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Fluorescent substrates for ADAM15 useful for assaying and high throughput screening
Marcia L Moss1, Miles A Miller2, Nikola Vujanovic3
1BioZyme Inc, 1513 Old White Oak Church Rd., Apex, NC 27523, USA.
Analytical Biochemistry
|September 18, 2016
Summary
New fluorescence resonance energy transfer (FRET) substrates effectively detect and characterize A disintegrin and metalloproteinase 15 (ADAM15) activity. These tools are crucial for developing selective ADAM15 inhibitors, addressing a significant gap in current therapeutic strategies.
Area of Science:
- Biochemistry and Molecular Biology
- Enzymology
- Drug Discovery
Background:
- A disintegrin and metalloproteinase 15 (ADAM15), also known as metargidin, is implicated in inflammation, wound healing, and neovascularization.
- ADAM15 represents an attractive therapeutic target due to its roles in various physiological and pathological processes.
- Existing metalloproteinase inhibitors often lack specificity, necessitating the development of targeted approaches for ADAM15.
Purpose of the Study:
- To identify and characterize novel fluorescence resonance energy transfer (FRET)-based peptide substrates for high-throughput screening of ADAM15 activity.
- To evaluate the efficacy of known metalloproteinase inhibitors against ADAM15.
- To establish tools for the development of potent and selective ADAM15 inhibitors.
Main Methods:
- Synthesis and testing of three FRET-based peptide substrates (PEPDAB011, PEPDAB017, PEPDAB022) for ADAM15 cleavage.
- Determination of specificity constants (kcat/Km) for ADAM15 with the developed FRET substrates.
- Screening of known metalloproteinase inhibitors (TAPI-2, marimastat, GI-254023, TIMP-1, TIMP-3) using the PEPDAB022 substrate.
Main Results:
- ADAM15 efficiently cleaved PEPDAB011 and PEPDAB017 with specificity constants of 5800 M⁻¹s⁻¹ and 4300 M⁻¹s⁻¹, respectively.
- ADAM15 also processed the physiological substrate mimic PEPDAB022 with a specificity constant of 5200 M⁻¹s⁻¹.
- Many broad-spectrum metalloproteinase inhibitors failed to inhibit ADAM15 activity at concentrations up to 50 μM, highlighting a lack of selectivity.
Conclusions:
- The developed FRET substrates are effective tools for assaying ADAM15 activity and facilitate high-throughput screening.
- There is a critical need for the development of novel, selective inhibitors targeting ADAM15.
- These FRET substrates will significantly advance research efforts aimed at discovering and developing specific ADAM15 therapeutics.
