance of water molecules in the binding of O-triazinones, developing a new method for computing dynamic pharmacophores, which allows for a more holistic insight into intermolecular recognition than contemporary methods. The developed method will also be of use in analyzing dynophore models for other protein-ligand systems. Our research has broadened our understanding of the structure-activity relationships of this compound class as well as aided in planning a more rational approach to the optimization of these promising catalytical inhibitors of topo IIα.

• Keywords: 4, 6-substituted-1, 3, 5-triazin-2(1H)-ones, catalytic inhibitors, anticancer drugs, molecular dynamics, drug design