Analysis of gamma secretase inhibitors as potential drugs for Alzheimer's disease on SH-SY5Y cells

Značaj: Alzheimerova bolest je smrtonosan neurodegenerativni poremećaj za kojeg još uvijek nije pronađen učinkovit lijek. Zbog stalnog porasta incidencije i jako skupe zdravstvene skrbi oboljelih, Alzheimerova bolest predstavlja jedan od najvećih izazova za farmaceutsku industriju 21. stoljeća. Postoji više različitih teorija o patogenezi bolesti, a do danas najbolje potvrđena je amiloidna hipoteza. Prema njoj neurodegenerativni procesi rezultat su poremećaja u metabolizmu amiloidnih proteina koji nastaju zbog promjena u djelovanju mebranske proteaze γ-sekretaze. Stoga, γ-sekretaza predstavlja glavni put u razvoju potencijalnih lijekova za Alzheimerovu bolest. Rezultati: Prvo smo optimizirali Sandwich ELISA-u za detekciju unutarstaničnog Aβ1-40 proteina u stanicama ljudskog neuroblastoma SH-SY5Y koje nisu genetički modificirane. Pripremljeni protokol koristili smo za ispitivanje potencijalnih lijekova za Alzheimerovu bolest koje razvija farmaceutska tvrtka Jiva Pharma. Svi JIVA spojevi pokazali su inhibiciju aktivnosti γ-sekretaze u koncentracijama od 10 μM i 100 μM. Najveća inhibicija zapažena je kod spojeva JIVA0028 i JIVA0029, koji su smanjili aktivnost enzima za više od 80%. Najveću razliku inhibicije između dvije ispitivane koncentracije pokazao je spoj JIVA0041, dok kod spoja JIVA0026 nije uočena značajna razlika. Niti jedan ispitivani spoj nije utjecao na morfologiju stanica niti je u danim koncentracijama bio letalan za stanice. Zaključak: Svi JIVA spojevi predstavljaju potencijalne lijekove za Alzheimerovu bolest. Međutim, najzanimljiviji su spojevi JIVA0028 i JIVA0029 koji su inhibirali aktivnost γ-sekretaze gotovo jednako kao jedan od najpoznatijih inhibitora γ- sekretaze semagacestat. Time predstavljaju obećavajuće strukture koje se mogu dalje modificirati kako bi se dobili novi, još potentniji spojevi.Significance: Alzheimer's disease is a fatal neurodegenerative disorder that currently has no effective treatment. Alzheimer's disease is one of the biggest challenges for the pharmaceutical industry of the 21st century as the most expensive disease in the current health care system in the industrialised world. There are several different theories on the pathogenesis of the disease, to this date the amyloid hypothesis appears to be the most credible. According to amyloid hypothesis, the neurodegenerative processes are the result of disorders in the metabolism of amyloid proteins that occur due to changes in the function of the membrane embedded protease, γ-secretase. Therefore, γ-secretase is currently the main target in the development of potential drug candidates for Alzheimer's disease. Results: We first optimized the Sandwich ELISA for the detection of intracellular Aβ1-40 protein in non-genetically modified human neuroblastoma cells SH-SY5Y. The created protocol was used to test potential drugs for Alzheimer's disease that are developed by the pharmaceutical company Jiva Pharma. All JIVA compounds showed inhibition of γ-secretase activity at concentrations of 10 μM and 100 μM. The highest inhibition was observed in JIVA0028 and JIVA0029, which reduced enzyme activity by more than 80%. The highest difference in inhibition between the two concentrations was shown in JIVA0041, while no significant difference was observed with JIVA0026. No JIVA compound had any effect on the morphology of the cells or was lethal for the cells at given concentrations. Conclusion: All JIVA compounds represent potential drugs for Alzheimer's disease. The most interesting compounds are JIVA0028 and JIVA0029 who showed inhibition of γ-secretase activity almost as good as semagacestat, one of the best known inhibitors of γ-secretase. Thus, their structures can be further modified to obtain new, more potent compounds