qHTS for Inhibitors of Tau Fibril Formation, Fluorescence Polarization
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: 1047
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 identify inhibitors of tau aggregation, a heparin-induced tau fibril formation assay was used that employed a recombinantly expressed fragment of tau, K18 (Q242-E372), bearing a P301L mutation (Gustke et al. 1994; Hong et al. 1998). This assay monitors tau fibrillization by fluorescence polarization (FP) of Alexa 594-labeled K18 P301L, which does not fibrillize readily but incorporates into growing filaments of unlabeled tau.
Gustke, N., B. Trinczek, et al. (1994). "Domains of tau protein and interactions with microtubules." Biochemistry 33(32): 9511-22.
Hong, M., V. Zhukareva, et al. (1998). "Mutation-specific functional impairments in distinct tau isoforms of hereditary FTDP-17." Science 282(5395): 1914-7.
Li, W. and V. M. Lee (2006). "Characterization of two VQIXXK motifs for tau fibrillization in vitro." Biochemistry 45(51): 15692-701.
von Bergen, M., S. Barghorn, et al. (2001). "Mutations of tau protein in frontotemporal dementia promote aggregation of paired helical filaments by enhancing local beta-structure." J Biol Chem 276(51): 48165-74.
NCGC Assay Protocol Summary:
Two K18 mutants were produced: P301L, which fibrillizes faster than the wild-type form (von Bergen et al. 2001) and K311D, which does not fibrillize (Li and Lee 2006) and was thus used as non-fibrillizing control in the assay. For screening, 2 uL/well human K18 P301L (15 uM unlabeled and 0.24 uM Alexa 594-labeled tau final concentrations) in reagent buffer (100 mM sodium acetate pH 7) was dispensed into black solid 1536-well plates (Grenier) using a solenoid-based dispenser. Following transfer of 23 nL compound or DMSO vehicle by a pin tool, 2 uL/well heparin (20 uM final concentration) in reagent buffer was added and the plate centrifuged 15 s at 1000 RPM. Plates were incubated 6 hr at 37 C and then 1 uL/well ThT (30 uM final concentration) was added. After a 1 hr ambient incubation, the plates were read by an Envision (Perkin Elmer) to monitor Alexa 594 FP (555 nm excitation and 632 nm emission).
Keywords: NIH Roadmap, MLPCN, MLI, MLSMR, NCGC, qHTS, tau, tauopathies, Alzheimer's disease, fibril, neurodegeneration, aggregation
In this assay we provide the fluorescence polarization (FP) data, which include % activity (the fluorescence intensity has been normalized to positive and negative controls) data for each concentration as well as potencies for the active compounds.
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.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)