Ugotavljanje vpliva izbranih izoflavonoidov na aktivnost kresničkine luciferaze

Vegelj, Jan (author), Sollner Dolenc, Marija (mentor), Kenda, Maša (comentor)

Kresničkina luciferaza je reporterski gen, ki se pogosto uporablja na področju raziskovanja vpliva spojin na transkripcijsko aktivnost genov in se njegovo izražanje ugotavlja preko bioluminiscenčne reakcije nastalega proteina. Izraženi protein je dovzeten za potencialni vpliv spojin, med drugim zaviranje in stabilizacijo, kar lahko vodi v lažno pozitivne rezultate. Luciferaza morske mačehe, ki se pogosto uporablja v kombinaciji s kresničkino luciferazo, predstavlja pomembno alternativo le-tej. Ker so izoflavonoidi kot ena izmed skupin naravnih spojin pogosto preiskovanih tudi v testnih sistemih s kresničkino luciferazo, je smiselno, da ovrednotimo njihov vpliv na le-to. V nalogi smo se osredotočili na 11 strukturno različnih izoflavonoidov in ovrednotili njihov vpliv na aktivnost kresničkine luciferaze in luciferaze morske mačehe. Pri tem smo potencialno interferenco kresničkine luciferaze v obliki zaviranja najprej ovrednotili s pomočjo QSAR programa InterPred, ki je napovedal veliko verjetnost zaviranja za 7 od 11 izoflavonoidov. Z novo metodo in vitro, ki temelji na osnovi luciferaznega testa na lizatu celične linije AR-EcoScreen, smo preverili zaviranje obeh reporterskih proteinov. Pri tem smo najprej testirali spojine pri 1, 10 in 100 µM koncentraciji, čemur je v primeru zaviralne aktivnosti sledilo še testiranje pri dodatnih koncentracijah in določitev IC50. Sedem od 11 izoflavonoidov je zaviralo kresničkino luciferazo, pri čemer smo pri primerjavi z rezultati programa InterPred prišli do zaključka, da program pravilno napove zaviralno aktivnost. Nobeden izmed izbranih izoflavonoidov ni zaviral luciferaze morske mačehe in vitro. Za najbolj močne zaviralce smo v obliki prirejenega testa in vitro vrednotili tudi temperaturno stabilizacijo kresničkine luciferaze. Vendar pa z gotovostjo težko potrdimo, da so pridobljene vrednosti posledica stabilizacije proteina, saj ima ta metoda nekaj pomanjkljivosti. Zaviranje kresničkine luciferaze smo raziskali še bolj podrobno s pomočjo molekulskega sidranja in potrdili, da se aktivni izoflavonoidi vežejo v luciferinski žep proteina ter preučili odnos med strukturo in aktivnostjo aktivnih izoflavonoidov. Z molekulskim sidranjem smo tudi potrdili, da izbrani izoflavonoidi ne zavirajo luciferaze morske mačehe. Potrdili smo zaviralno aktivnost nekaterih izbranih izoflavonoidov, kar lahko zaradi stabilizacije pojasni višje odzive celičnih linij s kresničkino luciferazo v preteklih študijah. Pri testiranju vpliva izoflavonoidov na transkripcijsko aktivnost izbranih genov bi bila tako bolj ustrezna uporaba luciferaze morske mačehe.

• Language: Slovenian
• Keywords: izoflavonoidi, kresničkina luciferaza, luciferaza morske mačehe
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2021
• PID: 20.500.12556/RUL-128303
• Publication date in RUL: 08.07.2021

Secondary language

• Language: English
• Title: Influence of selected isoflavonoids on firefly luciferase activity

Abstract

Firefly luciferase is a reporter gene, commonly used when determining the influence of selected compounds on transcriptional activity of genes and its expression is determined by the bioluminescent reaction of the protein product. Expressed protein can be influenced by studied compounds, mostly by inhibition or stabilisation, which can lead to false-positive results. Sea pansy luciferase, which is commonly used in combination with firefly luciferase, is its important alternative. Isoflavonoids are one of the groups of natural compounds, often studied in reporter gene assays with firefly luciferase, so it is important to evaluate their influence on firefly luciferase. In our thesis we focused on 11 structurally diverse isoflavonoids and evaluated their influence on the activity of firefly and sea pansy luciferase. At first we evaluated potential interference of firefly luciferase in the form of inhibition with QSAR programme InterPred, which predicted high likelihood of inhibition for 7 out of 11 isoflavonoids. By using a new, method in vitro, based on luciferase assay of lysate of cell line AR-EcoScreen, we evaluated inhibition of both reporter proteins. Firstly, we tested compounds in 1, 10 and 100 µM concentration, which was followed by testing at additional concentrations, in the case of inhibition, for the determination of IC50. Seven out of 11 isoflavonoids inhibited firefly luciferase, which in comparison with the results of InterPred confirmed its correct prediction of interference. None of the isoflavonoids inhibited sea pansy luciferase in vitro. The most potent inhibitors were also tested in a modified in-vitro assay for their potential thermal stabilisation of firefly luciferase. We cannot confirm that obtained values are the consequence of stabilisation, due to shortcomings of the method. Firefly luciferase inhibition was further explored with molecular docking, which confirmed that active isoflavonoids bind to luciferin pocket, followed by determination of structure-activity relationship for the active isoflavonoids. With molecular docking we also confirmed, that the selected isoflavonoids do not inhibit sea pansy luciferase. We confirmed inhibitory activity of some isoflavonoids, which can due to stabilisation explain previously reported higher responses in cell lines with firefly luciferase. When studying the influence of isoflavonoids on transcriptional activity of selected genes it would be more appropriate to use sea pansy luciferase as reporter gene.

• Keywords: isoflavonoids, firefly luciferase, sea pansy luciferase