Biološko vrednotenje himernih molekul kot modulatorjev O-GlcNAc transferaze in vitro

Buhin, Ana (author), Gobec, Martina (mentor), Smrdel, Lara (co-mentor)

Prenos N-acetilglukozamina na akceptorske proteine katalizira encim N-acetil-β-glukozaminil transferaza (OGT), ki uravnava delovanje številnih proteinov ter na ta način omogoča vzdrževanje znotrajcelične homeostaze. Neuravnana aktivnost encima OGT je povezana z različnimi bolezni, kot sta rak in sladkorna bolezen, zaradi česar se je pojavila potreba po razvoju orodja za preučevanje vloge encima OGT pri patoloških procesih. Himerni razgrajevalci proteinov (angl. Proteolysis Targeting Chimeras- PROTAC), so molekule sestavljene iz dveh strukturnih ligandov, pri čemer je eden namenjen vezavi na E3 ligazo, drugi pa vezavi na tarčni protein. Le-ti so se v zadnjih letih izkazali kot obetavna alternativa klasičnim zaviralcem, saj ne zavirajo samo delovanja tarčnega proteina, ampak ga s pomočjo naravno prisotnega ubikvitin-proteasomskega sistema tudi usmerjajo v razgradnjo. Številne objavljene študije so pokazale uspešno razgradnjo različnih tarčnih proteinov s pomočjo himernih razgrajevalcev. Na podlagi tega smo sklepali, da bi molekule PROTAC načrtovane za OGT kot tarčni protein lahko razgrajevale le-tega. Z namenom preverjanja naše domneve smo najprej na različnih celičnih linijah vrednotili izražanje encima OGT in E3 ligaz za katere so načrtovani ligandi molekul PROTAC. Na podlagi izražanja encima OGT in E3 ligaz smo za poskuse izbrali celično linijo MDA-MB-231. Morebitno razgradnjo encima OGT smo želeli vrednotiti pri netoksičnih koncentracijah spojin. Zato smo najprej naredili test metabolne aktivnosti na celicah MDA-MB-231, ki so bile 48 ur izpostavljene himernim razgrajevalcem. Ugotovili smo, da molekule PROTAC do 10 µM koncentracije ne delujejo citotoksično. Nato je sledilo vrednotenje učinka razgradnje molekul PROTAC na OGT pri različnih koncentracijah. Rezultati so pokazali, da so na celični liniji MDA-MB-231 le spojine z ligandom za E3 ligazo IAP vodile v delno razgradnjo encima OGT. Najbolj učinkovita je bila spojina SAB417, ki je povzročila razgradnjo OGT že pri 0,1µM koncentraciji. Ta spojina je vodila tudi v znižan nivo N-acetilglukozaminiliranih proteinov, kar dodatno potrjuje, da je prišlo do znižanega delovanja encima OGT. Rezultati kažejo, da smo identificirali himerne razgrajevalce, ki razgrajujejo OGT, a bodo potrebne dodatne modifikacije teh spojin, s katerimi bomo dosegli še bolj učinkovito razgradnjo tarčnega proteina. Le takšne spojine bodo namreč dobra orodja za proučevanje vloge OGT v bioloških in patoloških procesih.

• Language: Slovenian
• Keywords: N-acetilglukozaminilacija, encim OGT, himerni razgrajevalci, molekule PROTAC, celična linija MDA-MB-231
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2023
• PID: 20.500.12556/RUL-145134
• Publication date in RUL: 07.04.2023

Secondary language

• Language: English
• Title: In vitro biological evaluation of chimeric O-GlcNAc transferase modulators

Abstract

The enzyme O-GlcNAc transferase (OGT) is responsible for adding O-linked N-acetylglucosamine to proteins, which is important for maintaining cell homeostasis and regulating the functions of several proteins. However, alterations in OGT function have been linked to various diseases, including cancer and diabetes. As a result, there is a need to develop tools to study the role of OGT in these diseases. Proteolysis Targeting Chimeras (PROTACs) are bifunctional molecules consisting of two structural ligands, one that binds E3 ligase and other that binds protein of interest. PROTAC technology has emerged as a promising alternative to small molecule inhibitors as PROTACs not only inhibit the activity of the target protein, but also have the ability to induce degradation of targeted proteins through the ubiquitin–proteasome system. In numerous studies PROTACs have shown successful degradation of various target proteins. At the Department of Pharmaceutical Chemistry 12 PROTACs targeting OGT were designed. Herein we aimed to evaluate if these molecules efficiently degrade OGT. In first experiment, we investigated the expression of OGT and E3 ligases in different cell lines. We chose the breast cancer cell line MDA-MB-231 for further experiments, since it expressed OGT as well as targeted E3 ligases. Since we wanted to evaluate the potential OGT degradation at non-toxic concentrations of the compounds, we first performed a metabolic activity assay on MDA-MB-231 cells exposed to PROTAC molecules for 48 h. We conclude that PROTAC molecules at concentration up to 10 µM did not exhibit cytotoxic effects on tested cells. Next, effects of PROTACs on OGT degradation were determined and revealed that only PROTACs designed for E3 ligase IAP led to partial degradation of OGT in cell line MDA-MB-231. The most effective compound was SAB417 leading to OGT degradation already at 0.1 µM concentration. This compound also caused a drop in the level of O-GlcNAc proteins, which further confirms reduced OGT activity. Our results confirm that we have identified chimeric molecules for partial OGT degradation. However, for even more efficient degradation of target protein, further modifications of compounds are needed. Only such compounds have opportunity to become tools for studying OGT's role in biological and pathological processes.

• Keywords: O-GlcNAcylation, enzyme OGT, Proteolysis Targeting Chimeras, PROTAC molecules, cell line MDA-MB-231