Razvoj bioloških testova za identifikaciju liganada steroidnih receptora i ispitivanje aktivnosti steroidogenog enzima aromataze

Abstract

U ovoj doktorskoj disertaciji razvijen je fluorescentni test u kvascu za identifikaciju potencijalnih prirodnih ili sintetičkih liganada ERα, ERβ ili AR i kvantifikaciju njihovog afiniteta vezivanja sa mogućnošću testiranja čitavih biblioteka modifikovanih steroida i ksenoestrogena. Takođe, opisana je primena optimizovanog biosenzora za procenu estrogenog potencijala sintetskih steroida i odabranih biljnih ekstrakata bogatih jedinjenjima fitoestrogenih osobina. U cilju potpunijeg sagledavanja mehanizma delovanja odabranih modifikovanih steroida ispitana je njihova antiproliferativna aktivnost prema ćelijskim linijama estrogen receptor pozitivnog kancera dojke (MCF-7) i kancera prostate (PC-3), dok su in silico metodom molekularnog dokinga predviđene energije i geometrije vezivanja ovih jedinjenja za ligand-vezujuće domene ERα i ERβ. Drugi deo ovog rada obuhvata razvoj testa za ispitivanje aktivnosti humanog enzima aromataze, heterologno eksprimiranog u ćelijama kvasca Saccharomyces cerevisiae i/ili bakterija Escherichia coli, u prisustvu ili odsustvu inhibitora. Interakcije modifikovanih steroida, odabranih na osnovu strukture, sa aromatazom ispitane su osetljivim spektroskopskim metodama, praćenjem promene spinskog stanja Fe iz hem grupe ili promene fluorescencije ostatka triptofana iz aktivnog centra usled konformacione promene proteina, izazvane interakcijom sa ligandom. Razvijeni in vitro testovi bez upotrebe radioaktivnih izotopa su, osim visoke efikasnosti i bezbednosti po korisnika i okolinu, pokazali specifičnost i ekonomičnost u preliminarnom skriningu liganada steroidnih receptora i inhibitora aromataze. Jedinjenja kod kojih je detektovana značajna biološka aktivnost mogu potencijalno poslužiti kao osnova za razvoj terapeutika u lečenju hormon-zavisnih bolesti i stanja, koja danas predstavljaju globalni zdravstveni problem.

In this doctoral dissertation, a fluorescent assay in yeast was developed for identification of potential natural or synthetic ligands of ERα, ERβ or AR and quantification of their binding affinity, as well asevaluation of the estrogenic potential of synthetic steroids and selected plant extracts rich in phytoestrogen content. The assay could be used to screen libraries of modified steroids and xenoestrogens. In order to better understand the biomedical potential of selected modified steroids, results were compared to antiproliferative activity against estrogen receptor positive breast cancer (MCF-7) and prostate cancer (PC-3) cell lines. Binding energies and the geometry of binding of these compounds for ERα and ERβ ligand binding domains were predicted in silico by molecular docking methods. The second part of this study includes development of an assay for study of aromatase activity in the presence or absence of inhibitors by heterologous expression of human aromatase in Saccharomyces cerevisiae and/or Escherichia coli cells, as model-organisms. Furthermore, interactions between modified steroids, selected according to their structure, and aromatase were tested using sensitive spectroscopic methods based on ligand-induced changes in the spin state of Fe from the heme group or changes in the fluorescence of a tryptophan residue in the active site. The non-radioactive in vitro assays developed here, besides high efficiency, user and environmental safety, also have greater specificity and are more cost-effective for preliminary screening of steroid receptor ligands and aromatase inhibitors. Additionally, compounds identified to express significant biological activity can serve as a basis for the development of potential therapeutics in the treatment of hormone-dependent diseases and conditions, a global health issue today.