Maretzky Thorsten, Swendeman Steven, Mogollon Elin, Weskamp Gisela, Sahin Umut, Reiss Karina, Blobel Carl P
Arthritis and Tissue Degeneration Program, Hospital for Special Surgery at Weill Cornell Medicine, New York, NY 10021, U.S.A.
Boston Children's Hospital, Boston, MA 02115, U.S.A.
Biochem J. 2017 Apr 13;474(9):1467-1479. doi: 10.1042/BCJ20170075.
ADAM9 (A Disintegrin And Metalloprotease 9) is a membrane-anchored metalloproteinase that has been implicated in pathological retinal neovascularization and in tumor progression. ADAM9 has constitutive catalytic activity in both biochemical and cell-based assays and can cleave several membrane proteins, including epidermal growth factor and Ephrin receptor B4; yet little is currently known about the catalytic properties of ADAM9 and its post-translational regulation and inhibitor profile in cell-based assays. To address this question, we monitored processing of the membrane-anchored Ephrin receptor B4 (EphB4) by co-expressing ADAM9, with the catalytically inactive ADAM9 E > A mutant serving as a negative control. We found that ADAM9-dependent shedding of EphB4 was not stimulated by three commonly employed activators of ADAM-dependent ectodomain shedding: phorbol esters, pervanadate or calcium ionophores. With respect to the inhibitor profile, we found that ADAM9 was inhibited by the hydroxamate-based metalloprotease inhibitors marimastat, TAPI-2, BB94, GM6001 and GW280264X, and by 10 nM of the tissue inhibitor of metalloproteinases (TIMP)-3, but not by up to 20 nM of TIMP-1 or -2. Additionally, we screened a non-hydroxamate small-molecule library for novel ADAM9 inhibitors and identified four compounds that selectively inhibited ADAM9-dependent proteolysis over ADAM10- or ADAM17-dependent processing. Taken together, the present study provides new information about the molecular fingerprint of ADAM9 in cell-based assays by showing that it is not stimulated by strong activators of ectodomain shedding and by defining a characteristic inhibitor profile. The identification of novel non-hydroxamate inhibitors of ADAM9 could provide the basis for designing more selective compounds that block the contribution of ADAM9 to pathological neovascularization and cancer.
ADAM9（一种解聚素和金属蛋白酶9）是一种膜锚定金属蛋白酶，与病理性视网膜新生血管形成和肿瘤进展有关。在生化和基于细胞的实验中，ADAM9都具有组成性催化活性，并且可以切割多种膜蛋白，包括表皮生长因子和Ephrin受体B4；然而，目前对于ADAM9的催化特性及其在基于细胞的实验中的翻译后调控和抑制剂谱了解甚少。为了解决这个问题，我们通过共表达ADAM9来监测膜锚定的Ephrin受体B4（EphB4）的加工过程，将催化无活性的ADAM9 E>A突变体作为阴性对照。我们发现，ADAM9依赖性的EphB4脱落不受三种常用的ADAM依赖性胞外域脱落激活剂的刺激：佛波酯、过钒酸盐或钙离子载体。关于抑制剂谱，我们发现ADAM9受到基于异羟肟酸的金属蛋白酶抑制剂马立马司他、TAPI-2、BB94、GM6001和GW280264X的抑制，以及10 nM的金属蛋白酶组织抑制剂（TIMP）-3的抑制，但不受高达20 nM的TIMP-1或-2的抑制。此外，我们筛选了一个非异羟肟酸小分子文库以寻找新型ADAM9抑制剂，并鉴定出四种化合物，它们对ADAM9依赖性蛋白水解的抑制作用优于对ADAM10或ADAM17依赖性加工的抑制作用。综上所述，本研究通过表明ADAM9不受胞外域脱落强激活剂的刺激并定义了其特征性抑制剂谱，提供了关于ADAM9在基于细胞的实验中的分子指纹的新信息。新型非异羟肟酸ADAM9抑制剂的鉴定可为设计更具选择性的化合物提供基础，这些化合物可阻断ADAM9对病理性新生血管形成和癌症的作用。
