Concentration-Response Counterscreen for Tau: Redox Active Inhibitors of Caspase-1
The microtubule-associated protein tau is an abundant protein in the axons of neurons that stabilizes microtubules. With its ability to modulate microtubule dynamics, tau contributes directly or indirectly, to key structural and regulatory cellular functions. Particularly important is the influence tau exerts on axonal transport, which allows signaling molecules, trophic factors and other more ..
BioActive Compounds: 2
NIH Molecular Libraries Probe Production Network [MLPCN]
NIH Chemical Genomics Center [NCGC]
MLPCN Grant: X01 MH083262-01
Assay Provider: Carlo Ballatore, University of Pennsylvania
NCGC Assay Overview:
The microtubule-associated protein tau is an abundant protein in the axons of neurons that stabilizes microtubules. With its ability to modulate microtubule dynamics, tau contributes directly or indirectly, to key structural and regulatory cellular functions. Particularly important is the influence tau exerts on axonal transport, which allows signaling molecules, trophic factors and other essential cellular constituents to travel along the axons. Under pathological conditions, tau becomes sequestered into insoluble aggregates called neurofibrillary tangles. This phenomenon is believed to have pathological consequences by promoting axonal transport deficits that ultimately lead to synaptic dysfunction and neuronal loss. To examine the selectivity of tau fibrillization inhibitors, compounds were tested in an in vitro assay for caspase-1 activity. This assay is a counterscreen for tau fibrillization inhibitors to identify non-specific oxidizing agents; an active site cysteine in caspase-1 must remain in a reduced state and the enzyme is inactivated by oxidative compounds. Caspase-1 activity was monitored using a profluorescent substrate that upon enzymatic cleavage yields a fluorescent probe that is detectable at 405 nm excitation and 525 nm emission.
Scheer, J. M., Wells, J. A., and Romanowski, M. J. (2005) Protein Expression and Purification 41, 148-153.
NCGC Assay Protocol Summary:
Caspase-1, kindly provided by Dr. James Wells (University of California, San Francisco), was purified and assayed at 100 nM in buffer containing 50 mM HEPES pH 7.5, 50 mM KCl, 200 mM NaCl, and 0.1% CHAPS. Caspase-1 was dispensed at 3 uL/well into black solid 1536-well plates and incubated 5 min with various concentrations of test compound, with subsequent addition of 1 ul/well Ac-WEHD-AFC substrate (20 uM final concentration). Within 1 minute of substrate addition, fluorescence intensity (405 nm excitation and 525 nm emission) was measured on a ViewLux (PerkinElmer) every 30 s for 10 min. The first 3 minutes of fluorescence values were linear and used to calculate the slope of substrate conversion as a measure of enzyme activity.
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)