Development of bisubstrate inhibitors for protein kinase CK2
Date
2019-02-13
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Abstract
Proteiinkinaasid (PK) on ensüümide perekond, millesse kuulub üle 530 erineva kinaasi. Nende ensüümide ülesandeks on katalüüsida reaktsiooni, kus ATP-lt kantakse fosforüülrühm üle sihtvalgule, mille tõttu antud valgu aktiivsus muutub.
Üheks PK-de perekonna liikmeks on CK2, mis on rakus olulise tähtsusega PK kuna vastutab hinnanguliselt vähemalt 10% kõigis rakus toimuvate fosforüleerimiste eest. CK2 kõrgenenud aktiivust seostatakse mitmete haigustega, sealhulgas erinevat tüüpi vähktõbedega. Aastate jooksul on CK2-le arendatud mitmeid ATP-konkurentseid inhibiitoreid, mis seonduvad ATP sidumistaskuga ning pärsivad ensüümi tavapärast tööd. Kuna suurem osa kinaase kasutavad fosforüülrühma allikana ATP pole seda tüüpi inhibiitorid tihti väga selektiivset ühe kinaasi suhtes.
Uurimustöö käigus arendati välja bisubstraatseid inhibiitorid CK2-le, mis seostuvad korraga nii ATP sidumistaskuga kui ka valksubstraadi sidumistaskuga. Sellist tüüpi inhibiitorid koosnevad ATP-konkurentsest ja valksubstraati mimikeerivast fragmendist ning neid osi ühendavast linkerist. Inhibiitorite arendusel kasutati nelja erinevat ATP-konkurentset osa: varasemalt kirjeldatud CK2 inhibiitoreid TBBi ja CX-4945 ning pika luminestsents elueaga 4-(2-amino-1,3-tiasool-5-üül)bensoe hapet (ATB) ning tema seleeni sisaldavat analoogi. Lisaks neile variatsioonidele optimeeriti inhibiitorite arenduse käigus ka valksubstraadi mimeetiku pikkust ning neid kaht osa ühendava linkeri koostist ja pikkust. Osasid inhibiitoreid konjugeeriti fluorestsents värvidega. Uurimustöö tulemusena sünteesiti uudseid CK2 inhibiitoreid, mis omasid kõrget afiinsust ja head selektiivsust. Nende inhibiitoritega on võimalik määrata CK2 aktiivsust ning karakteriseerida uusi potentsiaalseid CK2 inhibiitoreid. Pika elueaga fotoluminestsentsiga ühendite rakendamine bioloogilistes süsteemides võimaldab kasutada aegviivitusega mõõtmisemeetodeid, mis parandab signaal-müra suhet ning tagab parema mõõtmismeetodite tundlikkuse.
Protein kinases (PKs) are a protein superfamily having more than 530 enzymes. Protein kinases are transferases that catalyze the transfer of the terminal phosphoryl group from nucleoside triphosphates to target substrate proteins/peptides, which thereafter change of activaty. Since protein kinases have important rolls in cell regulation, it has been found that misregulation of the signal transduction networks of kinases has shown to be related with different diseases and that is why many of protein kinases are target molecules of novel drugs and for compounds in current drug development. By now there are 48 FDA (Food and Drug Administration) approved protein kinase inhibitors used in treatment of different diseases. One of the members of protein kinase family is CK2, which is responsible of about 10% of all protein phosphorylations in cells. Because of the overexpression and elevated activity of CK2 and unbalanced expression of its subunits, CK2 is reported to be related to number of different diseases but is mostly associated with different types of cancers. Throughout the years many ATP-competitive inhibitors have developed for CK2 that bind to the ATP binding site and restrain the normal functioning of the enzyme. Since most of the protein kinases use ATP as the source of the phosphoryl group, this type of inhibitors have problem with substrate selectivity. During this work, bisubstrate inhibitors were developed for the protein kinase CK2. Bisubstrate inhibitors bind simultaneously to both active sites (ATP and protein substrate binding sites) of the enzyme. Bisubstrate inhibitors consist of an ATP-competitive moiety, a protein substrate mimic and a linker. Turing the development of the inhibitors, four different ATP-competitive fragments were used: previously known CK2 inhibitors TBBi and CX-4945 and 4-(2-amino-1,3-thiazol-5-yl)benzoic acid (ATB) and its selenium-containing counterpart, which both possessed long lifetime luminescence properties. In addition to these variations, also the length of protein substrate mimetic and the composition and length of the linker were optimized. Some of the inhibitors were also conjugated with fluorescence dyes. As a result of this research, novel CK2 inhibitors with higher binding affinity and also better selectivity toward the target enzyme were synthesized. These inhibitors allow to measure the activity of the CK2 and characterize novel, potent inhibitors of CK2. Also, compounds possessing long lifetime luminescence properties have advantages in biological samples, where the time gated measurements eliminate the autofluorescence background of biological components and leads to higher signal sensitivity.
Protein kinases (PKs) are a protein superfamily having more than 530 enzymes. Protein kinases are transferases that catalyze the transfer of the terminal phosphoryl group from nucleoside triphosphates to target substrate proteins/peptides, which thereafter change of activaty. Since protein kinases have important rolls in cell regulation, it has been found that misregulation of the signal transduction networks of kinases has shown to be related with different diseases and that is why many of protein kinases are target molecules of novel drugs and for compounds in current drug development. By now there are 48 FDA (Food and Drug Administration) approved protein kinase inhibitors used in treatment of different diseases. One of the members of protein kinase family is CK2, which is responsible of about 10% of all protein phosphorylations in cells. Because of the overexpression and elevated activity of CK2 and unbalanced expression of its subunits, CK2 is reported to be related to number of different diseases but is mostly associated with different types of cancers. Throughout the years many ATP-competitive inhibitors have developed for CK2 that bind to the ATP binding site and restrain the normal functioning of the enzyme. Since most of the protein kinases use ATP as the source of the phosphoryl group, this type of inhibitors have problem with substrate selectivity. During this work, bisubstrate inhibitors were developed for the protein kinase CK2. Bisubstrate inhibitors bind simultaneously to both active sites (ATP and protein substrate binding sites) of the enzyme. Bisubstrate inhibitors consist of an ATP-competitive moiety, a protein substrate mimic and a linker. Turing the development of the inhibitors, four different ATP-competitive fragments were used: previously known CK2 inhibitors TBBi and CX-4945 and 4-(2-amino-1,3-thiazol-5-yl)benzoic acid (ATB) and its selenium-containing counterpart, which both possessed long lifetime luminescence properties. In addition to these variations, also the length of protein substrate mimetic and the composition and length of the linker were optimized. Some of the inhibitors were also conjugated with fluorescence dyes. As a result of this research, novel CK2 inhibitors with higher binding affinity and also better selectivity toward the target enzyme were synthesized. These inhibitors allow to measure the activity of the CK2 and characterize novel, potent inhibitors of CK2. Also, compounds possessing long lifetime luminescence properties have advantages in biological samples, where the time gated measurements eliminate the autofluorescence background of biological components and leads to higher signal sensitivity.
Description
Väitekirja elektrooniline versioon ei sisalda publikatsioone
Keywords
enzymes, protein kinases, inhibitors