Fluorescent substrates for the proteinases ADAM17, ADAM10, ADAM8, and ADAM12 useful for high-throughput inhibitor screening
Fluorescent substrates for the proteinases ADAM17, ADAM10, ADAM8, and ADAM12 useful for high-throughput inhibitor screening
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DOI：
10.1016/j.ab.2007.04.043
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发表时间：
2007-07-15
影响因子：
2.9
通讯作者：
Rasmussen, Fred H.
中科院分区：
文献类型：
--
作者：
Moss, Marcia L.;Rasmussen, Fred H.
在这篇文章中，我们描述了人类ADAM家族成员ADAM17、ADAM10、ADAM8和ADAM12的新型荧光底物，这些底物具有良好的特异性常数，有助于高通量筛选抑制剂。荧光共振能量转移底物含有4-(4-二甲氨基苯偶氮)苯甲酰和5-羧基荧光素(Dabcyl/Fam)对，并基于前体肿瘤坏死因子-α(TNF-α)和CD23的已知切割序列。以肿瘤坏死因子-α为基础的前体底物Dabcyl-Leu-Ala-Gln-Ala-Homophe-Arg-Ser-Lys(Fam)-NH2，是所有受试ADAMS的良好底物，包括尚未报道的荧光底物ADAM12。以CD23为底物的Dabeyl-His-Gly-Asp-Gln-Met-Ala-Gln-Lys-Ser-Lys(Fam)-NH2，具有更高的选择性，仅能被ADAM8和ADAM10有效地降解。用底物测定了TMI-1、GM6001、GW9471和TAPI-2四种常用抑制剂的抑制常数。惠氏Aerst化合物TMI-1是一种对所有测试的ADAMS有效的抑制剂，与ADAM17结合缓慢。(C)2007 Elsevier Inc.保留所有权利。
In this paper we describe novel fluorescent substrates for the human ADAM family members ADAM17, ADAM10, ADAM8, and ADAM12 that have good specificity constants and are useful for high-throughput screening of inhibitors. The fluorescence resonance energy transfer substrates contain a 4-(4-dimethylaminophenylazo)benzoyl and 5-carboxyfluorescein (Dabcyl/Fam) pair and are based on known cleavage sequences in precursor tumor necrosis factor-alpha (TNF-alpha) and CD23. The precursor TNF-alpba-based substrate, Dabcyl-Leu-Ala-Gln-Ala-Homophe-Arg-Ser-Lys(Fam)-NH2, is a good substrate for all the ADAMs tested, including ADAM12 for which there is no reported fluorescent substrate. The CD23-based substrate, Dabeyl-His-Gly-Asp-Gln-Met-Ala-Gln-Lys-Ser-Lys(Fam)-NH2, is more selective, being hydrolyzed efficiently only by ADAM8 and ADAM10. The substrates were used to obtain inhibition constants for four inhibitors that are commonly used in shedding assays: TMI-1, GM6001, GW9471, and TAPI-2. The Wyeth Aerst compound, TMI-1, is a potent inhibitor against all of the ADAMs tested and is slow binding against ADAM17. (c) 2007 Elsevier Inc. All rights reserved.