Department of Chemistry, Molecular Sciences Research Hub, Imperial College London, London W12 0BZ, U.K.
Mater Research Institute-The University of Queensland, Translational Research Institute, Woolloongabba, QLD 4102, Australia.
J Am Chem Soc. 2021 Jun 16;143(23):8911-8924. doi: 10.1021/jacs.1c03950. Epub 2021 Jun 4.
Kallikrein-related peptidases (KLKs) are a family of secreted serine proteases, which form a network (the KLK activome) with an important role in proteolysis and signaling. In prostate cancer (PCa), increased KLK activity promotes tumor growth and metastasis through multiple biochemical pathways, and specific quantification and tracking of changes in the KLK activome could contribute to validation of KLKs as potential drug targets. Herein we report a technology platform based on novel activity-based probes (ABPs) and inhibitors enabling simultaneous orthogonal analysis of KLK2, KLK3, and KLK14 activity in hormone-responsive PCa cell lines and tumor homogenates. Importantly, we identifed a significant decoupling of KLK activity and abundance and suggest that KLK proteolysis should be considered as an additional parameter, along with the PSA blood test, for accurate PCa diagnosis and monitoring. Using selective inhibitors and multiplexed fluorescent activity-based protein profiling (ABPP), we dissect the KLK activome in PCa cells and show that increased KLK14 activity leads to a migratory phenotype. Furthermore, using biotinylated ABPs, we show that active KLK molecules are secreted into the bone microenvironment by PCa cells following stimulation by osteoblasts suggesting KLK-mediated signaling mechanisms could contribute to PCa metastasis to bone. Together our findings show that ABPP is a powerful approach to dissect dysregulation of the KLK activome as a promising and previously underappreciated therapeutic target in advanced PCa.
激肽释放酶相关肽酶（KLKs）是一组分泌的丝氨酸蛋白酶，它们形成了一个具有重要蛋白水解和信号作用的网络（KLK 激活组）。在前列腺癌（PCa）中，KLK 活性的增加通过多种生化途径促进肿瘤生长和转移，对 KLK 激活组变化的特异性定量和跟踪可能有助于验证 KLKs 作为潜在的药物靶点。在此，我们报告了一个基于新型活性探针（ABP）和抑制剂的技术平台，能够在激素反应性 PCa 细胞系和肿瘤组织匀浆中同时进行 KLK2、KLK3 和 KLK14 活性的正交分析。重要的是，我们发现 KLK 活性和丰度存在显著的解耦，并且表明 KLK 蛋白水解应该被视为除 PSA 血液检测之外的另一个参数，用于准确诊断和监测 PCa。使用选择性抑制剂和多重荧光活性探针蛋白谱分析（ABPP），我们在 PCa 细胞中剖析了 KLK 激活组，并表明 KLK14 活性的增加导致迁移表型。此外，使用生物素化的 ABP，我们表明在成骨细胞刺激后，PCa 细胞将活性 KLK 分子分泌到骨微环境中，这表明 KLK 介导的信号机制可能有助于 PCa 向骨骼转移。总之，我们的研究结果表明，ABPP 是一种剖析 KLK 激活组失调的有力方法，作为晚期 PCa 中一种有前途且以前未被充分认识的治疗靶点。
