Sinteza in biološko vrednotenje novih piperidinskih zaviralcev proteina Hsp90 s protitumornim delovanjem

Vižintin, Urška (author), Tomašič, Tihomir (mentor), Gramec Skledar, Darja (comentor)

Rakava obolenja predstavljajo poleg kardiovaskularnih bolezni najpomembnejši javnozdravstveni problem v svetovnem merilu. Gre za široko skupino bolezni, pri kateri nekontrolirana delitev celic in njihovo razraščanje v okoliška tkiva zaradi odpovedi kontrolnih sistemov povzročita nastanek tumorja. Protein toplotnega šoka 90 (Hsp90) je od ATP odvisni šaperon, ki v celicah pomaga pri zvijanju in stabilizaciji več kot 200 substratnih proteinov. Ti nadzorujejo celični cikel in celično preživetje ter so vpleteni v številne signalne poti. V rakavih celicah so substrati Hsp90 pogosto mutirani, njihovo delovanje in stabilizacija pa sta odvisni od povečane aktivnosti proteina Hsp90, kar lahko vodi v neoplastične spremembe in nastanek tumorja. Raziskovalci so pri iskanju novih načinov zdravljenja raka odkrili, da lahko z zaviranjem delovanja proteina Hsp90 dosežemo protitumorno delovanje. Prvi zaviralci so interagirali z vezavnim mestom za ATP na N-končni domeni proteina, vendar je zaradi njihove toksičnosti in sprožitve odziva toplotnega šoka njihov terapevtski potencial omejen. Z odkritjem novobiocina, ki se veže v C-končno domeno Hsp90 in ne inducira odziva toplotnega šoka, je C-končna domena postala nova tarča za razvoj protitumornih učinkovin. V okviru magistrske naloge smo sintetizirali trinajst potencialnih piperidinskih zaviralcev C-končne domene Hsp90, ki smo jih pripravili z modifikacijo referenčne spojine. Vlogo hidroksilne skupine na obroču piperidina smo proučili z vpeljavo amidne vezi, ki smo jo pri nekaterih analogih vpeljali tudi med osrednja obroča distančnika. Proučevali smo tudi vpliv menjave različnih lipofilnih substituentov na aromatskem obroču in raziskali vpliv bazičnega centra na morfolinu. S testom MTS smo v nadaljevanju določili vpliv sintetiziranih spojin na proliferacijo celic raka dojke MCF-7. Najaktivnejše spojine smo nato testirali v širšem koncentracijskem območju na celičnih linijah MCF-7 in HepG2 ter jim določili vrednost IC50. Ugotovili smo, da štiri spojine vplivajo na znižanje proliferacije celic, le dve pa sta imeli močnejše delovanje v primerjavi z referenčno spojino. Rezultati magistrske naloge imajo pomemben doprinos k razumevanju odnosa med strukturo in delovanjem ter so osnova za nadaljnjo optimizacijo do spojin z izboljšanim protitumornim delovanjem.

• Language: Slovenian
• Keywords: rak, protein toplotnega šoka, C-končni zaviralci Hsp90, MCF-7, piperidin
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2019
• PID: 20.500.12556/RUL-112926
• Publication date in RUL: 22.11.2019

Secondary language

• Language: English
• Title: Synthesis and biological evaluation of new piperidine-based Hsp90 inhibitors displaying anticancer activity

Abstract

Cancer is alongside cardiovascular diseases one of the main public-health problems worldwide. It is characterised by uncontrolled cell division and cell proliferation into surrounding tissues, where tumour formation begins. Heat shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone, which has an important role in regulation of folding and stability of more than 200 client proteins. Clients control cell cycle and cell survival, and assist in many signalling pathways. In cancer cells, Hsp90 activity is overexpressed to stabilize these clients, which are often mutated and responsible for neoplastic changes. Inhibition of Hsp90 represents a new therapeutic target for cancer treatment. First Hsp90 inhibitors targeted N-terminal ATP-binding site of the protein. They displayed limited therapeutic potential in anticancer treatment and caused side effects – toxicity and induction of heat shock response. Discovery that novobiocin, first identified Hsp90 C-terminal inhibitor, does not induce heat shock response started a new era of developing novel anticancer inhibitors targeting Hsp90. Experimental work of this master’s thesis contains synthesis of thirteen potential Hsp90 C-terminal inhibitors with piperidine scaffold based on the reference Hsp90 inhibitor. We studied the role of hydroxyl group on piperidine scaffold by replacing it with an amide bond. We studied structure-activity relationship by changing different lipophilic substituents on the benzene ring, and importance of the basic centre on the morpholine ring. We tested activity of synthesized analogues with MTS test of cell proliferation in human breast cancer cells MCF-7 and liver cancer HepG2 cell line. For the most active compounds, we determined IC50 values in MCF-7 and HepG2 cell lines. Four compounds showed effect on cell proliferation, but only two analogues displayed better activity than reference compound. Results of this master’s thesis have an important contribution to understanding of the structure-activity relationship, which is a basic for further optimization of analogues with improved anticancer activity and higher Hsp90 binding affinity.

• Keywords: cancer, heat shock proteins, Hsp90 C-terminal inhibitors, MCF-7, piperidine