Dobijanje aminokiselinskih derivata avarona i tert-butilhinona i ispitivanje njihove biološke aktivnosti

Abstract

U okviru ove doktorske disertacije je sintetisana serija aminokiselinskih derivata seskviterpenskog hinona avarona, izolovanog iz morskog sunđera Dysidea avara, kao i model jedinjenja tert-butilhinona. Određena je in vitro antitumorska aktivnost svih jedinjenja prema humanim tumorskim ćelijskim linijama (HeLa, A549, Fem-X, K562, MDA-MB-453) i zdravoj MRC-5 ćelijskoj liniji. Utvrđeno je da aminokiselinski derivati avarona pokazuju jače dejstvo nego odgovarajući aminokiselinski derivati tert-butilhinona, sa IC50 vrednostima pojedinih derivata nižim od 10 μM. Rezultati analize ćelijskog ciklusa na HeLa ćelijama ukazuju da je apoptoza jedan od mogućih mehanizama dejstva derivata. Ispitan je uticaj kaspaza 3, 8 i 9 na ćelijsku smrt korišćenjem specifičnih kaspaznih inhibitora. Ispitano je i antimikrobno dejstvo derivata prema nizu Gram-pozitivnih i Gramnegativnih bakterija, kao i na tri soja gljivica, pri čemu je zabeležena vrlo dobra aktivnost derivata avarona. Ispitane su interakcije derivata sa biomakromolekulima. SDS gel-ektroforezom i masenom spektrometrijom je potvrđeno da sintetisana jedinjenja ne modifikuju lizozim. Spektrofotometrijskim ispitivanjima je zabeležena pojava hiperhromizma kao posledica interakcija između hinona i CT-DNA. Utvrđeno je da dolazi do smanjenja intenziteta fluorescencije interkalatora etidijum-bromida i boje Hoechst 33258 koja se vezuje u malu brazdu u kompleksu derivata sa CT-DNA. Spektri cirkularnog dihroizma su ukazali na izostanak interkalacije hinona u CT-DNA. Rezultati molekulskog modelovanja su sugerisali da se derivati smeštaju u malu brazdu DNA. Elektroforetska ispitivanja su potvrdila da ne dolazi do cepanja plazmida pUC19 u prisustvu derivata. Na kraju je utvrđen stepen antioksidativne aktivnosti dobijenih aminokiselinskih derivata hinona.

In this doctoral dissertation, a series of amino acid derivatives of the sesquiterpene quinone avarone, isolated from the marine sponge Dysidea avara, as well as the model compund tert-butylquinone has been synthesized. In vitro cytotoxic activity of all compounds toward human cancer cell lines (HeLa, A549, Fem-X, K562, MDA-MB-453) and normal MRC-5 cell line was determined. It was found that the amino acid derivatives of avarone exhibit a stronger activity than the corresponding amino acid derivatives of tert-butylquinone, with IC50 values of some derivatives less than 10 μM. The results of the cell cycle analysis of HeLa cells indicate that apoptosis is one of the possible mechanisms of action of derivatives. The effect of caspases 3, 8, and 9 on cell death by using specific caspase inhibitors was examined. The antimicrobial effect of the derivatives on the panel of Gram-positive and Gram-negative bacteria, as well as on three fungal species, were investigated, whereby very good activity of avarone derivatives was observed. Interactions of the derivatives with biomacromolecules were studied. SDS gelelectrophoresis and mass spectrometry confirmed the absence of modifications of lysozyme by any of the synthesized compounds. Spectrophotometric studies have recorded the occurrence of hyperchromism as a result of interactions between the quinone and CT-DNA. The quenching of the fluorescence of the intercalator ethidium bromide and of the minor groove binder Hoechst 33258 in the complex with CT-DNA by the derivatives was determined. Circular dichroism spectra demonstrated a nonintercalative binding mode of the quinone derivatives to CT-DNA. The results of molecular modeling suggested that the derivatives bind to the minor groove. The electrophoretic pattern showed no cleavage of the pUC19 plasmid in the presence of any derivatives. Finally, the degree of antioxidant activity of the obtained amino acid derivatives of the quinones was determined.