Faculty of Chemistry, University of Gdansk, 80-308 Gdansk, Poland.
Malopolska Centre of Biotechnology, Jagiellonian University, 30-387 Krakow, Poland.
Int J Mol Sci. 2019 Mar 28;20(7):1557. doi: 10.3390/ijms20071557.
Kallikrein 13 (KLK13) was first identified as an enzyme that is downregulated in a subset of breast tumors. This serine protease has since been implicated in a number of pathological processes including ovarian, lung and gastric cancers. Here we report the design, synthesis and deconvolution of libraries of internally quenched fluorogenic peptide substrates to determine the specificity of substrate binding subsites of KLK13 in prime and non-prime regions (according to the Schechter and Berger convention). The substrate with the consensus sequential motive ABZ-Val-Arg-Phe-Arg-ANB-NH₂ demonstrated selectivity towards KLK13 and was successfully converted into an activity-based probe by the incorporation of a chloromethylketone warhead and biotin bait. The compounds described may serve as suitable tools to detect KLK13 activity in diverse biological samples, as exemplified by overexpression experiments and targeted labeling of KLK13 in cell lysates and saliva. In addition, we describe the development of selective activity-based probes targeting KLK13, to our knowledge the first tool to analyze the presence of the active enzyme in biological samples.
激肽释放酶 13（KLK13）最初被鉴定为在一组乳腺癌肿瘤中下调的酶。此后，这种丝氨酸蛋白酶已被牵连到许多病理过程中，包括卵巢癌、肺癌和胃癌。在这里，我们报告了内部猝灭荧光肽底物文库的设计、合成和解卷积，以确定 KLK13 在主要和非主要区域（根据 Schechter 和 Berger 公约）的底物结合亚基的特异性。具有共识序列基序 ABZ-Val-Arg-Phe-Arg-ANB-NH₂的底物对 KLK13 具有选择性，并通过引入氯甲基酮弹头和生物素诱饵成功转化为活性探针。所描述的化合物可以作为在各种生物样品中检测 KLK13 活性的合适工具，如过表达实验和 KLK13 在细胞裂解物和唾液中的靶向标记所例示的。此外，我们还描述了针对 KLK13 的选择性活性探针的开发，据我们所知，这是第一个用于分析生物样品中活性酶存在的工具。
