Inhibitors and photoluminescent probes for in vitro studies on protein kinases PKA and PIM
Kuupäev
2021-11-03
Autorid
Ajakirja pealkiri
Ajakirja ISSN
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Kirjastaja
Abstrakt
Proteiinkinaasid (PK-d) katalüüsivad valkude fosforüülimist. PK-de ebanormaalne aktiivsus rakkudes on korrelatsioonis keeruliste haigustega. Seetõttu teeb farmaatsiatööstus märkimisväärseid jõupingutusi, et reguleerida PK-de aktiivsust inhibiitoritega ja jälgida nende aktiivsust luminestsents-sondidega.
Käesolevas töös kasutatud ARC-inhibiitorid on keemiliselt struktuurilt adenosiini matkivate heteroaromaatsete fragmentide ja peptiidide analoogide konjugaadid, neid ühendeid on pikemalt uuritud Tartu Ülikooli keemia instituudis. Käesolevas uuringus näidati, et PK PKA katalüütilise alaühiku α-isovormi (PKAcα) monoklonaalse antikeha (kloon D38C6) seondumine sihtvalguga on konkurentne ARC-Lum(Fluo) sondiga ja see antikeha inhibeerib substraadi fosforüülimist. Proovi järjestikust töötlemist nende konkureerivate PKAcα ligandidega kasutati tundliku AbARC immuunanalüüsi-meetodi väljatöötamiseks, mis võimaldas määrata väikseid koguseid (alates 93 pg) PKAcα rakulüsaatides. Hiljuti avastati Cushingi sündroomiga patsiendil S54L mutatsiooniga PRKACB geen. See mutatsioon viib PK glütsiinirikka aasa struktuuri muutumiseni. Käesolevas uuringus konstrueeriti muteerunud PK suhtes kuuekordse selektiivsusega inhibiitor. Lisaks töötati välja luminestsents-meetod PKAcβ-valgu kaubanduslike ja väljatöötatud inhibiitorite afiinsuse määramiseks.
Koostöös Oxfordi ülikooliga viidi läbi ARC-inhibiitorite ja proteiinkinaasi PIM-1 komplekside röntgenstruktuuranalüüs. Saadud struktuurimudelitest lähtuvalt konstrueeriti lihtsustatud keemilise ehitusega ained. Uued inhibiitorid derivatiseeriti biotiiniga või fluorestsentsvärviga Cy5 ja neid aineid kasutati PIM-kinaasidetuvastamiseks biokeemilistes lahustes ja bioloogilistes proovides. Analüüsimeetod, milles kasutati ARC-sonde koos PIM-2-selektiivse antikehaga , võimaldas määrata sihtvalgu väikseid koguseid (alates 44 pg PIM-2). Konfokaalmikroskoopia abil tuvastati, et uued fluorestsents-sondid tungivad kiiresti U2OS-rakkudesse, kus nende paiknemine kattub PIM-1 ja fluorestsentsvalgu konjugaadi paiknemisega.
Protein kinases (PKs) catalyze the phosphorylation of proteins. Abnormal activity of PKs in cells is correlated to complex diseases. Therefore, the pharma industry is making significant efforts to regulate the activity of PKs with inhibitors and to monitor the activity of PKs with luminescent probes. ARC inhibitors are conjugates of adenosine analogues and peptide mimetic moieties; they have been studied at length at the Institute of Chemistry of the University of Tartu. In the present study, it was shown that the binding of monoclonal antibody (clone D38C6) to α-isoform of the catalytic subunit of PKA (PKAcα) was competitive with binding of ARC-Lum(Fluo) probes. Sequential treatment of a sample with these competing PKAcα ligands was used to develop a sensitive AbARC immunoassay that allowed the determination of small amounts (from 93 pg) of PKAcα in cell lysates. Recently, PRKACB gene with the S54L mutation was discovered in a patient with the Cushing's syndrome. This mutation leads to a change in the structure of the glycine-rich loop of the PK. In the present study, an inhibitor with six-fold selectivity for the mutated PK was developed. In addition, a luminescence method was worked out for determination of affinity of both commercial and developed inhibitors of the PKAcβ protein. X-ray structure analysis of complexes of ARC inhibitors and PK PIM-1 was performed in collaboration with the University of Oxford. Based on the obtained structural models, compounds with simplified chemical structures were constructed. New inhibitors were derivatized with biotin or fluorescent dye Cy5 and applied for the detection of PIM PKs in biochemical solutions and complex biological samples. The sandwich assay utilizing a PIM-2-selective detection antibody featured a low limit of quantification (44 pg of PIM-2). A confocal microscopy study showed that the fluorescent probes were efficiently taken up by U2OS cells and the probes revealed high extent of co-localization with PIM-1-fused fluorescent proteins.
Protein kinases (PKs) catalyze the phosphorylation of proteins. Abnormal activity of PKs in cells is correlated to complex diseases. Therefore, the pharma industry is making significant efforts to regulate the activity of PKs with inhibitors and to monitor the activity of PKs with luminescent probes. ARC inhibitors are conjugates of adenosine analogues and peptide mimetic moieties; they have been studied at length at the Institute of Chemistry of the University of Tartu. In the present study, it was shown that the binding of monoclonal antibody (clone D38C6) to α-isoform of the catalytic subunit of PKA (PKAcα) was competitive with binding of ARC-Lum(Fluo) probes. Sequential treatment of a sample with these competing PKAcα ligands was used to develop a sensitive AbARC immunoassay that allowed the determination of small amounts (from 93 pg) of PKAcα in cell lysates. Recently, PRKACB gene with the S54L mutation was discovered in a patient with the Cushing's syndrome. This mutation leads to a change in the structure of the glycine-rich loop of the PK. In the present study, an inhibitor with six-fold selectivity for the mutated PK was developed. In addition, a luminescence method was worked out for determination of affinity of both commercial and developed inhibitors of the PKAcβ protein. X-ray structure analysis of complexes of ARC inhibitors and PK PIM-1 was performed in collaboration with the University of Oxford. Based on the obtained structural models, compounds with simplified chemical structures were constructed. New inhibitors were derivatized with biotin or fluorescent dye Cy5 and applied for the detection of PIM PKs in biochemical solutions and complex biological samples. The sandwich assay utilizing a PIM-2-selective detection antibody featured a low limit of quantification (44 pg of PIM-2). A confocal microscopy study showed that the fluorescent probes were efficiently taken up by U2OS cells and the probes revealed high extent of co-localization with PIM-1-fused fluorescent proteins.
Kirjeldus
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Märksõnad
inhibitors, photoluminesence, sondes, in vitro