Fluorescent substrates for the proteinases ADAM17, ADAM10, ADAM8, and ADAM12 useful for high-throughput inhibitor screening
Section snippets
Materials and methods
Recombinant human TACE, ADAM10, and ADAM8 were obtained from R & D Systems. Human ADAM12 was prepared as described [17]. The catalytic domain of human ADAM17 was the kind gift of Marcos Milla, Roche Bioscience. Matrix metalloproteinases (MMPs) were the kind gifts of Gillian Murphy, Cambridge University, Hideaki Nagase, Imperial College of London, Chris Overall, University of British Colombia, Canada, and William Stetler-Stevenson, National Cancer Institute. Fluorescent substrates were obtained
Substrates for TACE, ADAM10, ADAM8, and ADAM12
We searched for substrates for TACE, ADAM10, ADAM8, and ADAM12 which are fluorescent and sensitive and have reasonably large specificity constants; these qualities are useful for high-throughput screening. The first substrate, Dabcyl-Pro-Cha-Gly-Cys(Me)-His-Ala-Lys(Fam)-NH2, was chosen because its peptide sequence was reported to be good for an ADAM family member, even though it was originally designed for collagenase 1 and other matrix metalloproteinases [7], [20]. TACE and ADAM10, 8, and 12
Conclusion
TACE and ADAM10, 8, and 12 represent potential therapeutic targets for investigation. TACE inhibition could lead to new treatments for cancer and/or arthritis [25]. ADAM10 is able to release membrane-bound CD23 or betacellulin and epidermal growth factor to generate soluble forms, suggesting that inhibitors of this family member could be useful for allergic responses and cancer, respectively [22], [24], [26]. ADAM8 is also a CD23 sheddase [23], so targeting it may also treat allergic responses,
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