Department of Biotechnology, Faculty of Biological Sciences, Alzahra University, Tehran, Iran.
Pharmaceutical Sciences Research Center, Health Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran.
Biophys Chem. 2021 Feb;269:106522. doi: 10.1016/j.bpc.2020.106522. Epub 2020 Dec 15.
Neurodegenerative disease is caused by the abnormal build-up of proteins in and around cells called amyloid. The amyloid fibril formation and its mechanism have been investigated with various techniques, including dye-binding assay. Thioflavin T (ThT) has been one of the most widely used dyes for quantifying amyloid deposits, but ThT has a weak fluorescence signal especially at low concentration of amyloid fibrils, low lipophilicity and positive charge that makes it unable to cross the blood-brain barrier (BBB) to detect amyloid fibrils in vivo. Hence, there is a strong motivation for designing and developing the new compounds for in vitro amyloid quantification and in vivo amyloid imaging. The need for new probes to detect amyloid fibrils, especially within the cell, is highlighted by the fact that an accurate understanding of the molecular details of amyloid fibril formation is required to design and develop strategies for controlling the amyloid formation, and this needs more reliable probes for amyloid identification. In this work, we synthesized and applied barbituric and thiobarbituric acid-based chromene derivatives, as new fluorescent dyes to quantitatively detect the amyloid fibrils of bovine serum albumin (BSA) and human insulin in comparison with native soluble proteins or amorphous aggregation. Our results showed that among the 14 synthesized compounds, five compounds 4a, 4h, 4j, 4k, and 4l could selectively and specifically bind to amyloid fibrils while other compounds demonstrated a low-affinity binding. Furthermore, according to the cell viability experiment, compounds 4a, 4j and 4l at low concentration of compounds are not toxic, especially compound 4j which could be used as a suitable candidate for in vivo study. Further studies are needed to determine all the properties of compounds, especially in vivo experiments.
神经退行性疾病是由细胞内和细胞周围称为淀粉样蛋白的蛋白质异常堆积引起的。已经使用各种技术，包括染料结合测定法，研究了淀粉样纤维的形成及其机制。噻唑蓝（ThT）是用于定量淀粉样沉积物的最广泛使用的染料之一，但 ThT 的荧光信号很弱，尤其是在淀粉样纤维的低浓度、低脂溶性和正电荷的情况下，使其无法穿过血脑屏障（BBB）来检测体内的淀粉样纤维。因此，强烈需要设计和开发新的化合物来进行体外淀粉样定量和体内淀粉样成像。需要新的探针来检测淀粉样纤维，特别是在细胞内，这突出表明需要更可靠的淀粉样鉴定探针，以准确了解淀粉样纤维形成的分子细节，从而设计和开发控制淀粉样形成的策略。在这项工作中，我们合成并应用了巴比妥酸和硫代巴比妥酸为基础的色烯衍生物，作为新的荧光染料，与天然可溶性蛋白质或无定形聚集物相比，定量检测牛血清白蛋白（BSA）和人胰岛素的淀粉样纤维。我们的结果表明，在合成的 14 种化合物中，有 5 种化合物 4a、4h、4j、4k 和 4l 可以选择性和特异性地与淀粉样纤维结合，而其他化合物则表现出低亲和力结合。此外，根据细胞活力实验，在低浓度的化合物下，化合物 4a、4j 和 4l 没有毒性，特别是化合物 4j 可以作为体内研究的合适候选物。需要进一步研究以确定化合物的所有特性，特别是体内实验。
