Elsevier

Analytical Biochemistry

Volume 366, Issue 2, 15 July 2007, Pages 144-148
Analytical Biochemistry

Fluorescent substrates for the proteinases ADAM17, ADAM10, ADAM8, and ADAM12 useful for high-throughput inhibitor screening

https://doi.org/10.1016/j.ab.2007.04.043Get rights and content

Abstract

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-alpha-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, Dabcyl-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.

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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