Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC, Leiden, The Netherlands.
Department of Drug Discovery and Development, Harrison School of Pharmacy, Auburn University, Auburn, AL 36849, USA.
Chembiochem. 2020 Nov 16;21(22):3220-3224. doi: 10.1002/cbic.202000375. Epub 2020 Jul 29.
Proteasomes are established therapeutic targets for hematological cancers and promising targets for autoimmune diseases. In the past, we have designed and synthesized mechanism-based proteasome inhibitors that are selective for the individual catalytic activities of human constitutive proteasomes and immunoproteasomes: β1c, β1i, β2c, β2i, β5c and β5i. We show here that by taking the oligopeptide recognition element and substituting the electrophile for a fluorogenic leaving group, fluorogenic substrates are obtained that report on the proteasome catalytic activity also targeted by the parent inhibitor. Though not generally applicable (β5c and β2i substrates showing low activity), effective fluorogenic substrates reporting on the individual activity of β1c, β1i, β2c and β5i subunits in Raji (human B cell) lysates and purified 20S proteasome were identified in this manner. Our work thus adds to the expanding proteasome research toolbox through the identification of new and/or more effective subunit-selective fluorogenic substrates.
蛋白酶体是血液系统癌症的既定治疗靶点，也是自身免疫性疾病的有前途的靶点。过去，我们设计并合成了基于机制的蛋白酶体抑制剂，这些抑制剂对人组成型蛋白酶体和免疫蛋白酶体的各个催化活性具有选择性：β1c、β1i、β2c、β2i、β5c 和 β5i。我们在这里表明，通过采用寡肽识别元件并将亲电试剂替换为荧光离去基团，可以获得报告蛋白酶体催化活性的荧光底物，该活性也是亲本抑制剂的靶标。虽然不是普遍适用的（β5c 和 β2i 底物显示低活性），但通过这种方式在 Raji（人 B 细胞）裂解物和纯化的 20S 蛋白酶体中鉴定出了有效报告β1c、β1i、β2c 和 β5i 亚基的个别活性的荧光底物。因此，我们的工作通过鉴定新的和/或更有效的亚基选择性荧光底物，为不断扩大的蛋白酶体研究工具包增添了内容。
