Identification of inhibitors using a cell-based assay for monitoring Golgi-resident protease activity.

Abstract

Noninvasive real-time quantification of cellular protease activity allows monitoring of enzymatic activity and identification of activity modulators within the protease's natural milieu. We developed a protease activity assay based on differential localization of a recombinant reporter consisting of a Golgi retention signal and a protease cleavage sequence fused to alkaline phosphatase (AP). When expressed in mammalian cells, this protein localizes to Golgi bodies and, on protease-mediated cleavage, AP translocates to the extracellular medium where its activity is measured. We used this system to monitor the Golgi-associated protease furin, a pluripotent enzyme with a key role in tumorigenesis, viral propagation of avian influenza, ebola, and HIV as well as in activation of anthrax, pseudomonas, and diphtheria toxins. This technology was adapted for high-throughput screening of 39,000-compound small molecule libraries, leading to identification of furin inhibitors. Furthermore, this strategy was used to identify inhibitors of another Golgi protease, the beta-site amyloid precursor protein (APP)-cleaving enzyme (BACE). BACE cleavage of the APP leads to formation of the Abeta peptide, a key event that leads to Alzheimer's disease. In conclusion, we describe a customizable noninvasive technology for real-time assessment of Golgi protease activity used to identify inhibitors of furin and BACE.

• Authors: Julia M Coppola, Christin A Hamilton, Mahaveer S Bhojani, Martha J Larsen, Brian D Ross, Alnawaz Rehemtulla
• Journal: Analytical biochemistry (Anal Biochem) Vol. 364 Issue 1 Pg. 19-29 (May 01 2007) ISSN: 0003-2697 [Print] United States
• PMID: 17316541 (Publication Type: Evaluation Study, Journal Article, Research Support, N.I.H., Extramural)

Chemical References

• Indicators and Reagents
• Protease Inhibitors
• Alkaline Phosphatase
• Amyloid Precursor Protein Secretases
• Furin

Topics

• Alkaline Phosphatase (metabolism)
• Amyloid Precursor Protein Secretases (antagonists & inhibitors, metabolism)
• Animals
• Biological Assay
• Cells, Cultured
• Drug Evaluation, Preclinical (instrumentation, methods)
• Feasibility Studies
• Furin (antagonists & inhibitors, metabolism)
• Golgi Apparatus (enzymology)
• Humans
• Indicators and Reagents (metabolism)
• Protease Inhibitors (analysis, metabolism)
• trans-Golgi Network (metabolism)