Načrtovanje in sinteza zaviralcev stresnega proteina Hsp90 z različnimi mehanizmi delovanja

Gradišek, Nina (avtor), Tomašič, Tihomir (mentor), Dernovšek, Jaka (komentor)

Stresni protein Hsp90 kot eden najpomembnejših šaperonov odgovornih za homeostazo celic in zvitje številnih - tudi onkogenih - proteinov predstavlja pomemben dejavnik v napredovanju raka in zato obetavno terapevtsko tarčo. Številni neželeni učinki zaviralcev ATP-vezavnega mesta na N-končni domeni (NKD) proteina Hsp90, ki so bili tudi posledica neselektivnega zaviranja vseh njegovih štirih izoform, so vodili v razvoj zaviralcev z alternativnimi mehanizmi delovanja. Tako so začeli raziskovati selektivne zaviralce posameznih izoform Hsp90, zaviralce alosteričnega vezavnega mesta na C-končni domeni (CKD) in zaviralce medproteinskih interakcij med Hsp90 in ko-šaperonom Cdc37. V okviru eksperimentalnega dela smo na podlagi literaturnih podatkov pripravili knjižnice selektivnih zaviralcev posameznih izoform Hsp90. Na podlagi struktur selektivnih zaviralcev Hsp90β smo razvili farmakoforne modele, s katerimi smo izvedli virtualno rešetanje in nato ovrednotili dobljene virtualne zadetke na podlagi ujemanja z modelom. Nadalje smo načrtovali in sintetizirali enajst zaviralcev CKD s 4,5,6,7-tetrahidrobenzotiazolnim ogrodjem, pri čemer smo preverjali vpliv aromatskega substituenta, distančnika in substitucije na bazičnem centru na zaviralno aktivnost. Dvema znanima zaviralcema CKD Hsp90 smo nato za doseganje selektivnega zaviranja mitohondrijske izoforme Hsp90 (TRAP1) pripeli tudi trifenilfosfonijev ion (TPP+) na različnih distančnikih in opazovali spremembo aktivnosti pred in po uvedbi tega mitohondrijskega dostavnega sistema. Na koncu smo sintetizirali še šestnajst zaviralcev medproteinskih interakcij med Hsp90 in Cdc37, s katerimi smo želeli pridobiti informacije o odnosu med strukturo in delovanjem (SAR). Protirakavo delovanje zaviralcev smo vrednotili na celicah raka dojke in Ewingovega sarkoma. Rezultati celičnih testov so pri zaviralcih interakcije Hsp90-Cdc37 nakazali pomen para-substitucije za orientacijo ostalih strukturnih elementov. Pripenjanje TPP+ je po pričakovanjih izboljšalo jakost delovanja zaviralcev TRAP1. Poleg tega smo pri zaviralcih CKD Hsp90 s celičnimi testi potrdili pomen aromatskega obroča na mestu 6 (spojine 8a-d) in ustreznega distančnika do bazičnega centra (spojine 11a-d in 12-14). Najobetavnejša spojina 11b je tako zavirala rast rakave celične linije MCF-7 z IC50 = 0,43 ± 0,10 µM. Nova spoznanja magistrske naloge pomembno prispevajo k boljšemu razumevanju alosteričnega vezavnega mesta na CKD Hsp90 in predstavljajo izhodišče za nadaljnje načrtovanje tako C-končnih kot tudi selektivnih zaviralcev posameznih izoform Hsp90 in zaviralcev medproteinskih interakcij Hsp90 s Cdc37.

• Jezik: Slovenski jezik
• Ključne besede: alosterični zaviralec, Hsp90, rak, selektivnost, zaviralec Cdc37
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2023
• PID: 20.500.12556/RUL-150349
• Datum objave v RUL: 16.09.2023

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Design and synthesis of heat shock protein 90 inhibitors with various mode of action

Izvleček

The stress protein Hsp90 plays a pivotal role in maintaining cellular homeostasis and facilitating the folding of many – including oncogenic – proteins, identifying it as an important factor in cancer progression and therefore a promising therapeutic target. The numerous adverse effects of N-terminal ATP-binding domain (NTD) inhibitors, resulting from nonselective inhibition of all four Hsp90 isoforms, have led to the development of inhibitors with alternative mechanisms of action. Thus, selective inhibitors of individual Hsp90 isoforms, inhibitors of the allosteric binding site on the C-terminal domain (CTD) and inhibitors of protein-protein interactions between Hsp90 and its co-chaperone Cdc37 were investigated. In the experimental phase, libraries of selective inhibitors of individual Hsp90 isoforms were prepared based on literature data. Utilizing the structures of selective Hsp90β inhibitors, pharmacophore models for high-throughput virtual screening were developed and obtained virtual hits were assessed based on pharmacophore matching. Further on, eleven CTD inhibitors with the 4,5,6,7-tetrahydrobenzothiazole scaffold were designed and synthesized, exploring the impact of the aromatic substituents, spacers, and substitution at the basic center on the inhibitory activity. To achieve selective inhibition of the mitochondrial Hsp90 isoform (TRAP1), triphenylphosphonium ion (TPP+) was attached to two known Hsp90 CTD inhibitors on different spacers and the change in activity before and after the introduction of this mitochondria-targeting moiety was observed. In the end, sixteen inhibitors of protein-protein interactions between Hsp90 and Cdc37 were synthesized to obtain information on the structure-activity relationship (SAR). The anticancer activity of the inhibitors was evaluated in breast cancer and Ewing sarcoma cell lines. The results of cell-based assays indicated the importance of the para-substitution for the orientation of other structural elements in Hsp90-Cdc37 interaction inhibitors. As predicted, the potency of TRAP1 inhibitors was enhanced by TPP+ attachment. In addition, in Hsp90 CTD inhibitors, the importance of the aromatic ring at position 6 (compounds 8a-d) and the corresponding spacer to the basic center (compounds 11a-d and 12-14) were confirmed by cell-based assays. The most promising compound 11b thus inhibited the growth of the breast cancer cell line MCF-7 with an IC50 value of 0.43 ± 0.10 µM. The new findings of the master's thesis significantly contribute to a better understanding of the allosteric binding site on Hsp90 CTD and represent a starting point for the further design of both C-terminal inhibitors and selective inhibitors of individual Hsp90 isoforms and inhibitors of Hsp90 protein-protein interactions with Cdc37.

• Ključne besede: allosteric inhibitor, Hsp90, cancer, selectivity, Cdc37 inhibitor