Sinteza in vrednotenje zaviralcev indolamin 2,3-dioksigenaze 1 z izoksazolo[5,4-d]pirimidin-4(5H)-onskim osnovnim skeletom

Poljanšek Bitenc, Eva (author), Sova, Matej (mentor)

Indolamin 2,3-dioksigenaza 1 (IDO1) je citoplazemski encim, ki igra pomembno vlogo pri rakotvornosti in njenem napredovanju. Sodeluje v katabolizmu esencialne aminokisline triptofan. Imunomodulatorno delovanje IDO1 je predvsem posledica pomanjkanja triptofana v tumorskem mikrookolju in povečanja presnovkov, ki izvirajo iz triptofana. Aktivirani encim IDO1 namreč zniža raven triptofana v okolici tumorja, zmanjšanje pa povzroči imunosupresivne učinke. Zaviralci IDO1 predstavljajo enega izmed potencialnih terapevtskih pristopov za zaviranje rasti tumorja. V sklopu magistrske naloge smo sintetizirali enajst končnih spojin, čistost in strukturo vmesnih ter končnih spojin smo preverili z različnimi analitskimi tehnikami. Pri načrtovanju spojin smo izhajali iz strukture že znanega zaviralca IDO1, 3-(4-fluorofenil)izoksazolo[5,4-d]pirimidin-4(5H)-ona, ki je bil odkrit z virtualnim rešetanjem. Z biokemijskim testiranjem smo na osnovi merjenja fluorescence ovrednotili zaviralno delovanje vseh končnih spojin, aktivnim spojinam z močnejšim zaviralnim delovanjem pa določili in izračunali še vrednost IC50. Glede na dobljene rezultate smo ugotovili, da najmočnejše zaviralno delovanje kaže spojina N-(4-aminofenil)-2-(3-(4-fluorofenil)-4-oksoizoksazolo[5,4-d]pirimidin-5(4H)-il)acetamid (21) z IC50 = 26,3 µM. Z namenom napovedi potencialnih interakcij z vezavnim mestom IDO1 smo izvedli še sidranje spojine 21 v aktivno mesto encima. Izmed vseh sintetiziranih končnih spojin ima pet spojin pri koncentraciji 100 µM rezidualno aktivnost nižjo od 60 %, zato smo jim lahko določili in izračunali še vrednost IC50. Od teh tri spojine (15, 19 in 21) močneje zavirajo encim, dve spojini (20 in 24) pa šibkeje. Zaključimo lahko, da spojina 21 predstavlja pomembno izhodišče za nadaljnje raziskovanje zaviralcev IDO1, kot eno izmed obetavnih terapevtskih možnosti za zaviranje rasti tumorja.

• Language: Slovenian
• Keywords: indolamin 2, 3-dioksigenaza, imunomodulacija, tumor, zaviralci
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2020
• PID: 20.500.12556/RUL-121245
• Publication date in RUL: 02.10.2020

Secondary language

• Language: English
• Title: Synthesis and evaluation of indoleamine 2,3-dioxygenase 1 inhibitors with isoxazolo[5,4-d]pyrimidin-4(5H)-one scaffold

Abstract

Indoleamine 2,3-dioxygenase 1 (IDO1) is a cytoplasmic enzyme, which plays an important role in carcinogenicity and its progress. It takes part in the catabolism of the essential amino acid tryptophan. Immunomodulatory activity of IDO1 is caused by the lack of tryptophan in the tumor microenvironment and the increase of the metabolites, which originate from tryptophan. The activated enzyme IDO1 lowers the levels of tryptophan in the surrounding of the tumor, which causes immunosuppressive effects. IDO1 inhibitors represent one of the potential therapeutic approaches to inhibit the growth of the tumor. As part of the Master’s thesis, we synthesized eleven final compounds. We checked the purity and confirmed the structure of the intermediates and final compounds with different analytical techniques. The design was based on the already known inhibitor 3-(4-fluorophenyl)isoxazole[5,4-d]pyrimidine-4(5H)-one, which was discovered by virtual screening. With the biochemical testing, based on the fluorescence measurement, we evaluated the inhibitory activity of all final compounds: Furthermore, we also determined and calculated the IC50 value of the most potent compounds. Based on the results obtained, the most potent inhibitory activity was determined for the compound N-(4-aminophenyl)-2-(3-(4-fluorophenyl)-4-oxoisoxazole[5,4-d]pyrimidine-5(4H)-il)acetamide (21) with IC50 = 26.3 µM. In order to predict the potential interactions with the IDO1 binding site, we also performed the docking of the compound 21 in the active site of the enzyme. Among all synthesized final compounds, five of them showed a residual activity lower than 60 % at a concentration of 100 µM; thus, we could determine and calculate the IC50 value. Among those five compounds, three of them (15, 19 in 21) exhibited potent inhibition of the enzyme, whereas two of them (20 in 24) were only weak inhibitors. We can conclude that the compound 21 represents an important starting point for further research of the inhibitors of IDO1, as one of the promising therapeutic possibilities to inhibit the growth of the tumor.

• Keywords: indoleamine 2, 3-dioxygenase, immunomodulation, tumor, inhibitors