Vezenkov Lubomir, Honson Nicolette S, Kumar Nag S, Bosc Damien, Kovacic Suzana, Nguyen Thanh G, Pfeifer Tom A, Young Robert N
Department of Chemistry, Simon Fraser University, Burnaby, BC, Canada.
Centre for Drug Research and Development, Vancouver, BC, Canada.
Bioorg Med Chem. 2015 Jul 1;23(13):3237-47. doi: 10.1016/j.bmc.2015.04.064. Epub 2015 Apr 28.
An efficient assay for monitoring the activity of the key autophagy-initiating enzyme ATG4B based on a small peptide substrate has been developed. A number of putative small fluorogenic peptide substrates were prepared and evaluated and optimized compounds showed reasonable rates of cleavage but required high enzyme concentrations which limited their value. A modified peptide substrate incorporating a less sterically demanding self-immolative element was designed and synthesized and was shown to have enhanced properties useful for evaluating inhibitors of ATG4B. Substrate cleavage was readily monitored and was linear for up to 4h but enzyme concentrations of about ten-fold higher were required compared to assays using protein substrate LC3 or analogs thereof (such as FRET-LC3). Several known inhibitors of ATG4B were evaluated using the small peptide substrate and gave IC50 values 3-7 fold higher than previously obtained values using the FRET-LC3 substrate.
基于一种小肽底物开发了一种用于监测关键自噬起始酶ATG4B活性的高效检测方法。制备并评估了多种假定的小荧光肽底物，优化后的化合物显示出合理的切割速率，但需要高酶浓度，这限制了它们的价值。设计并合成了一种包含空间位阻较小的自裂解元件的修饰肽底物，该底物显示出对评估ATG4B抑制剂有用的增强特性。底物切割易于监测，长达4小时呈线性，但与使用蛋白质底物LC3或其类似物（如FRET-LC3）的检测相比，所需的酶浓度要高约十倍。使用小肽底物评估了几种已知的ATG4B抑制剂，其IC50值比先前使用FRET-LC3底物获得的值高3至7倍。
