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| | In Vitro Enzyme Inhibition Assay and Bone-Resorption Assay |
| Description: | Prior to the addition of substrate, different concentrations of the inhibitor ranging from 100 uM to 0.2 nM were preincubated for 15 min with enzyme to allow the establishment of the enzyme-inhibitor complex. The proteolytic cleavage of N-acyl aminocoumarins by cathepsins was conducted in 96-well plate format and the plate read using a Spectramax (Molecular Devices) plate reader. The excitation wavelength was 355 nm and the emission wavelength was 460 nm for peptidyl-AMC substrates. The percent inhibition of the reaction was calculated from a control reaction containing only vehicle. IC50 curves were generated by fitting percent inhibition values to a four parameter logistic model (SOFTMAX PRO, Molecular Devices). EC50/IC50 values were obtained from bone-resorption assay. The long bones were aseptically isolated from a 10-day-old New Zealand White hare (Covance Research Products, Denver, PA) and the soft tissue removed. |
| Affinity data for this assay | |
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