Development of protein kinase-responsive photoluminescent probes and cellular regulators of protein phosphorylation
Date
2016-07-07
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
Proteiinkinaasid (PK-d) on olulised rakkude elutegevust reguleerivad ensüümid. Katalüüsides valkude fosforüülimist on nad tähtsaks lüliks rakusisestes signaalülekande radades. Häired PK-de normaalses funktsioneerimises, mis on tingitud nende liigsest aktiivsusest, üleekspressioonist või geenimutatsioonidest, on seotud mitmete haigustega, nagu vähkkasvajad, diabeet ja südame-veresoonkonna haigused. Sellest tulenevalt on PK-dest saanud viimase 15 aasta jooksul olulised ravimiarenduse sihtmärgid ning üle 30 PK inhibiitori on jõudnud kasutusele vähiravimitena. Lisaks on intensiivselt toimunud erinevate analüüsimeetodite arendamine uute efektiivsete ravimikandidaatide võimalikult kiireks väljatöötamiseks, PK-de toimemehhanismide ja signaalradade uurimiseks ning PK-de kui oluliste biomarkerite tuvastamiseks.
Töös testiti uute ARC-ühendite võimet inhibeerida erinevaid PK-sid ning selgitati ARC-de nukleosiid-osa struktuuri mõju nende ainete inhibeerimispotentsiaalile erinevate PKde (PKAc, PKBγ and ROCK-II) suhtes. Leiti, et selle seeria ühenditest on just tiofeeni-sisaldavad ained suurima inhibeerimisvõimega. Edasistel biokeemilistel uuringutel avastati, et tiofeeni-sisaldavate ARC-de märgistamisel fluorestsentsvärvidega saadud fotoluminestsents-sondid omavad optilisi omadusi, mida pole varem orgaaniliste ainete puhul kirjeldatud. Selgus, et need ARC-Lum(Fluo)-sondid on PK-de aktiivsust registreerivad fotoluminestsents-sondid. Nende ühendite seostumisel PK-dega ning selle kompleksi ergastamisel kiirgusega lähis-UV alas emiteerivad nad spektri oranžis või punases piirkonnas aeglase sumbumisega (20–250 μs) valgust. Seega on need sondid kasutatavad PKde kontsentratsiooni määramiseks biokeemilistes ja bioloogilistes lahustes ning PK-de aktiivsuse seireks ja kaardistamiseks rakkudes.
Käesolevas töös uuriti ka uute ARC-inhibiitorite kasutusvõimalusi valkude rakusiseste fosforüülimistasakaalude reguleerimisel. Näidati, et ARC-de suutlikkus mõjutada PK-de aktiivsust rakutuumades sõltub nii ainete inhibiitoromadustest kui ka nende võimest läbida raku plasmamembraani. Töös näidati, et arendatud ARC-inhibiitorid on rakendatavad PK-de aktiivsuse reguleerimiseks rakkudes ning rakkude signaalradade uurimiseks.
Protein kinases (PKs) are important cellular signalling molecules. Aberrant activity of PKs can lead to several complex diseases, for instance, different forms of cancer, diabetes, and cardiovascular diseases. This relevance has made PKs into important therapeutic targets: more than 30 small-molecule PK inhibitors have reached the drug market during recent 15 years. In addition, PK inhibitors are useful tools for studying both physiological roles of PKs and molecular mechanisms behind human diseases. Therefore, considerable effort has been put into developing reliable biochemical and cell-based assays for screening of PK inhibitors and for monitoring cellular functioning of PKs as potential diagnostic and prognostic biomarkers. In this thesis the effect of the structure of nucleoside-mimicking moiety of ARCs on their activity towards basophilic PKs (PKAc, PKBγ, ROCK-II) was demonstrated. The thiophene-containing compounds had the highest activity, revealing inhibitory potency in the subnanomolar region. Further biochemical studies with fluorescently labelled thiophene-containing ARCs led to the discovery of novel protein binding-responsive photoluminescent probes [ARC-Lum(Fluo)] whose binding to PK induces orange or red luminescence with long (20–250 μs) decay after pulse-excitation at near-UV radiation. The novel probes are useful for determining PK concentration in biochemical and biological solutions, and for monitoring and mapping the activity of PKs in living cells. Moreover, for the first time, inhibitory potency of ARCs in the living cells was demonstrated, whereas the cellular activity of the compounds was influenced on both cell-penetration capability and their affinity. These results support the applicability of ARCs to clarify the roles of PKs in signalling pathways, which make ARCs powerful tools in this research.
Protein kinases (PKs) are important cellular signalling molecules. Aberrant activity of PKs can lead to several complex diseases, for instance, different forms of cancer, diabetes, and cardiovascular diseases. This relevance has made PKs into important therapeutic targets: more than 30 small-molecule PK inhibitors have reached the drug market during recent 15 years. In addition, PK inhibitors are useful tools for studying both physiological roles of PKs and molecular mechanisms behind human diseases. Therefore, considerable effort has been put into developing reliable biochemical and cell-based assays for screening of PK inhibitors and for monitoring cellular functioning of PKs as potential diagnostic and prognostic biomarkers. In this thesis the effect of the structure of nucleoside-mimicking moiety of ARCs on their activity towards basophilic PKs (PKAc, PKBγ, ROCK-II) was demonstrated. The thiophene-containing compounds had the highest activity, revealing inhibitory potency in the subnanomolar region. Further biochemical studies with fluorescently labelled thiophene-containing ARCs led to the discovery of novel protein binding-responsive photoluminescent probes [ARC-Lum(Fluo)] whose binding to PK induces orange or red luminescence with long (20–250 μs) decay after pulse-excitation at near-UV radiation. The novel probes are useful for determining PK concentration in biochemical and biological solutions, and for monitoring and mapping the activity of PKs in living cells. Moreover, for the first time, inhibitory potency of ARCs in the living cells was demonstrated, whereas the cellular activity of the compounds was influenced on both cell-penetration capability and their affinity. These results support the applicability of ARCs to clarify the roles of PKs in signalling pathways, which make ARCs powerful tools in this research.
Description
Väitekirja elektrooniline versioon ei sisalda publikatsioone.
Keywords
ensüümid, proteiinkinaasid, inhibiitorid, fotoluminestsents, sondid, enzymes, protein kinases, inhibitors, photoluminescence, sondes