Crystal structure-guided development of bisubstrate-analogue inhibitors of mitotic protein kinase Haspin

Abstract

Rakujagunemine (sh mitoos) on ülitäpselt reguleeritud teatud ensüümide – proteiinkinaaside – koostöö poolt. Rakutsükli regulatsioonis oluliste proteiinkinaaside aktiivsuse muutumisel võivad rakud hakata kontrollimatult jagunema ning tagajärjeks on erinevate haiguste teke, millest tuntuimad on vähkkasvajad. Sellest lähtuvalt püütakse arendada ühendeid (inhibiitoreid), mis suudaksid kontrollida proteiinkinaaside aktiivsust ning oleksid kasutatavad vähivastaste ravimitena. Paralleelselt ravimiarendusega on oluline välja töötada lihtsasti kasutatavaid, kiireid ning täpseid diagnostilisi vahendeid, mis võimaldaksid määrata proteiinkinaaside kui biomarkerite hulka eri kudedes, sh kehavedelikes, ning seeläbi diagnoosida haigusi juba võimalikult varajases etapis. Proteiinkinaas kui ensüüm on ruumiline molekul, milles paiknevad teatud keemilise koostise ja kujuga „taskud“, kuhu saavad seostuda erinevad ühendid (sh inhibiitorid). Inhibiitorite disainil soovitakse konstrueerida sobiva kujuga ühend, mis sobituks võimalikult täpselt ensüümil oleva(te)sse „tasku(te)sse“. Selle tulemusel on võimalik saada kõrge: • afiinsusegasega (st tugeva seostumisvõimega just temale mõeldud „taskusse“, kusjuures mida täpsemalt ühend „taskusse“ paigutub, seda kauem ta seal püsib ja seda suurem on mõju) ja • selektiivsusega ühendeid (tunneb teiste seast ära just temale mõeldud „tasku“; siinjuures vajab ära märkimist, et inimorganismis on kokku 538 erinevat proteiinkinaasi). Antud uurimistöös võeti sihtmärgiks proteiinkinaas Haspin, mida on suhteliselt vähe uuritud, kuid on näidatud tema olulisus rakujagunemise regulatsioonis. Doktoritöö käigus arendati bisubstraatseid inhibiitoreid Haspinile, mille puhul ühendati linkeriga kaks erinevat Haspiniga seostuvat fragmenti (omavad ensüümil erinevaid „taskuid“ seostumiseks). Töö kõige silmapaistvamaks tulemiks on väga kõrge afiinsusega ning selektiivsusega (võrreldes teiste proteiinkinaasidega) Haspinile suunatud bisubstraatsed inhibiitorid. Lisaks sellele suudavad uudsed ühendid olla Haspiniga seotud mitmeid tunde, pikendades seeläbi oma mõju ensüümi aktiivsusele. Arendatud ühendite väljundiks on nende kasutamine Haspini signaaliradade uurimisel ja mõjutamisel ning Haspini kui potentsiaalse biomarkeri määramisel diagnostikas.The cell division (including mitotic events) is precisely regulated by the crosstalk of certain enzymes – protein kinases. Abnormal changes in the activity of protein kinases that have a role in the regulation of the cell cycle may lead to uncontrollable cell division and hence development of various diseases (including cancerous tumors). Accordingly, it is essential to develop compounds (inhibitors), which are able to block the activity of protein kinases and which can be used as anticancer drugs. In parallel, it is of utmost importance to work out ‘easy-to-use’ diagnostic tools, which would allow to determine the changed profile of protein kinases as biomarkers and thus start the suitable treatment as early as possible. Protein kinase as an enzyme is a 3D-molecule that has ‘pockets’ with a certain chemical composition and characteristic shape. To these ‘pockets’, various compounds (including inhibitors) can bind. During the design of an inhibitor, it is attempted to construct a compound which would fit as precisely as possible into the enzymatic ‘pocket(s)’. As a result, it is possible to obtain the compound with high • affinity (i.e., strong binding ability to the ‘pocket’ intended for it, wherein the longer the compound stays in this site, the greater is its effect), and • selectivity towards its target (ability to distinguish the ‘pocket’ intended for it among others; in this context, it should be noted that there are 538 different protein kinases in the human body). In this dissertation, the novel target represented by protein kinase Haspin was taken under investigation. Haspin has been relatively little studied, but its importance in the regulation of cell division has been shown. During the study, bisubstrate inhibitors for Haspin were synthesized by connecting two different fragments targeting separate but still adjacent ‘pockets’ on Haspin by a linker. The most outstanding result of this work was development of bisubstrate inhibitors with high affinity and selectivity towards Haspin (as compared to other protein kinases). In addition, the novel compounds are able to stay bound to Haspin for many hours, thereby extending their effect on the activity of enzyme. The field of practical applications of the developed Haspin-targeting compounds includes investigation of and intervention with the signalling pathways of Haspin in natural systems, as well as detection of Haspin as potential biomarker in biological specimen.