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P2063
A Novel Fluorimetric Assay for Detection of TACE (Ą-secretase) Activity Using a Long Wavelength FRET Peptide Substrate
Presenter Cecilia Po, AnaSpec
Additional Authors: Xiaohe Tong, Vera Rakhmanova, Xudong Zhu, Fengying Li, Xiaofen Zhong, Andrei Kozyrev, and Anita Hong
TACE (TNF-a converting enzyme), also called ADAM17 or a-secretase, is involved in myogenesis, neurogenesis, and fertilization through the process of shedding of cell surface proteins. TACE is the predominant 'sheddase' responsible for the generation of soluble mature TNF. Considerable efforts have been made for the research and development of anti-TNF-a agents for the purpose of reducing the severity of inflammatory responses in disease states. The inhibition of TACE by a pharmacological agent may represent an alternative approach to modulate the effect of TNF-a.
To facilitate high-throughput screening of TACE inhibitors, we synthesized a novel peptide substrate for TACE based on QXLTM 520/5-FAM FRET pair. Active TACE cleaves the FRET substrate into two separate fragments resulting in an increase of 5-FAM fluorescence, which is monitored at excitation/emission = 490 nm/520 nm. Based on this FRET substrate, we have developed a new SensoLyte™ 520 TACE Activity Assay that can be used to detect the activity of the enzyme and for screening of new TACE inhibitors. The assay is highly sensitive and can detect subnanogram amounts of enzyme. To validate the assay for inhibitor screening, we used a previously described TACE inhibitor. 
The SensoLyte™ 520 TACE Activity Assay has a convenient homogeneous format and is less interfered by the autofluorescence of cell components and test compounds due to the long wavelength fluorescence of 5-FAM.