Fluorescent Substrates Useful as High Throughput Screening Tools for ADAM9

Marcia      L. Moss; Fred      H. Rasmussen; Raphael      Nudelman; Peter      J. Dempsey; Jason      Williams

Abstract

Fluorescence resonance energy transfer substrates were designed and tested as substrates for ADAM9. The donor/quencher pair used were 5-carboxyfluorescein (Fam) and 4-(4-dimethyl-aminophenylazo)benzoyl (Dabcyl) since they have been well studied sensitive fluorescent probes. The peptides based on precursor TNF-alpha, Dabcyl-Ser-Pro- Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Lys(Fam)-NH2 and Dabcyl-Leu-Ala-Gln-Ala-HomoPhe-Arg-Ser-Lys(Fam)-NH2, and C-terminal TGF-alpha, Dabcyl-Glu-His-Ala-Asp-Leu-Leu-Ala-Val-Val-Ala-Ala-Lys(Fam)-NH2 cleavage sites were effectively processed by ADAM9 with turnover numbers of 100 ± 20 x 10-2 min-1, 20 ± 10 x 10-2 min-1, and 10 ± 3 x 10-2 min-1. In addition, a peptide based on the 33kDa cleavage site of the low affinity receptor for IgE, CD23, Dabcyl-Leu- Arg-Ala-Glu-Gln-Gln-Arg-Leu-Lys-Ser-Lys(Fam)- NH2 was processed as well but with less efficiency. A more selective substrate for ADAM9 was found based on the betacellulin cleavage site. However, the valine containing precursor TNF-alpha based substrate was used to measure IC50 values of metalloproteinase inhibitors against ADAM9 since it was processed most efficiently. The tightest binding inhibitor was the Wyeth Aerst compound, TMI-1, with an IC50 of 2.1 ± 0.3 nM. In addition, GI254023, previously identified as a selective inhibitor of ADAM10, also inhibited ADAM9 with an IC50 of 280 ± 110 nM. These results demonstrate that sensitive substrates for ADAM9 can be developed that are useful in high throughput screening assays for ADAM9.

Keywords: ADAM8, ADAM9, ADAM10, ADAM12, ADAM17, TACE, fluorescent, substrate, inhibitor, TMI-1, GI254023, GM6001, disintegrin, metalloproteinase, FRET, CD23, TGF-alpha