Vrednotenje spektralnih lastnosti derivatov kumarina, stilbena in pirola ob vezavi na fibrile amiloida β

Grbić, Anamarija (avtor), Knez, Damijan (mentor), Pajk, Stane (komentor)

Najpogostejši vzrok demence je kronična nevrodegenerativna bolezen, imenovana Alzheimerjeva bolezen, med značilne patološke spremembe le-te sodi kopičenje amiloida β (Aβ). Ker se spremembe v možganih začnejo razvijati že pred pojavom izrazitih kliničnih simptomov, kot so motnje spomina in koncentracije, je njihovo zgodnje prepoznavanje pomembno za pravočasno in zanesljivejšo diagnozo. Fluorescenčne sonde v bližnjem infrardečem območju predstavljajo enega izmed pristopov za neinvazivno detekcijo patoloških sprememb, povezanih z Alzheimerjevo boleznijo, in bi lahko prispevale k zgodnejši postavitvi diagnoze bolezni. V magistrskem delu smo za strukturno raznolike fluorescenčne sonde proučevali povezavo med njihovo strukturo, intenziteto odziva fluorescene, navidezno vezavno afiniteto in selektivnostjo do različnih proteinov v nativnem oziroma agregiranem stanju. Emisijske spektre sond smo najprej izmerili v pufru HEPES in v prisotnosti fibril Aβ1-42 ter na podlagi povečanja intenzitete fluorescence za nadaljnje vrednotenje izbrali sonde SP-1Z-1, SP-sp5, SP-ES-17 in SP-sp9. Njihov fluorescenčni odziv smo nato primerjali z odzivom sond v prisotnosti fibril inzulina, neagregiranega Aβ1–42 ter humanega in govejega serumskega albumina, izbranim štirim sondam pa smo določili tudi navidezno vezavno afiniteto do fibril Aβ1–42 in fibril inzulina. Med preiskovanimi sondami je najizrazitejši odziv fluorescence ob prisotnosti fibril Aβ1–42 imel kumarin SP-1Z-1, ki je imel tudi največjo navidezno vezavno afiniteto med sondami z določljivo vrednostjo Kd. Vse štiri sonde so se vezale tudi na fibrile inzulina, pri čemer so opažene spremembe emisijskih maksimumov razkrile, da na njihove spektralne lastnosti vpliva lokalno mikrookolje vezavnega mesta. V prisotnosti neagregiranega Aβ1–42 je bil fluorescenčni odziv večine sond nizek, kar kaže, da je bil odziv povezan z vezavo na fibrilarno obliko peptida. Izjema je bila sonda SP-ES-17, ki je imela izrazitejši odziv emisije fluorescence tudi v prisotnosti neagregiranega Aβ1–42 ter serumskih albuminov, s čimer je pokazala njeno večjo tendenco za nespecifično vezavo, torej manjšo selektivnost. Med preiskovanimi sondami je imela sonda SP-1Z-1 najugodnejši profil za zaznavanje fibrilarnega Aβ1–42, kljub temu pa izrazit odziv emisije fluorescence in velika navidezna afiniteta do fibril inzulina kažeta, da ni selektivna izključno za fibrile Aβ1–42.

• Jezik: Slovenski jezik
• Ključne besede: Alzheimerjeva bolezen, amiloid beta, fibrile, fluorescenčne sonde
• Vrsta gradiva: Magistrsko delo/naloga
• Tipologija: 2.09 - Magistrsko delo
• Organizacija: FFA - Fakulteta za farmacijo
• Založnik: [A. Grbić]
• Leto izida: 2026
• PID: 20.500.12556/RUL-188907
• UDK: 616.894(043.2)
• COBISS.SI-ID: 293769475
• Datum objave v RUL: 30.09.2026

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Evaluation of spectral properties of coumarin, stilbene, and pyrrole derivatives upon binding to β-amyloid fibrils

Izvleček

The most common cause of dementia is a chronic neurodegenerative disease known as Alzheimer's disease. One of its characteristic pathological changes is the accumulation of amyloid β (Aβ). Because these changes in the brain begin to develop before the onset of pronounced clinical symptoms, such as memory loss and concentration impairment, their early detection is important for a timely and more reliable diagnosis. Near-infrared fluorescent probes represent one approach for non-invasive detection of pathological changes associated with Alzheimer's disease and could contribute to its earlier diagnosis. In the master's thesis, we investigated the relationships between the structure of structurally diverse fluorescent probes and their fluorescence response intensity, apparent binding affinity, and selectivity towards various proteins in their native or aggregated states. The emission spectra of the probes were first measured in HEPES buffer and in the presence of Aβ1–42 fibrils. Based on the increase in fluorescence intensity, probes SP-1Z-1, SP-sp5, SP-ES-17 and SP-sp9 were selected for further evaluation. Their fluorescence responses were subsequently compared with that observed in the presence of insulin fibrils, non-aggregated Aβ1–42, human and bovine serum albumin. The apparent binding affinity of the four selected probes for Aβ1–42 and insulin fibrils were also determined. Among the probes investigated, the coumarin derivative SP-1Z-1 exhibited the most pronounced fluorescence response to Aβ1–42 fibrils and the highest apparent binding affinity among the probes for which a Kd value could be determined. All four probes also bound to insulin fibrils, and the observed shifts in their emission maxima revealed that their spectral properties were influenced by the local microenvironment of the binding site. In the presence of non-aggregated Aβ1–42, most probes exhibited only a weak fluorescence response, indicating that their response was associated with binding to the fibrillar form of the peptide. The exception was SP-ES-17, which also displayed a more pronounced response in the presence of non-aggregated Aβ1–42 and serum albumins, demonstrating a greater tendency towards nonspecific binding and, consequently, lower selectivity. Among the probes investigated, SP-1Z-1 showed the most favorable profile for the detection of fibrillar Aβ1–42 Nevertheless, its pronounced fluorescence response and high apparent affinity for insulin fibrils indicate that it is not exclusively selective for Aβ1–42 fibrils.

• Ključne besede: Alzheimer's disease, amyloid beta, fibrils, fluorescent probes