Vrednotenje vezave fluorescenčnih sond s fenilnim in difenilacetilenskim skeletom na fibrile inzulina, amiloida β in lizocima

Bedek, Ana (avtor), Knez, Damijan (mentor)

Trenutno najpogostejša nevrodegenerativna bolezen je Alzheimerjeva bolezen (AB), ki je glavni razlog za demenco. Za njo je značilna izguba spomina, težave z govorom in vsakdanjimi opravili. Terapija ta hip je precej omejena in neučinkovita, bi pa k boljšim rezultatom zdravljenja lahko pripomogla zgodnejša diagnoza. Eden od pokazateljev AB pred prvimi simptomi je prisotnost amiloidnih plakov v možganski skorji, zato ima zgodnja detekcija le-teh dober potencial za uspešnejše zdravljenje. Pri tem so uporabne nekatere fluorescenčne sonde, ki ob vezavi na tovrstne fibrile povečajo emisijo fluorescence. Te sonde imajo značilno strukturo: ena elektrondonorska in ena elektronakceptorska skupina, ki ju povezuje distančnik – različni π-sistemi, ki omogočajo vzpostavitev »push-pull« sistema. Za uspešno diagnozo so torej zaželene spojine z visoko občutljivostjo in specifičnostjo za amiloidne plake, zato smo v ta namen okarakterizirali novo serijo pironskih sond in preverili njihove vezavne lastnosti. Pomerili smo absorbanco in ojačenje signala emisije v prisotnosti fibril Aβ1–42 ter vezavno afiniteto sond. Ugotovili smo, da pri vezavi nekaterih sond na fibrile Aβ1–42 pride do sprememb v okolju, kar vodi v omejeno intramolekularno vrtljivost, s tem pa se poveča emisija fluorescence. Največje ojačenje signala so pokazale sonde ALZ177, AnaBr-14a ter AnaBr-16a, precej manjše povečanje signala pa smo pomerili za AnaBr-4b ter ALZ153, ki imata visok odziv že v pufrni raztopini. Razlog je verjetno v samoasociaciji zaradi slabše vodotopnosti. Fluorescenčnim sondam smo pomerili spektralne lastnosti tudi po vezavi na fibrile inzulina. Pri tem so se rezultati precej ujemali z meritvami vezave na fibrile Aβ1–42, to pa nakazuje na slabšo specifičnost vezave. Pri podrobni analizi maksimumov emisije fluorescence smo opazili, da so le-ti po vezavi na fibrile Aβ1–42 ter inzulina malenkost drugačni, zato bi lahko s pomočjo fluorescenčne mikroskopije razlikovali med tema dvema vrstama fibril. Glede na pomerjene vrednosti vezavne afinitete je najbolj optimalna spojina AnaBr-16a, ki ima v strukturi številne funkcionalne skupine, s katerimi lahko poleg nespecifičnih interakcij tvori tudi usmerjene vodikove vezi ter dipol-dipol interakcije. Za nadaljnji razvoj je poleg sonde AnaBr-16a perspektivna tudi ALZ177, saj imata obe visoko ojačenje intenzitete emisije fluorescence ter visoko vezavno afiniteto.

• Jezik: Slovenski jezik
• Ključne besede: amiloid β, emisija, fibrile, inzulin, lizocim, vezavna afiniteta
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2024
• PID: 20.500.12556/RUL-161714
• Datum objave v RUL: 13.09.2024

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Evaluation of fluorescent probes with phenyl and diphenylacetylene scaffolds binding to insulin, β-amyloid and lysozyme fibrils

Izvleček

Currently, the most common neurodegenerative disease is Alzheimer's disease (AD), which is characterized by the loss of memory, impairment in language and execution of everyday tasks, and is the main cause of dementia. Therapy at this stage is quite limited and ineffective, but earlier diagnosis could help to improve treatment outcomes. One of the indicators of AD prior to the onset of the symptoms is the presence of amyloid plaques in the cerebral cortex, and their early detection offers good potential for a more successful treatment. Fluorescent probes, which increase the emission of fluorescence when bound to such fibrils, would be of great value in the diagnostics. These probes have a characteristic structure: an electron-donor and an electron-acceptor group connected by linkers – π-systems that allow the establishment of a "push-pull" system. Compounds with high sensitivity and specificity for amyloid plaques are therefore desirable for successful diagnosis. To this end, we have characterized a new series of pyrone-based probes and tested their binding properties. We measured the absorbance and amplification of the emission signal in the presence of Aβ1–42 fibrils as well as the binding affinity of the probes. The binding of the probes to Aβ1–42 fibrils is associated with changes in the environment that result in limited intramolecular rotation, thereby increasing the fluorescence emission. Probes ALZ177, AnaBr-14a and AnaBr-16a showed the highest signal enhancement, while a much lower signal enhancement was measured for AnaBr-4b and ALZ153, which already show high responses in the buffer. The reason for this is presumably self-association due to the poor aqueous solubility. The spectral properties of the fluorescent probes were also measured after binding to fibrils of insulin. Here, the results agreed well with the measurements of binding to Aβ1–42 fibrils, indicating lower binding specificity. When we analyzed the fluorescence emission maxima in detail, we found that they were slightly different after binding to Aβ1–42 and insulin fibrils, which would allow to distinguish between these two using fluorescence microscopy. According to the measured binding affinity values, the most optimal probe is AnaBr-16a, which has a number of functional groups that can in addition to nonspecific interactions form directed hydrogen bonds and dipole-dipole interactions. In addition to the AnaBr-16a, ALZ177 is also promising for further development, since they both display high amplification of fluorescence emission intensity and have high binding affinities.

• Ključne besede: amyloid β, emission, fibrils, insulin, lysozyme, binding affinity