In silico analysis and antiproliferative effect of novel pyrimidine-2,4-dione-1,2,3-triazole hybrids

Cilj rada je predviđanje mogućih bioloških meta hibrida 1,2,3-trizola metodama molekulskog modeliranja te utvrđivanje mjesta i načina vezivanja aktivnih spojeva u odabranoj meti in silico metodama. U radu je ispitana antiproliferativna aktivnost osam derivata hibrida 1,2,3-triazola na pet različitih tumorskih staničnih linija, i svi su pokazali antiproliferativni učinak in vitro sa značajnom citostatskom aktivnošću. Između N,N-1,3-bis-(1,2,3-triazol)-5-bromouracil hibrida značajnu citostatsku aktivnost imaju p-fluor(1), o-fluor(3) i p-klorfenil(2) supstituirani 1,2,3- triazoli koji su pokazali jaki inhibitorni utjecaj na rast stanica ispitanih na staničnim linijama HepG2 i HeLa. Inhibicija proliferacije zapažena je pri mikromolarnoj koncentraciji ispitanih spojeva, i bila je jača od inhibicije proliferacije stanica istih staničnih linija inkubiranih pod istim uvjetima s 5-fluoruracilom kao referentnom supstancom. Kod ispitanih spojeva nije uočena mitohondrijska toksičnost. Osim metoda stanične biologije u radu je rabljen program PASS za ispitivane spojeve kako bi se odredila moguća biološka meta. Detekcija biološke mete zasniva se na strukturi ispitivanih spojeva te na strukturi bioloških makromolekula iz proteinskih baza. Analiza je pokazala da je za snažno antitumorsko djelovanje spojeva 1, 2 i 3 vrlo vjerojatno odgovorna inhibicija tirozin kinaze Wee1. Uporabom programa Autodock Vina spojevi 2 i 3 uklopljeni su u kristalnu strukturu kinaze Wee1 koja je preuzeta iz proteinske baze podataka [PDB: 5v5y]. Analiza molekulskog uklapanja pokazala je da se spojevi 2 i 3 vežu u ATP vezno mjesto kinaze Wee1 jednako kao i spoj MK-1775 koji je snažni i specifični inhibitor kinaze Wee1 (IC50 = 5,2 nM). Citostatski učinci spojeva 2 i 3 mogu se pripisati inhibiciji kinaze Wee1, "čuvara" zastoja G2-M kontrolne točke staničnog ciklusa. Osim toga, za spojeve 2, 3 i MK-1775 metodom Western blota ispitan je učinak na fosforilaciju proteina, mogućih supstrata kinaze Wee1. Tako je u konačnici dokazano da se ukupna fosforilacija izoliranih proteina nakon stimulacije perifernih mononuklearnih stanica znatno smanjuje u prisutnosti specifičnog inhibitora MK-1775 kao i ispitivanih spojeva 2 i 3, što jasno ukazuje na moguću inhibiciju kinaze Wee1. S obzirom da je ciklin ovisna kinaza Cdk1 supstrat za kinazu Wee1, u radu se molekulskim modeliranjem analizirao utjecaj enzimske aktivnosti kinaze Wee1 na fosforilaciju kinaze Cdk1.The aim of this work is to use molecular modeling methods to predict the possible biological target of the 1,2,3-triazole hybrid molecules and using in silico methods to determine the site and the way of binding of active compounds to the selected target. Antiproliferative activity of eight hybrid 1,2,3-triazole derivatives were tested on five different tumor cell lines, and all of them have shown antiproliferative effect in vitro, with significant cytostatic activity. Between N, N-1,3-bis(1,2,3-triazole)-5-bromouracil hybrids p-fluoro(1), o-fluoro(3) and pchlorophenyl( 2) substituted 1,2,3-triazoles which showed a strong inhibitory effect on HepG2 and HeLa cell line growth. Inhibition of cell proliferation was observed in the presence of micromolar concentration of tested compounds. The inhibition of the cell proliferation was even stronger if compared with the inhibition of the same cells under the same conditions in the presence of the reference substance of 5-fluorouracil. No mitochondrial toxicity was observed for compounds tested. In addition to above mentioned methods, the PASS analysis was used to make predictions of possible biological targets for tested compounds. The selection of the biological target(s) was based on the structure of compounds and on known structure of biological macromolecules from protein bases. PASS analysis predicted that strong antitumor activity of compounds 1, 2, and 3 is due the inhibition of the Wee1 tyrosine kinase. Using the Autodock Vina program and in silico screening, compounds 2 and 3 were docked to Wee1 kinase crystal structure downloaded from the protein database [PDB: 5v5y]. Molecular fit analysis has shown that compounds 2 and 3 specifically bind to the pocket of ATP binding site of Wee1 kinase, and that the binding domain of the tested compounds is the same as the binding site of specific inhibitor of Wee1 kinase MK-1775 (IC50 = 5,2 nM). Cytostatic effects of compounds 2 and 3 can be attributed to the inhibition of the G2-M control of the cell cycle, which is a control point of Wee1 kinase. In addition, compounds 2, 3, and MK-1775 have shown decreased phosphorylation of the isolated proteins by Western blot after the stimulation of peripheral mononuclear cells. Considering that the cyclin-dependent kinase Cdk1 is one of the major substrates for Wee1 kinase, molecular modeling and in silico docking has been undertaken to better explain the influence of Wee1 kinase activity on phosphorylation of Cdk1 kinase