prikaz prve stranice dokumenta Optimization of the solvent-induced protein precipitation method for determining erlotinib's specificity toward its biological target, EGFR
Embargo period 2026-09-25
master's thesis
Optimization of the solvent-induced protein precipitation method for determining erlotinib's specificity toward its biological target, EGFR
2024. urn:nbn:hr:193:709187
Moržan, Sara
University of Rijeka
Faculty of Biotechnology and Drug Development

Cite this document

Moržan, S. (2024). Optimization of the solvent-induced protein precipitation method for determining erlotinib's specificity toward its biological target, EGFR (Master's thesis). Rijeka: University of Rijeka. Retrieved from https://urn.nsk.hr/urn:nbn:hr:193:709187

Moržan, Sara. "Optimization of the solvent-induced protein precipitation method for determining erlotinib's specificity toward its biological target, EGFR." Master's thesis, University of Rijeka, 2024. https://urn.nsk.hr/urn:nbn:hr:193:709187

Moržan, Sara. "Optimization of the solvent-induced protein precipitation method for determining erlotinib's specificity toward its biological target, EGFR." Master's thesis, University of Rijeka, 2024. https://urn.nsk.hr/urn:nbn:hr:193:709187

Moržan, S. (2024). 'Optimization of the solvent-induced protein precipitation method for determining erlotinib's specificity toward its biological target, EGFR', Master's thesis, University of Rijeka, accessed 05 November 2024, https://urn.nsk.hr/urn:nbn:hr:193:709187

Moržan S. Optimization of the solvent-induced protein precipitation method for determining erlotinib's specificity toward its biological target, EGFR [Master's thesis]. Rijeka: University of Rijeka; 2024 [cited 2024 November 05] Available at: https://urn.nsk.hr/urn:nbn:hr:193:709187

S. Moržan, "Optimization of the solvent-induced protein precipitation method for determining erlotinib's specificity toward its biological target, EGFR", Master's thesis, University of Rijeka, Rijeka, 2024. Available at: https://urn.nsk.hr/urn:nbn:hr:193:709187

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Metadata
TitleOptimization of the solvent-induced protein precipitation method for determining erlotinib's specificity toward its biological target, EGFR
Title (croatian)Optimizacija metode precipitacije proteina inducirane otapalom u određivanju specifičnosti erlotiniba prema njegovoj biološkoj meti EGFR
AuthorSara Moržan
MentorMilan Mesić (mentor)
MentorIvana Ratkaj (komentor)
Committee memberToni Todorovski (predsjednik povjerenstva)
Committee memberKatarina Kapuralin (član povjerenstva)
GranterUniversity of Rijeka
(Faculty of Biotechnology and Drug Development)
Rijeka
Defense date and country2024-09-25, Croatia
Scientific / art field,
discipline and subdiscipline		BIOTECHNICAL SCIENCES
Biotechnology
Abstract
In recent years, label-free methods have become important in drug discovery for studying drug-protein interactions by observing conformational changes in target proteins after drug binding. These methods overcome the limitations of conventional techniques that rely on chemical modifications, which can affect a drugs’ specificity or affinity and lead to misidentification of targets. Identifying all protein interactions, whether the drug is designed for a specific target or discovered by phenotypic screening, is crucial for understanding both intended and off-target effects. This thesis uses solvent-induced protein precipitation (SIP) to assess erlotinibs’ specificity for its biological target, the epidermal growth factor receptor (EGFR). Additionally, it aims to identify any additional proteins, known as off-targets, that erlotinib may interact with. SIP is based on the principle that drug binding stabilizes proteins, making them more resistant to precipitation and denaturation by organic solvents. By comparing erlotinib-treated samples to untreated controls, we aimed to verify whether erlotinib specifically binds to EGFR, which would be indicated by increased stabilization compared to untreated samples. In experiments with A431 and HeLa cell lysates, Western blotting confirmed EGFR as erlotinibs’ biological target with differences in band intensity between erlotinib-treated and control samples validating its specificity. Additionally, certain experimental conditions required optimization. The initial use of DMSO and the lack of a loading control led to inconsistent results. Replacing DMSO and precipitating the lysates with acetone improved the results. Furthermore, β-actin may be sensitive to higher concentrations of organic solvents, suggesting the need for alternative controls, such as GAPDH. As this thesis focused on the Western blotting component of the SIP method, further studies should incorporate mass spectrometry to gain better insight into these interactions, and potentially confirm the off-target of erlotinib.
Abstract (croatian)
Metode bez kemijskog označavanja molekula (engl. label-free methods) postale su važne u proučavanju interakcija između lijekova i ciljnih proteina, promatrajući konformacijske promjene koje nastaju nakon vezanja lijeka. Ove metode prevladavaju ograničenja prethodnih tehnika koje kemijskim modifikacijama mogu promijeniti specifičnost ili afinitet lijeka što može dovesti do pogrešne identifikacije njegovih meta. Identifikacija svih proteina je ključna za razumijevanje učinka nekog lijeka te njegovih potencijalnih nuspojava, bez obzira na to je li lijek dizajniran za specifičan ciljni protein ili otkriven fenotipskim skeniranjem (engl. phenotypic screening). Ovaj rad usmjeren je na korištenje metode precipitacije proteina inducirane otapalom (engl. solvent-induced protein precipitation; SIP) u određivanju specifičnosti erlotiniba prema njegovoj biološkoj meti, receptoru epidermalnog faktora rasta (engl. epidermal growth factor receptor; EGFR). Također, još jedan cilj ovog rada je identifikacija dodatnih proteina (engl. off-targets) s kojima erlotinib može biti u interakciji. SIP metoda temelji se na načelu da vezanje lijeka za ciljni protein povećava njegovu stabilnost, čineći ga otpornijim na precipitaciju i denaturaciju organskim otapalima. Uspoređujući uzorke tretirane erlotinibom s netretiranim kontrolama, htjeli smo potvrditi specifično vezanje erlotiniba za EGFR, vidljivo povećanom stabilnošću EGFR-a nakon vezanja erlotiniba. U eksperimentima sa lizatima stanica A431 i HeLa, Western blotting tehnika potvrdila je EGFR kao biološku metu erlotiniba. Razlike u intenzitetu linija između uzoraka tretiranih erlotinibom i netretiranih kontrola su potvrdile njegovu specifičnost. U radu je bilo potrebno i optimizirati eksperimentalne uvjete. Početna upotreba DMSO-a i nedostatak kontrole doveli su do nepouzdanih rezultata. Zamjena DMSO-a i precipitacija lizata acetonom poboljšala je pouzdanost rezultata. Također, β-aktin se pokazao osjetljivim na visoke koncentracije organskih otapala, što sugerira potrebu za alternativnim kontrolama, poput GAPDH. Budući da je rad fokusiran na Western blot dio SIP metode, buduća istraživanja trebala bi uključivati masenu spektrometriju kako bi se dobio širi uvid u interakcije između erlotiniba i EGFR-a i identifikaciju potencijalnog off-target proteina.
Keywords
Keywords (croatian)
Languageenglish
URN:NBNurn:nbn:hr:193:709187
Study programmeTitle: Medicinal chemistry
Study programme type: university
Study level: graduate
Academic / professional title: magistar/magistra medicinske kemije (magistar/magistra medicinske kemije)
Type of resourceText
File originBorn digital
Access conditionsEmbargoed access
Embargo expiration date: 2026-09-25
Terms of useLicence In copyright icon
Created on2024-09-28 15:45:49