ARC-inhibitors: from reliable biochemical assays to regulators of physiology of cells

Date

2018-05-11

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Abstract

Inimrakk on keeruline süsteem, mis koosneb paljudest molekulidest ning rangelt reguleeritud molekulidevahelisest kommunikatsioonist. Proteiinkinaasid on valgud, mis rakus olulisi protsesse kontrollivad. Häireid proteiinkinaaside töös on seostatud nt vähkkasvajate ja südameveresoonkonna haigustega. Üheks võimaluseks nende haigustega võidelda on piirata proteiinkinaaside liigset aktiivsust molekulidega, mida nimetatakse inhibiitoriteks. Selleks, et teha kindlaks, milline inhibiitor sobib proteiinkinaasi aktiivsuse reguleerimiseks rakkudes, tuleb inhibiitorite omadusi võrrelda. Antud doktoritöös optimeeriti kaks meetodit. Esimene võimaldab mõõta proteiinkinaasi ja inhibiitori omavahelist sobivust rakuvabas keskkonnas (katseklaasis). Arendatud meetod on võrreldes varasemate käsitlusviisidega märgatavalt tundlikum. Selleks, et rakku sisenenud inhibiitori koguse kohta aimu saada, töötati doktoritöö käigus välja kvantitatiivne meetod. Teades, kui palju inhibiitorimolekule on rakkudesse sisenenud, oli võimalik ennustada, kui võimekas inhibiitor rakkudes on. Järgmisena arendati kahele olulisele proteiinkinaasile inhibiitorid, mille omadusi hinnati muuhulgas ka rakendades eelmainitud meetodeid. Näidati, et arendatud inhibiitorid sisenesid edukalt rakkudesse, seostusid tugevasti sihtmärk-kinaasile ning mõjutasid nende aktiivsust. Võrreldes kommertsiaalselt saadavate inhibiitorite ning kliinilistes katsetustes olevate ühenditega, näitasid uued inhibiitorid rakkudes märgatavalt paremat tõhusust. Proteiinkinaaside aktiivsuse mõjutamine inhibiitoriga võimaldas selektiivselt tappa vähirakud jättes samadel tingimustel normaalsed rakud elujõuliseks. Katsed vereproovidega näitasid, et inhibiitorid aitasid efektiivselt vältida vereproovides agregaatide ehk trombi algete tekkimist. Doktoritöö tulemused toetavad uute meetodite kasutamist inhibiitorite analüüsimiseks ning uut tüüpi inhibiitorite edasi arendamist ravimiarenduses ning proteiinkinaaside rollide uurimises.
Human cell is a complex system comprising variety of molecules, each playing its own role in the life cycle of a cell. Members of the protein kinase super-family play an important role in regulation of vital processes in cells. Aberrant activity of protein kinases is related to genesis and progression of several complex diseases, for example cancer and cardiovascular diseases. One possibility to fight these diseases is to suppress excessive activity of protein kinases with molecules called inhibitors. In order to define which inhibitors are most efficient for regulating activity of protein kinases in cells, one should determine and compare the properties of the inhibitors. In the present thesis two methods were for developed. First assay enables the evaluation of the compatibility of inhibitors and protein kinases. The method was significantly more sensitive than other previously known methods. The second method enables the determination of uptake efficiency of the compound by the cell. Knowing how many molecules of inhibitor has entered the cell aids the prediction of the effectiveness of the inhibitor in the cellular milieu. Next, new inhibitors (called ARCs) for two important protein kinases were developed. The aforementioned methods were applied for analysing the properties of these inhibitors. The developed inhibitors were capable of penetrating the cell plasma membrane, binding to the target kinase, and affecting its activity in cells. The inhibitors revealed significantly better effect in cells compared to a commercially available inhibitor and a compound currently under clinical trials. Regulating the activity of protein kinases resulted in selective killing of cancer cells while the normal cells were unaffected in the same conditions. Experiments with blood samples showed that treatment with new inhibitors avoided the formation of aggregates (pristine blood clots). The results of this doctoral thesis introduce new methods for analysing protein kinase inhibitors, and justify further development of ARC-inhibitors with the aim of their implementation for disease treatment and diagnosis.

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Keywords

protein kinases, inhibitors, ligands, enzyme activity, substrate, afinity, drug design

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