Combining chemical and genetic approaches for photoluminescence assays of protein kinases
Date
2016-02-23
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Abstract
Doktoritöös kasutati geneetilisi ja keemilisi meetodeid sensorsüsteemide loomiseks proteiinkinaaside analüüsiks.
Proteiinkinaasid katalüüsivad valkude fosforüülimist, see on oluline reaktsioon valkude modifitseerimisel, mille kaudu reguleeritakse valkude aktiivsust rakkudes. Valkude fosforüülimine võimaldab suurendada proteoomi mitmekesisust, seeläbi mõjutatakse rakkude normaalset ja patoloogilist toimimist. Valkude fosforüülimistasakaalude nihkumine PKde häiritud aktiivsuse tõttu on paljude raskete haiguste (vähktõve erinevad vormid, diabeet jne) põhjuseks või märguandeks nende tekkimise kohta. Sellest tuleneval on proteiinkinaasid olnud 21. sajandi olulisteks ravimiarenduse sihtmärkideks. Viimase 15 aasta jooksul on 30 proteiinkinaaside inhibiitorit jõudnud vähiravimitena haiglatesse.
Lisaks olulisele rollile ravimiarenduses on proteiinkinaasid tähtsad biomarkerid vähktõve erinevate vormide ning teistele haiguste tuvastamisel. Sellest tuleneb ka suur nõudmine analüüsimeetodite järele, mis võimaldaksid määrata kinaaside kontsentratsiooni ja aktiivsust kliinilistes proovides, näiteks kehavedelikes ja biopsiaproovides.
Viimasel aastakümnel on mõistetud, et just kahe uurimisala, sünteetilise keemia ja valkude konstrueerimise geneetiliste meetodite kooskasutamine on vajalik uudsete omadustega molekulide ja nende komplekside konstrueerimiseks. Need struktuurid lisaksid uudseid võimalusi biomeditsiiniliste uuringute läbiviimiseks, ravimiarenduseks ning haiguste tuvastamiseks.
Käesolevas töös ühitati keemilised ja geneetilised meetodid selleks, et välja arendada uudsed sensorsüsteemid PKde ja nend inhibiitoride uurimiseks. Ühelt poolt toodeti imetajate rakkudes mitmete proteiinkinaaside (PKAc, CK2α, and PIM-kinaasid) liitvalke fluorestseeruvate valkudega. Teisalt konstrueeriti ARC-inhibiitoritest lähtuvalt uudsete luminestsentsomadustega kõrge afiinsusega sondid kinaasidede jaoks [ARC-Lum(Fluo)-sondid].
Seoses PIM-kinaaside (PIM1, PIM2 ja PIM3) olulise tähtsusega nii ravimarenduse sihtmärkidena kui ka vähktõve biomarkeritena, on abivahendite väljatöötamine nende kinaaside uurimiseks suure praktilise väärtusega. PIM2 liitvalke punase fluorestseeruva valguga (TagRFP) ekspresseeriti imetajate rakkudes kahel erineval kujul (TagRFP-PIM2 ja PIM2-TagRFP). Töös näidati, need erineva märgise paigutusega liitvalgud omavad kompleksis ARC-Lum(Fluo)-sondidega erinevaid luminestsentsomadusi, mis tuleneb erinevast Försteri-tüüpi energiaülekandest vastasmõjus olevate luminofooride vahel. Töös näidati esmakordselt, et kompleksis on võimalik energiaülekanne seleeni-sisaldava aromaatse süsteemi ergastatud tripletest olekust kinaasiga seotud fluorestseeruvale valgule, mille tulemusena fluorestsentsvärvilt kiiratav valgus on küll värvile omase kiirgusspektriga, kuid enam kui 10000 korda pikema elueaga.
See tulemus loob eelduse rakusiseste sensorsüsteemide loomiseks raku plasmamembraani läbivate ARC-Lum(Fluo)-sondide ja rakkudes ekspresseeritud proteiinkinaasi ja TagRFP liitvalkude baasil, mis võimaldaks kaardistada proteiinkinaaside aktiivsust elusrakkudes.
Phosphorylation reaction is an important post-translational protein modification procedure in cells, which is carried out by protein kinases (PKs). This modification leads to increase in the diversity of the proteome that influences various aspects of normal and pathological physiology. Dysregulation of protein phosphorylation balances, caused by the aberrant activity of PKs is a cause or consequence of several complex diseases such as cancers, inflammatory disorders, cardio-vascular diseases and diabetes. Therefore PKs have become important drug targets in the 21st century. In recent 15 years, 30 small-molecule PK inhibitors have been approved for use in clinical practice. More than 500 PKs, constituting almost 2.5 % proteins coded by the human genome, are carrying out the phosphorylation of various proteins. In addition to being a potential drug targets, PKs also serve as biomarkers for cancers and other diseases, as altered expression level of various PKs is observed in a variety of malignancies. Thus there is high demand for analytical methods that enable determination of expression and activity levels of specific PKs in clinical samples, such as bodily fluids and cancerous tissues. Thus high throughput assays for screening PK inhibitors also form an important component of drug development pipeline. During the last decade, it has been understood that merging of two disciplines, synthetic chemistry and protein engineering, is needed to construct molecules and their complexes with new functionalities that can create novel opportunities for biomedical research, drug development and disease diagnostics. In this thesis combination of chemical and genetic approaches was used for the development of analytical tools for the characterization of PKs and PK inhibitors. PKs (PKAc, CK2α, and PIM kinases) were fused with fluorescent proteins (FPs). On the other hand, ARC-based small-molecule PK inhibitors were developed into protein binding-responsive ARC-Lum probes possessing unique photoluminescent properties. Joint application of PKs fused with fluorescent proteins and ARC-Lum probes enabled the construction of sensor systems that can be used for specific and sensitive determination of PKs in biological samples and as research tools for mapping and monitoring PK activity in living cells.
Phosphorylation reaction is an important post-translational protein modification procedure in cells, which is carried out by protein kinases (PKs). This modification leads to increase in the diversity of the proteome that influences various aspects of normal and pathological physiology. Dysregulation of protein phosphorylation balances, caused by the aberrant activity of PKs is a cause or consequence of several complex diseases such as cancers, inflammatory disorders, cardio-vascular diseases and diabetes. Therefore PKs have become important drug targets in the 21st century. In recent 15 years, 30 small-molecule PK inhibitors have been approved for use in clinical practice. More than 500 PKs, constituting almost 2.5 % proteins coded by the human genome, are carrying out the phosphorylation of various proteins. In addition to being a potential drug targets, PKs also serve as biomarkers for cancers and other diseases, as altered expression level of various PKs is observed in a variety of malignancies. Thus there is high demand for analytical methods that enable determination of expression and activity levels of specific PKs in clinical samples, such as bodily fluids and cancerous tissues. Thus high throughput assays for screening PK inhibitors also form an important component of drug development pipeline. During the last decade, it has been understood that merging of two disciplines, synthetic chemistry and protein engineering, is needed to construct molecules and their complexes with new functionalities that can create novel opportunities for biomedical research, drug development and disease diagnostics. In this thesis combination of chemical and genetic approaches was used for the development of analytical tools for the characterization of PKs and PK inhibitors. PKs (PKAc, CK2α, and PIM kinases) were fused with fluorescent proteins (FPs). On the other hand, ARC-based small-molecule PK inhibitors were developed into protein binding-responsive ARC-Lum probes possessing unique photoluminescent properties. Joint application of PKs fused with fluorescent proteins and ARC-Lum probes enabled the construction of sensor systems that can be used for specific and sensitive determination of PKs in biological samples and as research tools for mapping and monitoring PK activity in living cells.
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Keywords
proteiinkinaasid, sensorid, luminestsentsanalüüs, fotoluminestsents, protein kinases, sensors, luminescence analysis, photoluminescence