Načrtovanje in sinteza himernih razgrajevalcev proteina Bcl-2

Bricelj, Aleša (author), Sosič, Izidor (mentor)

Področje farmacevtske kemije se sooča z izzivom, saj je le 25 % človeškega proteoma dostopnih z vidika klasičnega načrtovanja zdravilnih učinkovin, medtem ko na preostali del proteinov zaenkrat težko vplivamo z majhnimi molekulami. Obstajajo alternativni pristopi načrtovanja novih zdravilnih učinkovin, kot so stabilizirane peptidne učinkovine, monoklonska protitelesa, gensko zdravljenje in terapija s spojinami, ki interagirajo z nukleinskimi kislinami. Kljub izrazitemu napredku omenjeni pristopi ne pokrijejo celotnega biološkega prostora in niso uporabni pri velikem številu tarč in bolezni. Kot izjemno obetaven se je v zadnjih letih izkazal razvoj himernih molekul, ki izkoriščajo ubikvitin-proteasomski sistem za proteolitično razgradnjo tarčnih proteinov. Te himerne oziroma bifunkcionalne razgrajevalce imenujemo 'proteolysis-targeting chimeras' (PROTACs), sestavljeni pa so iz treh delov: liganda za izbrano E3 ubikvitin ligazo, ustreznega distančnika in liganda, ki se veže na tarčni protein. Zaradi svojega mehanizma delovanja so te molekule sposobne v nizkih koncentracijah z visoko selektivnostjo doseči razgradnjo tarčnega proteina. Tovrsten farmakološki pristop se je že izkazal za boljšo alternativo farmakološkemu modelu zasedenosti predvsem pri rakavih obolenjih, pri katerih se pogosto razvije rezistenca na klasične zaviralce proonkogenih proteinov, bodisi zaradi njihove mutacije ali pa kompenzatornega povečanja njihovega izražanja. Primer farmakološko izjemno uporabnega proteina, ki lahko razvije odpornost na uporabljane zaviralce, je tudi antiapoptotični protein Bcl-2, ki zavira programirano celično smrt in ima ključno vlogo pri patogenezi folikularnega limfoma, difuznih velikoceličnih B limfomov, multiplih mielomov in kroničnih limfocitnih levkemij. Na osnovi znanega zaviralca Bcl-2, tj. venetoklaksa, smo sintetizirali šest PROTAC spojin, ki vsebujejo ligand za cereblon kot E3 ligazo, ter po eno spojino, ki vsebuje ligand za VHL oziroma za IAP kot E3 ligazo. Vsem osmim spojinam smo ovrednotili njihovo afiniteto za vezavo na Bcl-2 ter sposobnost indukcije razgradnje proteina Bcl-2. Kljub dokazani afiniteti do Bcl-2 nobena izmed spojin pri dveh različnih koncentracijah ni izkazovala razgradnje tarče. Nadalje smo molekule testirali še na treh različnih celičnih linijah in ugotovili, da so nekatere izmed njih povzročile celično smrt. To nakazuje, da mehanizem delovanja teh molekul ne vključuje razgradnje Bcl-2, temveč najverjetneje le njegovo zaviranje, hkrati pa zaenkrat ne moremo izločiti tudi drugih mehanizmov delovanja.

• Language: Slovenian
• Keywords: himerni razgrajevalci, proteasom, venetoklaks, protein Bcl-2, apoptoza
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2019
• PID: 20.500.12556/RUL-108331
• Publication date in RUL: 28.06.2019

Secondary language

• Language: English
• Title: Design and synthesis of Bcl-2 proteolysis targeting chimeras

Abstract

The drug discovery industry is facing a challenge since more than 75 % of the human proteome is not easily modulated by small molecules and is therefore considered undruggable. Some alternative therapeutic approaches led to success, i.e. stabilized peptides, monoclonal antibodies, nucleic acid-based therapeutics, and gene therapy; however, these cannot be applied effectively to all biological targets and diseases. Recently, a novel approach using chimeric molecules that utilize the ubiquitin-proteasome system to induce proteolytic degradation of targeted proteins was discovered. These bifunctional compounds are termed proteolysis-targeting chimeras or PROTACs and are comprised of three elements: a ligand for the E3 ligase, a ligand binding to the protein of interest, and a linker connecting the two. The mechanism of action allows PROTACs to induce the degradation of the targeted protein in low concentrations and with high selectivity. This event-driven pharmacology model represents a better alternative to the occupancy-driven pharmacology model particularly in cancerous diseases, where resistance to classical inhibitors of prooncogenic proteins or their compensatory overexpression often develop. Bcl-2 is an example of a pharmacologically relevant anti-apoptotic protein that commonly becomes chemoresistant and is present in follicular lymphomas, diffuse B-cell lymphomas, multiple myeloma and in chronic lymphocytic leukemia. Building on the established Bcl-2 inhibitor venetoclax, we have synthesized six PROTAC molecules that bind to cereblon as the targeted E3 ligase, one that binds to VHL and another that binds to IAP as the E3 ligase. We determined the binding affinity of PROTACs for Bcl-2 and their ability to induce Bcl-2 degradation. The molecules expressed adequate affinity for Bcl-2 but failed to cause its proteolysis. We additionally tested the Bcl-2 – cereblon PROTACs on three different cell lines and found that some significantly reduced cell viability. The results indicate that the synthesized compounds are capable of inhibiting but not also degrading Bcl-2. However, further research is needed to fully explain their mechanism of action.

• Keywords: proteolysis-targeting chimeras, proteasome, venetoclax, Bcl-2 protein, apoptosis