Načrtovanje, sinteza in vrednotenje novih zaviralcev proteina toplotnega šoka 90 s protitumornim delovanjem

Dernovšek, Jaka (author), Tomašič, Tihomir (mentor), Zidar, Nace (comentor)

Proteini toplotnega šoka 90 (Hsp90) predstavljajo družino šaperonov odgovornih za pravilno zvijanje in zorenje t.i. proteinov klientov. Družino sestavljajo 4 izooblike: Hsp90?, Hsp90ß, Grp94 in TRAP1, z različno znotrajcelično lokalizacijo in vlogami. Od odkritja prvega N-končnega zaviralca Hsp90 družino Hsp90 intenzivno raziskujejo kot tarčo potencialnih učinkovin s protitumornim delovanjem. Protirakavo delovanje zaviralcev Hsp90 je posledica znižanih znotrajceličnih ravni več onkogenih proteinov, ki so odvisni od Hsp90. Odobritev prvih zaviralcev Hsp90 za klinično rabo pa je ustavilo njihovo sprožanje odziva toplotnega šoka (HSR), ki prek povečanja izražanja proteinov toplotnega šoka zaščiti celice in nasprotuje učinkom zdravljenja. Ob pomanjkanju učinkovitosti, ki je sledilo, je bil glavni vzrok neuspeha prvih N-končnih ATP-kompetitivnih zaviralcev Hsp90 tudi pojav toksičnih učinkov povezanih s povečevanjem odmerkov. Raziskave so se zato preusmerile v načrtovanje C-končnih zaviralcev, N-končnih izoformno selektivnih zaviralcev in zaviralcev medproteinskih interakcij med Hsp90 in proteini, ki modulirajo njegov šaperonski cikel - košaperoni. Vse tri alternativne pristope zaviranja delovanja Hsp90 brez sprožanja HSR smo v doktorskem delu naslovili z razvojem novih zaviralcev. Uspešno smo pripravili N-končne zaviralce Hsp90, ki selektivno vplivajo na izoformo Hsp90ß. Zasnovali in pripravili smo spojine, ki zavirajo interakcije med Hsp90 in košaperonom Cdc37, ki je odgovoren za dostavo raku pomembnih proteinskih kinaz do Hsp90. Največ časa pa smo posvetili razvoju novih C-končnih alosteričnih modulatorjev Hsp90, saj smo z različnimi pristopi načrtovanja in sinteze pripravili in optimizirali štiri nove strukturno različne razrede. Vsem pripravljenim spojinam smo ovrednotili njihovo protirakavo delovanje in vitro, najobetavnejše pa smo nato vrednotili še v kompleksnejših sistemih in vivo. Pokazali smo, da vsi trije alternativni tipi zaviralcev Hsp90 zaobidejo HSR, eno od ključnih težav obstoječih N-končnih zaviralcev Hsp90. Pripravljene spojine pomembno znižujejo ravni proteinov klientov Hsp90 v celici in posledično zavirajo rast različnih vrst rakavih celic tako v in vitro kot v in vivo okolju.

• Language: Slovenian
• Keywords: alosterija, Hsp90, protitumorno delovanje, rak, selektivnost
• Work type: Doctoral dissertation
• Organization: FFA - Faculty of Pharmacy
• Year: 2024
• PID: 20.500.12556/RUL-162202
• Publication date in RUL: 20.09.2024

Secondary language

• Language: English
• Title: Design, synthesis and evaluation of new heat shock protein 90 inhibitors with anticancer activity

Abstract

Heat shock protein 90 (Hsp90) is a family of chaperones responsible for the folding and maturation of the so-called client proteins. The family consists of 4 isoforms: Hsp90α, Hsp90β, Grp94 and TRAP1, with different intracellular localization and roles. Since the discovery of the first Hsp90 N-terminal inhibitor, the Hsp90 family has been intensively investigated as a target for the preparation of anticancer agents. Their anticancer activity results from decreased intracellular levels of several Hsp90-dependent oncoproteins. However, the approval of the first Hsp90 inhibitors for regular clinical use was halted due to their induction of the heat shock response (HSR), which protects the cells through increased heat shock protein expression and counteracts the effects of treatment. Along with the lack of efficacy that followed, the main reason for the failure of N-terminal ATPcompetitive Hsp90 inhibitors in the clinic was the occurrence of dose-escalating toxicities. Research has therefore shifted to the design of C-terminal inhibitors, N-terminal isoformselective inhibitors and inhibitors of protein-protein interactions (PPI) between Hsp90 and proteins that modulate its chaperone cycle - cochaperones. All three alternative approaches of inhibiting Hsp90 activity without triggering HSR were addressed in the doctoral thesis by the preparation of new inhibitors. We have successfully prepared N-terminal Hsp90 inhibitors that selectively target the Hsp90β isoform, inhibitors of the PPI between Hsp90 and its kinase-delivering cochaperone Cdc37, along with four structurally diverse classes of C-terminal allosteric modulators of Hsp90 function. All prepared compounds were evaluated for their in vitro anticancer activity, and the most promising ones were then evaluated in more complex in vivo systems. We show that all three alternative types of Hsp90 inhibitors bypass HSR, one of the key problems of existing Hsp90 N-terminal inhibitors. Our Hsp90 inhibitors significantly reduce the levels of Hsp90 client proteins in cells and consequently inhibit the growth of various types of cancer cells both in vitro and in vivo.

• Keywords: allostery, anticancer, cancer, Hsp90, selectivity