Kristallstruktuuridest juhinduv bisubstraatsete inhibiitorite arendamine mitootilisele proteiinkinaasile Haspin

Väitekirja elektrooniline versioon ei sisalda publikatsiooneRakujagunemine (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

Continuous cover forest management plan on the example of Kalda estate

Püsimetsandus on Eestis võrdlemisi vähelevinud metsa majandamise viis. Metsa majandamine püsiva metsa kattega on siiski asjakohane juhul, kui omanik soovib hoida metsa looduslikku ilmet ja mitmekesisust ning samas metsast saada mõningast majanduslikku kasu. Käesolev töö on koostatud autorile kuuluva metsakinnistu näitel, millel puudus eelnev metsamajandamise kava ja inventeerimise andmed. Töö käigus teostatud takseerimise andmete põhjal on analüüsitud standartset kava, kus teostatakse lageraiet, ning võrreldud püsimetsandusliku metsamajandusliku karakteristikutega koos nende majandamise viiside kombinatsiooniga. Vastavalt analüüsi tulemustele, on järeldatav, et majandamise strateegiate kombinatsioon on optimaalseim majandusliku kasumlikkuse ja töö autori metsadisaini nägemuse seisukohast. Antud töö leiab tulevikus reaalset kasutust autorile kuuluva Kalda metsakinnistu majandamisel.Continuous cover forestry is not very popular management system in Estonia. At this moment, it is appropriate for forest owners who wish to maintain the ecological diversity as well as the natural design of the forest and still earn profit from management process. This thesis is based on an example of Kalda estate that is owned by the author, the estate had no previous management plan or any inventory data. On the basis of the data, which was collected during the fieldwork, was a comparison between conventional clearcutting orientated management, continuous cover management and combination between these systems. By the results from analysis it can be concluded that the most suitable management system is the combined management system in terms of profit and forest design relationship. The thesis will find practice on the real management of Kalda estate

Bisubstrate Inhibitor Approach for Targeting Mitotic Kinase Haspin

During the past decade, the basophilic atypical kinase Haspin has emerged as a key player in mitosis responsible for phosphorylation of Thr3 residue of histone H3. Here, we report the construction of conjugates comprising an aromatic fragment targeted to the ATP-site of Haspin and a peptide mimicking the N-terminus of histone H3. The combination of effective solid phase synthesis procedures and a high throughput binding/displacement assay with fluorescence anisotropy readout afforded the development of inhibitors with remarkable subnanomolar affinity toward Haspin. The selectivity profiles of novel conjugates were established by affinity studies with a model basophilic kinase (catalytic subunit of cAMP-dependent protein kinase) and by a commercial 1-point inhibition assay with 43 protein kinases

Crystal structure-guided design of bisubstrate inhibitors and photoluminiscent probes for protein kinases of the PIM family

We performed an X-ray crystallographic study of complexes of protein kinase PIM-1 with three inhibitors comprising an adenosine mimetic moiety, a linker, and a peptide-mimetic (d-Arg)6 fragment. Guided by the structural models, simplified chemical structures with a reduced number of polar groups and chiral centers were designed. The developed inhibitors retained low-nanomolar potency and possessed remarkable selectivity toward the PIM kinases. The new inhibitors were derivatized with biotin or fluorescent dye Cy5 and then applied for the detection of PIM kinases in biochemical solutions and in complex biological samples. The sandwich assay utilizing a PIM-2-selective detection antibody featured a low limit of quantification (44 pg of active recombinant PIM-2). Fluorescent probes were efficiently taken up by U2OS cells and showed a high extent of co-localization with PIM-1 fused with a fluorescent protein. Overall, the developed inhibitors and derivatives represent versatile chemical tools for studying PIM function in cellular systems in normal and disease physiology

Crystal structure-guided design of bisubstrate inhibitors and photoluminescent probes for protein kinases of the PIM family

We performed an X-ray crystallographic study of complexes of protein kinase PIM-1 with three inhibitors comprising an adenosine mimetic moiety, a linker, and a peptide-mimetic (d-Arg)6 fragment. Guided by the structural models, simplified chemical structures with a reduced number of polar groups and chiral centers were designed. The developed inhibitors retained low-nanomolar potency and possessed remarkable selectivity toward the PIM kinases. The new inhibitors were derivatized with biotin or fluorescent dye Cy5 and then applied for the detection of PIM kinases in biochemical solutions and in complex biological samples. The sandwich assay utilizing a PIM-2-selective detection antibody featured a low limit of quantification (44 pg of active recombinant PIM-2). Fluorescent probes were efficiently taken up by U2OS cells and showed a high extent of co-localization with PIM-1 fused with a fluorescent protein. Overall, the developed inhibitors and derivatives represent versatile chemical tools for studying PIM function in cellular systems in normal and disease physiology