Bisubstrate luminescent probes, optical sensors and affinity adsorbents for measurement of active protein kinases in biological samples
Date
2012-02-15
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Abstract
Proteiinkinaasid osalevad paljudes rakusisestes signaaliülekanderadades. Kõrvalekalded proteiinkinaaside aktiivsuse normaalsest tasemest võivad viia mitmete raskete haiguste tekkeni. Teadmine, et protiinkinaaside aktiivsust on võimalik reguleerida madalmolekulaarsete ühendite abil, on muutnud proteiinkinaasid oluliseks ravimiarenduse sihtmärgiks, eriti vähiravimite väljatöötamisel. Haiguste tekkel oluliste kinaaside identifitseerimisel on vajalikud analüüsimeetodid, mis võimaldavad tuvastada proteiinkiniaaside aktiivsuse muutust patsientide koeproovides. Teisalt on kinaaside kiired ja odavad analüüsimeetodid vajalikud haiguste diagnoosimiseks nende varajases staadiumis.
Käesoleva doktoritöö eesmärgiks oli meetodite arendamine aktiivsete proteiinkinaaside hulga määramiseks koeproovides. ARC-tüüpi bisubstraatsete inhibiitorite immobiliseerimisel saadi afiinsusadsorbendid ja sensorpinnad, mis võimaldasid proteiinkinaaside kontsentreerimist bioloogilistest proovidest ning nende kinaaside iseloomustamist pinna plasmonresonantsspektoskoopiat kasutades. Järgnevalt töötati välja üheetapiline meetod proteiinkinaaside määramiseks otse koeproovides. Töö käigus avastati, et mõnede ARC-tüüpi ühendite (ARC-Lum) kompleksid proteiinkinaasidega omavad varemkirjeldamata optilisi omadusi. Nende komplekside ergastamisel kiirgusega lähis-UV alas emiteerivad nad punases spektriosas valgust, mille sumbumise poolestusaeg on vahemikus 14-170 mikrosekundit, 10000 korda pikem kui tavalistel fluorestsentsvärvidel. Töötati välja ülitundlik ARC-Lum-sondide kasutamisel põhinev meetod vähi biomarkeri, rakuvälise PKAc (ECPKA), määramiseks inimese vereplasmas. Väljatöötatud meetod on üheetapiline, automatiseeritav, odav, teostatav levinud aparatuuri kasutades ning võimaldab mõõta pikomolaarseid ECPKA kontsentratsioone eeltöötlemata plasmaproovides.
Protein kinases form a family of proteins that play a key role in signal transduction pathways inside the cells. The dysfunctioning of protein kinases may result in the progression of a disease. This and the fact that protein kinase activity can be regulated by small-molecule compounds have made protein kinases one of the most important protein targets for drug discovery of the past decade. Therefore the development of assays for protein kinase research and disease diagnostics is of great importance. Moreover, methods enabling the division of patients into groups based on proteomic information related to the special person support the understanding of the exact mechanism of the disease and enable the personalized tailoring of the therapy according to this information leading to better outcome of the treatment. The present thesis describes the development of bioanalytical methods for monitoring the activity of protein kinases in biological samples. High-affinity adsorbents were worked out and used for the enrichment of protein kinases. Novel non-metal long lifetime luminescence probes were discovered that were used for full thermodynamic and kinetic characterization of un-labelled inhibitors of protein kinases in biochemical assays and for monitoring kinase activity in live cells. A novel homogeneous assay based on the measurement of time-gated luminescence was used for the quantification of a putative cancer biomarker ECPKA in crude plasma samples.
Protein kinases form a family of proteins that play a key role in signal transduction pathways inside the cells. The dysfunctioning of protein kinases may result in the progression of a disease. This and the fact that protein kinase activity can be regulated by small-molecule compounds have made protein kinases one of the most important protein targets for drug discovery of the past decade. Therefore the development of assays for protein kinase research and disease diagnostics is of great importance. Moreover, methods enabling the division of patients into groups based on proteomic information related to the special person support the understanding of the exact mechanism of the disease and enable the personalized tailoring of the therapy according to this information leading to better outcome of the treatment. The present thesis describes the development of bioanalytical methods for monitoring the activity of protein kinases in biological samples. High-affinity adsorbents were worked out and used for the enrichment of protein kinases. Novel non-metal long lifetime luminescence probes were discovered that were used for full thermodynamic and kinetic characterization of un-labelled inhibitors of protein kinases in biochemical assays and for monitoring kinase activity in live cells. A novel homogeneous assay based on the measurement of time-gated luminescence was used for the quantification of a putative cancer biomarker ECPKA in crude plasma samples.
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Keywords
proteiinkinaasid, ensüümi aktiivsus, määramismeetodid, vereplasma, protein kinases, enzyme activity, fixed methods, blood plasma