Torrey Pines Institute for Molecular Studies, Port St. Lucie, Florida 34987, USA.
J Biol Chem. 2012 Oct 19;287(43):36473-87. doi: 10.1074/jbc.M112.389114. Epub 2012 Aug 27.
A disintegrin and metalloprotease (ADAM) proteases are implicated in multiple diseases, but no drugs based on ADAM inhibition exist. Most of the ADAM inhibitors developed to date feature zinc-binding moieties that target the active site zinc, which leads to a lack of selectivity and off-target toxicity. We hypothesized that secondary binding site (exosite) inhibitors should provide a viable alternative to active site inhibitors. Potential exosites in ADAM structures have been reported, but no studies describing substrate features necessary for exosite interactions exist. Analysis of ADAM cognate substrates revealed that glycosylation is often present in the vicinity of the scissile bond. To study whether glycosylation plays a role in modulating ADAM activity, a tumor necrosis factor α (TNFα) substrate with and without a glycan moiety attached was synthesized and characterized. Glycosylation enhanced ADAM8 and -17 activities and decreased ADAM10 activity. Metalloprotease (MMP) activity was unaffected by TNFα substrate glycosylation. High throughput screening assays were developed using glycosylated and non-glycosylated substrate, and positional scanning was conducted. A novel chemotype of ADAM17-selective probes was discovered from the TPIMS library (Houghten, R. A., Pinilla, C., Giulianotti, M. A., Appel, J. R., Dooley, C. T., Nefzi, A., Ostresh, J. M., Yu, Y., Maggiora, G. M., Medina-Franco, J. L., Brunner, D., and Schneider, J. (2008) Strategies for the use of mixture-based synthetic combinatorial libraries. Scaffold ranking, direct testing in vivo, and enhanced deconvolution by computational methods. J. Comb. Chem. 10, 3-19; Pinilla, C., Appel, J. R., Borràs, E., and Houghten, R. A. (2003) Advances in the use of synthetic combinatorial chemistry. Mixture-based libraries. Nat. Med. 9, 118-122) that preferentially inhibited glycosylated substrate hydrolysis and spared ADAM10, MMP-8, and MMP-14. Kinetic studies revealed that ADAM17 inhibition occurred via a non-zinc-binding mechanism. Thus, modulation of proteolysis via glycosylation may be used for identifying novel, potentially exosite binding compounds. The newly described ADAM17 inhibitors represent research tools to investigate the role of ADAM17 in the progression of various diseases.
解整合素金属蛋白酶（ADAM）蛋白酶与多种疾病有关，但目前尚无基于 ADAM 抑制作用的药物。迄今为止开发的大多数 ADAM 抑制剂都具有靶向活性位点锌的锌结合部分，这导致缺乏选择性和非靶毒性。我们假设次级结合位点（外位）抑制剂应该是活性位点抑制剂的可行替代品。ADAM 结构中的潜在外位已被报道，但没有描述底物特征对于外位相互作用是必要的研究。对 ADAM 同源底物的分析表明，糖基化通常存在于切口键附近。为了研究糖基化是否在调节 ADAM 活性中起作用，合成并表征了具有和不具有连接的聚糖部分的肿瘤坏死因子 α（TNFα）底物。糖基化增强了 ADAM8 和 -17 的活性，降低了 ADAM10 的活性。金属蛋白酶（MMP）活性不受 TNFα 底物糖基化的影响。使用糖基化和非糖基化底物开发了高通量筛选测定，并进行了位置扫描。从 TPIMS 文库中发现了一种新型的 ADAM17 选择性探针的化学型（Houghten，R. A.，Pinilla，C.，Giulianotti，M. A.，Appel，J. R.，Dooley，C. T.，Nefzi，A.，Ostresh，J. M.，Yu，Y.，Maggiora，G. M.，Medina-Franco，J. L.，Brunner，D.，和 Schneider，J.（2008）混合物基合成组合化学的使用策略。支架排名，直接体内测试和通过计算方法增强去卷积。J. Comb. Chem. 10, 3-19;Pinilla，C.，Appel，J. R.，Borràs，E.，和 Houghten，R. A.（2003）合成组合化学的最新进展。混合物库。自然医学。9, 118-122），它们优先抑制糖基化底物水解，并保留 ADAM10、MMP-8 和 MMP-14。动力学研究表明，ADAM17 的抑制作用是通过非锌结合机制发生的。因此，通过糖基化调节蛋白水解作用可能用于鉴定新型潜在的外位结合化合物。新描述的 ADAM17 抑制剂代表了研究工具，可用于研究 ADAM17 在各种疾病进展中的作用。
