Bifunctional inhibitors and photoluminescent probes for studies on protein complexes
Failid
Kuupäev
2017-07-06
Autorid
Ajakirja pealkiri
Ajakirja ISSN
Köite pealkiri
Kirjastaja
Abstrakt
Inimraku elutegevus on reguleeritud keeruliste protsesside kaudu. Ühed kõige olulisemad informatsiooni edasikandvad muutused on valkudevahelised interaktsioonid ja valkude fosforüülimine. Valkude fosforüülimist teostavad proteiinkinaasid, mis katalüüsivad fosforüülrühma ülekannet nukleotiidilt (milleks on enamasti ATP) sihtvalgule. Kõrvalekaldeid proteiinkinaaside normaalsest aktiivsuses on seostatud komplekssete ja sageli raskesti ravitavate haigustega, näiteks erinevad neurodegeneratiivsed haigused (Parkinson, Alzheimer), südame- ja veresoonkonnahaigused, diabeet ning vähkkasvajad. Seetõttu on ravimitööstuse kõrgendatud tähelepanu suunatud proteiinkinaaside aktiivsuse reguleerimisele inhibiitoritega. Tänaseks päevaks on kasutusloa saanud enam kui 30 proteiinkinaaside inhibiitorit, mis on märkimisväärselt aidanud parandada vähktõbe põdevate inimesete elukvaliteeti ja elulemust.
Käesoleva töö raames töötati välja ja iseloomustati mitmekülgseid ja tundlikke meetodeid, mis võimaldasid iseloomustada mitmete proteiinkinaaside aktiivsust biokeemilistes katsetes, inhibiitorite sidumise tugevust proteiinkinaasidele, proteiinkinaas:inhibiitor kompleksi lagunemise kiirust ja jälgida reaalajas proteiinkinaasi aktiivsust imetajarakkudes. Eriliseks teeb meetodid nendes rakendatud orgaaniline sond ARC-Lum, millel on unikaalsed optilised omadused. Esiteks, proovi ergastamisel kiirgub valgust vaid juhul, kui ARC-Lum sond on kompleksis proteiinkinaasiga. Teiseks, kiirgunud valgusel on pikk eluiga, mis võimaldab vähendada mittespetsiifilise signaali mõjusid. ARC-Lum sondi rakendamisel arendati uudse struktuuriga bifunktsionaalsed ARC-tüüpi inhibiitorid, mis saavutasid senikirjeldatud inhibiitoritest kõrgeima afiinsuse proteiinkinaasi PKAc suhtes (Kd < 10 pM). Võrreldes ravimina kasutatavate inhibiitoritega inhibeerivad uued ained proteiinkinaase oluliselt (kuni tuhat korda) madalamatel kontsentratsioonidel. Arendatud inhibiitorite võimekust demonstreeriti väga tugeva valkudevahelise interaktsiooni lõhkumisel ja pakuti välja uudne lahendus sihtida tugevaid valkudevahelisi interaktsioone bifunktsionaalsete inhibiitoritega
A living cell is highly complex and dynamic system, regulated by various signaling pathways. Interactions between proteins and phosphorylation of proteins are two of the most important changes in cells, carrying important information. The phosphorylation of proteins is aided by protein kinases, which catalyze the transfer of phosphoryl group from nucleotide (usually ATP) to target protein. Abnormal activity of protein kinases has been linked to complex and refractory diseases, for example different neurodegenerative diseases (Parkinson’s, Alzheimer’s), cardiovascular diseases, diabetes, and cancer. Therefore, pharmaceutical industry has directed considerable effort into regulating the activity of protein kinases with inhibitors. Currently 30 protein kinase inhibitors have been approved for therapeutics against various forms of cancer, immensely improving the life expectancy and quality of living of patients. In the current thesis, versatile and sensitive methods were developed and characterized for studying the activity of protein kinases in biochemical assays, determining interaction strength of inhibitors towards protein kinases, measuring dissociation kinetics of a complex between protein kinase and inhibitor, and monitoring cellular activity of protein kinases in real time. The novelty of these methods is based on organic ARC-Lum probes with unique optical properties. First, the specific emission of light is present only if ARC-Lum is in complex with protein kinase. Second, emitted light has a characteristic long lifetime, which allows to reduce the influence from non-specific signals. These properties of ARC-Lum probes supported the development of novel bifunctional ARC-type inhibitors with the highest affinity towards protein kinase PKAc to date (Kd < 10 pM). Compared to inhibitors in drug industry, these new compounds inhibit protein kinases at significantly (up to 1000-fold) lower concentrations. These potent inhibitors performed exceedingly well for disruption of a very strong protein- protein interaction (PPI) and led to a proposal of targeting strong PPIs by using bifunctional inhibitors.
A living cell is highly complex and dynamic system, regulated by various signaling pathways. Interactions between proteins and phosphorylation of proteins are two of the most important changes in cells, carrying important information. The phosphorylation of proteins is aided by protein kinases, which catalyze the transfer of phosphoryl group from nucleotide (usually ATP) to target protein. Abnormal activity of protein kinases has been linked to complex and refractory diseases, for example different neurodegenerative diseases (Parkinson’s, Alzheimer’s), cardiovascular diseases, diabetes, and cancer. Therefore, pharmaceutical industry has directed considerable effort into regulating the activity of protein kinases with inhibitors. Currently 30 protein kinase inhibitors have been approved for therapeutics against various forms of cancer, immensely improving the life expectancy and quality of living of patients. In the current thesis, versatile and sensitive methods were developed and characterized for studying the activity of protein kinases in biochemical assays, determining interaction strength of inhibitors towards protein kinases, measuring dissociation kinetics of a complex between protein kinase and inhibitor, and monitoring cellular activity of protein kinases in real time. The novelty of these methods is based on organic ARC-Lum probes with unique optical properties. First, the specific emission of light is present only if ARC-Lum is in complex with protein kinase. Second, emitted light has a characteristic long lifetime, which allows to reduce the influence from non-specific signals. These properties of ARC-Lum probes supported the development of novel bifunctional ARC-type inhibitors with the highest affinity towards protein kinase PKAc to date (Kd < 10 pM). Compared to inhibitors in drug industry, these new compounds inhibit protein kinases at significantly (up to 1000-fold) lower concentrations. These potent inhibitors performed exceedingly well for disruption of a very strong protein- protein interaction (PPI) and led to a proposal of targeting strong PPIs by using bifunctional inhibitors.
Kirjeldus
Väitekirja elektrooniline versioon ei sisalda publikatsioone
Märksõnad
proteiinkinaasid, inhibiitorid, biokeemilised protsessid, protein kinases, inhibitors, biochemical processes