Strukturno podprto načrtovanje in sinteza 4,6-substituiranih 1,3,5-triazin-2(1H)-onov kot katalitičnih zaviralcev človeške DNA-topoizomeraze IIα

Grošelj, Gašper (author), Sosič, Izidor (mentor), Perdih, Andrej (co-mentor)

Rak je eden najpogostejših vzrokov smrti, zato je razvoj protirakavih terapij ključen za uspešno obvladovanje te bolezni. Pomembna tarča kemoterapije je človeška DNA topoizomeraza IIα, ki regulira topološke spremembe molekule DNA. Večino klinično uporabnih učinkovin, ki ciljajo to tarčo, uvrščamo med topoizomerazne strupe, ki zavirajo encim preko stabilizacije kovalentnega kompleksa med DNA molekulo in encimom. Ta mehanizem delovanja je odgovoren za hude stranske učinke, npr. kardiotoksičnost ter nastanek sekundarnih oblik raka. Izziv rešujejo katalitični zaviralci, ki ne stabilizirajo omenjenega kompleksa. Podskupina teh zaviralcev deluje preko zaviranja vezave energijske molekule ATP, ki jo encim potrebuje za delovanje. V magistrski nalogi smo s pomočjo strukturno podprtega načrtovanja razreda optimizirali 4,6-substituirane-1,3,5-triazin-2(1H)-one, ki delujejo kot katalitični zaviralci encima. Za ovrednotenje novih možnih substituentov na mestu 6 triazinonskega obroča smo uporabili predhodno razvit vezavni model teh spojin v vezavno mesto za ATP ter program za optimizacijo spojin DeepFrag, ki temelji na metodah strojnega učenja. Sintezo izbranih spojin smo izvajali po razvitem postopku z ustrezno substituirano izotiosečnino in etoksikarbonil izotiocianatom za tvorbo triazinonskega obroča. V zadnjem koraku sinteze smo na mesto 4 nastalega obroča pripeli še najobetavnejše fragmente iz prejšnjih študij. Skupaj smo sintetizirali osem novih triazinonov, izmed katerih je najboljše rezultate zaviranja encima pokazala spojina 4, ki ima na mestu 6 uveden 2-metiltiazolni fragment. Uspešnost zaviranja je primerljiva s klinično uporabljenim topo strupom etopozidom. Za spojino 4 smo preko testa spodbujanja cepitve dokazali, da le-ta encima ne zavira kot topoizomerazni strup, ampak kot katalitični zaviralec. Vezavo spojin smo opredelili tudi z analizo dobljenih sidranih poz ter za aktivno spojino 4 opazili prisotnost nove interakcije med aminokislinskimi preostanki v ATP mestu in 2-metiltiazolnim substituentom. Rezultati naših raziskav razširjajo poznavanje odnosa med strukturo in delovanjem (SAR) teh spojin kot katalitičnih zaviralcev človeške DNA topoizomeraze IIα ter bodo koristni za potekajoči razvoj tega obetavnega razreda kot potencialnih protirakavih učinkovin.

• Language: Slovenian
• Keywords: rak, človeška DNA topoizomeraza IIα, katalitični zaviralci, 4, 6-substituirani-1, 3, 5-triazin-2(1H)-oni
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2023
• PID: 20.500.12556/RUL-144887
• Publication date in RUL: 21.03.2023

Secondary language

• Language: English
• Title: Structure-based design and synthesis of 4,6-substituted 1,3,5-triazin-2(1H)-ones as catalytic inhibitors of human DNA topoisomerase IIα

Abstract

Cancer is one of the leading causes of death, so the development of cancer therapies is critical to the successful treatment of this disease. An important target of chemotherapy is human DNA topoisomerase IIα, which regulates topological changes in the DNA molecule. Most clinically useful agents directed at this target are classified as topoisomerase poisons, which inhibit the enzyme by stabilizing the covalent complex between the DNA molecule and the enzyme. This mechanism of action is responsible for severe side effects, such as cardiotoxicity and formation of secondary forms of cancer. This challenge is addressed by catalytic inhibitors that do not stabilize the said complex. A subset of these inhibitors acts by inhibiting the binding of the ATP molecule that the enzyme requires for its function. In this Master’s thesis we optimized 4,6-substituted-1,3,5-triazin-2(1H)-ones acting as catalytic inhibitors of the enzyme using a structure-based design approach. To evaluate new possible substituents at position 6 of the triazinone ring, we used a previously developed binding model of these compounds in the ATP-binding site and the DeepFrag compound optimization program based on machine learning methods. The synthesis of the selected compounds was carried out according to the developed procedure using appropriately substituted isothiourea and ethoxycarbonyl isothiocyanate to form the triazinone ring. In the final step of the synthesis, the most promising fragments from previous studies were attached to the 4-position of the resulting ring. A total of eight new triazinones were synthesized, of which compound 4, containing a 2-methylthiazole fragment at position 6, showed the best topo IIα inhibition, comparable to the clinically used topo poison etoposide. For compound 4, we demonstrated by the cleavage assay that it inhibited the enzyme as a catalytic inhibitor rather than as a topoisomerase poison. The binding of the compounds was also determined by the analysis of the docking poses obtained, and for the active compound 4 we observed the presence of new interactions between the amino acid residues in the ATP site and the 2-methylthiazole substituent. The results of our research expand the knowledge of the structure-activity relationship (SAR) of these compounds as catalytic inhibitors of human DNA topoisomerase IIα and will be useful for the ongoing development of this chemical class as potential anticancer agents.

• Keywords: cancer, human DNA topoisomerase IIα, catalytic inhibitors, 4, 6-substituted-1, 3, 5-triazin-2(1H)-ones