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Optimization of a Medium‐Throughput Cathepsin K Inhibition Assay for NAAA Inhibitors
Author(s) -
Golden Emily Rose,
Miyabe Christina,
Wood JodiAnne,
Malamas Michael,
Alapafuja Shakiru,
Makriyannis Alexandros
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.609.3
Subject(s) - cathepsin k , chemistry , amidase , cathepsin , biochemistry , cathepsin h , cathepsin o , endocannabinoid system , ic50 , cathepsin c , cathepsin b , enzyme , microbiology and biotechnology , in vitro , biology , receptor , osteoclast
Cathepsin K and N‐acylethanolamine‐hydrolyzing acid amidase (NAAA) are members of the cysteine protease family. While NAAA is a metabolic enzyme in the endocannabinoid system, Cathepsin K is involved in bone reabsorption and the release of calcium into the bloodstream. Our interest in the endocannabinoid system has resulted in significant NAAA inhibitor development for the treatment of inflammation and the resulting chronic pain and has prompted our interest in Cathepsin K for off‐target selectivity screening. A medium‐throughput fluorescent assay to determine Cathepsin K inhibition was optimized to screen NAAA‐lead compounds using Omnicathepsin (aka Z‐Phe‐Arg‐AMC) as the fluorogenic substrate. The concentrations of enzyme (2 nM Cathepsin K) and substrate (25 μM Omnicathepsin) were optimized and the Km of Cathepsin K and Omnicathepsin was found to be 9.8 μM. Two published compounds, Odanacatib and BML‐244 with IC50 values of 0.54 ± 0.4 nM and 317 ± 41 nM respectively, were developed to be reference compounds in the inhibition 3‐point screen. The optimized assay was then used to screen NAAA‐inhibitors for competitive inhibition of Cathepsin K in order to create more potent, specific drugs for NAAA. Support or Funding Information This project was funded by NIH: 2‐R44‐DK104636, NAAA Inhibitors as Anti‐Inflammatory Agents, Shakiru Alapafuja, PI; Northeastern PI, JodiAnne T. Wood.