Development of fluorescence-based kinetic and binding assays for characterization of protein kinases and their inhibitors
Date
2010-07-29
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
Proteiinkinaaside (PK-de) poolt katalüüsitav valkude fosforüleerimine on üks tähtsamaid valkude aktiivsuse reguleerimise mehhanisme, mis võimaldab rakkudel reageerida väliskeskkonna muutustele ning suunata elutegevuse tagamiseks olulisi rakusiseseid protsesse. PK-de funktsioneerimishäiretega (eelkõige nende üleekspressiooni ning anomaalselt kõrge aktiivsususega) on seotud mitmed haigused, sealhulgas vähkkasvajad, suhkruhaigus ja Alzheimeri tõbi. Seetõttu on PK-de uurimine ja PK-de aktiivsust blokeerivate inhibiitorite arendamine muutunud oluliseks nii teadusasutustes kui ka ravimitööstustes. Käesoleva töö raames töötati välja fluorestentsi mõõtmisel põhinevad meetodid kinaaside aktiivsuse määramiseks ning kinaaside inhibiitorite iseloomustamiseks, mille rakendatavust demonstreeriti nii kinaase sisaldavates lahustes kui ka elusrakkudes. Esimene arendatud meetod põhineb fluorestsentsmärgistatud cAMP-sõltuva proteiinkinaasi (PKA) substraadi (5-TAMRA-kemptiid) lahutamisel fosforüleeritud produktist planaarkromatograafiliselt ning fosforüleerimisreaktsiooni komponentide kvantifitseerimisel fluorestsentsskänneriga. Antud meetodit rakendati edukalt inhibiitorite, sealhulgas ARC-tüüpi inhibiitorite, iseloomustamiseks. Teine meetod on fluorestentsanisotroopia mõõtmisel põhinev sidumismeetod, mis kasutab fluorestsentsvärviga märgistatud adenosiini analoogi ja arginiini-rikka peptiidi konjugaate, ARC-Photo sonde. Uudseid fluorestsentssonde kasutati kinaaside PKAc ja ROCKII inhibiitorite tuvastamiseks ning sidumisomaduste määramiseks. Tõestati ARC-Photo sondide bisubstraatne iseloom ning nende sobivus kinaaside aktiivsuse määramiseks ning rakendatavus sensorina cAMP kontsentratsiooni mõõtmisel. Järgmiseks sammuks oli sidumismeetodi ülekandmine elusrakkudesse, kuna just elusraku keskkond võimaldab adekvaatselt hinnata inhibiitorite omadusi ning proteiinkinaaside inhibeerimisest tingitud füsioloogilisi efekte. Näidati, et ARC-Photo sondid on võimelised läbima raku plasmamembraani. Sealjuures sõltus sondide võime rakku tungida ja nende lokalisatsioon rakus arginiinide arvust ARC-i peptiidses osas, ühendi kontsentratsioonist ning fluorestsentsvärvi keemilisest loomusest. ARC-Photo sondide struktuuride optimeerimine võimaldas välja töötada Försteri resonantsienergia ülekande (FRET) efektiivsuse mõõtmisel põhineva meetodi PKA aktiivsuse jälgimiseks elusrakkudes.
Protein kinases (PKs) play a key role in the regulation of protein functions in living cells. Many heavy human diseases (including cancer, diabetes, and Alzheimer’s disease) have been linked to the aberrant PK signalling, which has made PKs important drug targets. This has caused an increasing need for the elaboration and improvement of analytical methods for high-throughput screening and characterization of new compounds. This thesis describes the progress with the development of fluorescence-based assays for protein kinase research, starting with a biochemical kinetic method and getting at a binding assay applicable for monitoring of protein kinase activity in living cells. A kinetic assay based on the chromatographic separation of the fluorescently labelled substrate peptide TAMRA-Kemptide from its phosphorylated counterpart and quantification of the phosphorylation extent ratiometrically with a fluorescence imager was worked out. The assay was successfully applied for the characterization of the inhibitory properties of inhibitors of cAMP-dependent protein kinase (PKA). The fluorescent probe ARC-583 was developed and its bisubstrate character demonstrated. The probe was applied for the characterization of both ATP- and protein/peptide substrate-competitive inhibitors of PKs in an assay with fluorescence polarization readout. High affinity of ARC-583 (KD = 0.48 nM towards PKA) enabled its application for the characterization of inhibitors with nanomolar and micromolar potency. The effect of structural elements of ARC-Photo probes on the cellular internalization efficiency and intracellular localization was studied. It was demonstrated that the cellular uptake and localization of the labelled inhibitors were influenced by several factors such as the number of arginine residues in the peptide moiety, the concentration of extracellularly applied compounds and the origin of the fluorescent dye of the ARC-Photo probe. The ability ARC-Photo probes to bind with high affinity to the free catalytic subunit of PKA was used to construct a cellular sensor for monitoring PKA activity in living cells.
Protein kinases (PKs) play a key role in the regulation of protein functions in living cells. Many heavy human diseases (including cancer, diabetes, and Alzheimer’s disease) have been linked to the aberrant PK signalling, which has made PKs important drug targets. This has caused an increasing need for the elaboration and improvement of analytical methods for high-throughput screening and characterization of new compounds. This thesis describes the progress with the development of fluorescence-based assays for protein kinase research, starting with a biochemical kinetic method and getting at a binding assay applicable for monitoring of protein kinase activity in living cells. A kinetic assay based on the chromatographic separation of the fluorescently labelled substrate peptide TAMRA-Kemptide from its phosphorylated counterpart and quantification of the phosphorylation extent ratiometrically with a fluorescence imager was worked out. The assay was successfully applied for the characterization of the inhibitory properties of inhibitors of cAMP-dependent protein kinase (PKA). The fluorescent probe ARC-583 was developed and its bisubstrate character demonstrated. The probe was applied for the characterization of both ATP- and protein/peptide substrate-competitive inhibitors of PKs in an assay with fluorescence polarization readout. High affinity of ARC-583 (KD = 0.48 nM towards PKA) enabled its application for the characterization of inhibitors with nanomolar and micromolar potency. The effect of structural elements of ARC-Photo probes on the cellular internalization efficiency and intracellular localization was studied. It was demonstrated that the cellular uptake and localization of the labelled inhibitors were influenced by several factors such as the number of arginine residues in the peptide moiety, the concentration of extracellularly applied compounds and the origin of the fluorescent dye of the ARC-Photo probe. The ability ARC-Photo probes to bind with high affinity to the free catalytic subunit of PKA was used to construct a cellular sensor for monitoring PKA activity in living cells.
Description
Väitekirja elektrooniline versioon ei sisalda publikatsioone.
Keywords
doktoritööd, proteiinkinaasid, fluorestsentsanalüüs