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BioAssay: AID 624066

Late stage assay provider counterscreen for the probe development effort to identify IDE inhibitors: Fluorescence-based IDE activity assay using a fluorogenic peptide substrate (FRET1) to identify fluorescent artifacts

Name: Late stage assay provider counterscreen for the probe development effort to identify IDE inhibitors: Fluorescence-based IDE activity assay using a fluorogenic peptide substrate (FRET1) to identify fluorescent artifacts. ..more
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Active(9)
 
 
Inactive(43)
 
 
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Active(10)
 
 
Inactive(45)
 
 
 Related BioAssays
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AID: 624066
Data Source: The Scripps Research Institute Molecular Screening Center (FRET1-ARTIFACTS_INH_FLINT_3XIC50 MDCSRUN run by AP (WT))
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2012-04-11
Hold-until Date: 2013-04-09
Modify Date: 2013-04-10

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 9
Related Experiments
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AIDNameTypeComment
434962Fluorescence polarization-based cell-based primary high throughput screening assay to identify inhibitors of insulin-degrading enzyme (IDE)Screeningdepositor-specified cross reference: Primary screen (IDE inhibitors in singlicate)
434984Summary of probe development efforts to identify inhibitors of insulin-degrading enzyme (IDE)Summarydepositor-specified cross reference: Summary (IDE inhibitors)
435028Fluorescence polarization-based cell-based high throughput confirmation assay for inhibitors of insulin-degrading enzyme (IDE)Screeningdepositor-specified cross reference: Confirmation (IDE inhibitors in triplicate)
449730Counterscreen for IDE inhibitors: Luminescence-based cell-based high throughput assay to identify cytotoxic compounds using HEK cellsScreeningdepositor-specified cross reference: Counterscreen (Cytotoxicity in triplicate)
463220Dose Response: Fluorescence polarization-based cell-based high throughput dose response assay for inhibitors of insulin-degrading enzyme (IDE)Confirmatorydepositor-specified cross reference: Dose response (IDE inhibitors in triplicate)
463221Counterscreen for IDE inhibitors: Luminescence-based cell-based high throughput dose response assay to identify compounds that are cytotoxic to HEK cellsConfirmatorydepositor-specified cross reference: Dose response counterscreen (HEK cell cytotoxicity in duplicate)
588709Late stage counterscreen assay for the probe development effort to identify inhibitors of insulin-degrading enzyme (IDE): Luminescence-based cell-based dose response assay to identify compounds that are cytotoxic to HEK cellsConfirmatorydepositor-specified cross reference: Late stage dose response counterscreen (cytotoxicity to HEK cells in triplicate)
588711Late stage counterscreen assay for the probe development effort to identify inhibitors of insulin-degrading enzyme (IDE): Fluorescence polarization-based biochemical dose response assay for inhibitors of recombinant IDEConfirmatorydepositor-specified cross reference: Late stage dose response counterscreen (recombinant IDE inhibitors in duplicate)
588712Late stage assay for the probe development effort to identify inhibitors of insulin-degrading enzyme (IDE): Fluorescence polarization-based cell-based dose response assay for inhibitors of IDEConfirmatorydepositor-specified cross reference: Late stage dose response (IDE inhibitors in triplicate)
588718Late stage counterscreen assay for the probe development effort to identify inhibitors of insulin-degrading enzyme (IDE): fluorescence polarization-based biochemical dose response assay to identify fluorescent artifacts and/or optically active compoundsConfirmatorydepositor-specified cross reference: Late stage dose response counterscreen (fluorescent artifacts and/or optically active compounds in t
624306Late stage assay provider counterscreen for the probe development effort to identify IDE inhibitors: Fluorescence-based cysteine-free mutant IDE activity assay using a fluorogenic peptide substrate (FRET1) (reversibility)Otherdepositor-specified cross reference
624338Late stage assay provider counterscreen for the probe development effort to identify IDE inhibitors: Fluorescence-based cysteine-free mutant IDE activity assay using a fluorogenic peptide substrate (FRET1) (reversibility) (Round 2)Otherdepositor-specified cross reference
624340Late stage assay provider counterscreen for the probe development effort to identify IDE inhibitors: Fluorescence-based IDE activity assay using a fluorogenic peptide substrate (FRET1) to identify fluorescent artifacts (ROUND2)Confirmatorydepositor-specified cross reference
624353Late stage assay provider counterscreen for the probe development effort to identify IDE inhibitors: Fluorescence polarization-based biochemical dose-response assay for inhibitors of wild-type (WT) recombinant IDE (ROUND 2)Confirmatorydepositor-specified cross reference
624065Late stage assay provider counterscreen for the probe development effort to identify IDE inhibitors: TR-FRET-based IDE activity assay to identify inhibitors of recombinant IDE's degradation of insulinConfirmatorysame project related to Summary assay
624067Late stage assay provider counterscreen for the probe development effort to identify IDE inhibitors: Fluorescence polarization-based biochemical dose-response assay for inhibitors of wild-type (WT) recombinant IDEConfirmatorysame project related to Summary assay
Description:
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: Malcolm Leissring, Mayo Clinic College of Medicine
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R03 DA024888-01
Grant Proposal PI: Malcolm Leissring, Mayo Clinic College of Medicine
External Assay ID: FRET1-ARTIFACTS_INH_FLINT_3XIC50 MDCSRUN run by AP (WT)

Name: Late stage assay provider counterscreen for the probe development effort to identify IDE inhibitors: Fluorescence-based IDE activity assay using a fluorogenic peptide substrate (FRET1) to identify fluorescent artifacts.

Description:

Alzheimer's disease (AD) is characterized by accumulation of amyloid beta-protein (A-beta; Abeta) in brain regions involved in memory and cognition (1). The steady-state levels of AB reflect a balance between its production via beta- and gamma-secretases and its catabolism by proteolytic degradation (2-4). Because Abeta cleavage products are less neurotoxic than intact Abeta, enzymes that cleave Abeta are of therapeutic interest for AD. In fact, upregulation of Abeta-degrading proteases can prevent AD-like pathology in beta-amyloid precursor protein (APP) transgenic mice (5), suggesting that enhancing AB degradation may be therapeutic in human AD. Insulin-degrading enzyme (IDE) is an Abeta-degrading zinc metalloprotease that requires a free thiol and bivalent cations to degrade extracellular Abeta in neurons and other cell types (6-8). The deduced sequence of IDE shares little homology to other proteinases, including cysteine, metallo-, serine, or aspartic proteases (9). Most IDE is localized inside the cell (10), where it can degrade internalized insulin (11), insulin-like growth factors I and II (12), and amylin (13), which make IDE an attractive target for type-2 diabetes. However, since IDE has also been found in the extracellular space and at the plasma membrane (6), it can function as a principal protease in Abeta catabolism (5, 14, 15). IDE secretion is not dependent upon the classical secretion pathway (16). Studies showing reduced IDE levels in human AD patients (17, 18), combined with increased brain AB levels in IDE-deficient mice (14, 15), and association studies suggesting that IDE variants may be associated with AD severity (19-23), suggest that the identification of compounds that selectively modulate IDE activity will present as important tools for the study of IDE function, AD, and diabetes.

References:

1. Miners, JS, Baig, S, Palmer, J, Palmer, LE, Kehoe, PG and Love, S, Abeta-degrading enzymes in Alzheimer's disease. Brain Pathol, 2008. 18(2): p. 240-52.
2. Selkoe, DJ, Clearing the brain's amyloid cobwebs. Neuron, 2001. 32(2): p. 177-80.
3. Eckman, EA and Eckman, CB, Abeta-degrading enzymes: modulators of Alzheimer's disease pathogenesis and targets for therapeutic intervention. Biochem Soc Trans, 2005. 33(Pt 5): p. 1101-5.
4. Hersh, LB, The insulysin (insulin degrading enzyme) enigma. Cell Mol Life Sci, 2006. 63(21): p. 2432-4.
5. Leissring, MA, Farris, W, Chang, AY, Walsh, DM, Wu, X, Sun, X, Frosch, MP and Selkoe, DJ, Enhanced proteolysis of beta-amyloid in APP transgenic mice prevents plaque formation, secondary pathology, and premature death. Neuron, 2003. 40(6): p. 1087-93.
6. Qiu, WQ, Walsh, DM, Ye, Z, Vekrellis, K, Zhang, J, Podlisny, MB, Rosner, MR, Safavi, A, Hersh, LB and Selkoe, DJ, Insulin-degrading enzyme regulates extracellular levels of amyloid beta-protein by degradation. J Biol Chem, 1998. 273(49): p. 32730-8.
7. Qiu, WQ, Ye, Z, Kholodenko, D, Seubert, P and Selkoe, DJ, Degradation of amyloid beta-protein by a metalloprotease secreted by microglia and other neural and non-neural cells. J Biol Chem, 1997. 272(10): p. 6641-6.
8. Kurochkin, IV and Goto, S, Alzheimer's beta-amyloid peptide specifically interacts with and is degraded by insulin degrading enzyme. FEBS Lett, 1994. 345(1): p. 33-7.
9. Affholter, JA, Fried, VA and Roth, RA, Human insulin-degrading enzyme shares structural and functional homologies with E. coli protease III. Science, 1988. 242(4884): p. 1415-8.
10. Qiu, WQ and Folstein, MF, Insulin, insulin-degrading enzyme and amyloid-beta peptide in Alzheimer's disease: review and hypothesis. Neurobiol Aging, 2006. 27(2): p. 190-8.
11. Fawcett, J and Rabkin, R, Degradation of insulin by isolated rat renal cortical endosomes. Endocrinology, 1993. 133(4): p. 1539-47.
12. Misbin, RI and Almira, EC, Degradation of insulin and insulin-like growth factors by enzyme purified from human erythrocytes. Comparison of degradation products observed with A14- and B26-[125I]monoiodoinsulin. Diabetes, 1989. 38(2): p. 152-8.
13. Bennett, RG, Duckworth, WC and Hamel, FG, Degradation of amylin by insulin-degrading enzyme. J Biol Chem, 2000. 275(47): p. 36621-5.
14. Farris, W, Mansourian, S, Chang, Y, Lindsley, L, Eckman, EA, Frosch, MP, Eckman, CB, Tanzi, RE, Selkoe, DJ and Guenette, S, Insulin-degrading enzyme regulates the levels of insulin, amyloid beta-protein, and the beta-amyloid precursor protein intracellular domain in vivo. Proc Natl Acad Sci U S A, 2003. 100(7): p. 4162-7.
15. Miller, BC, Eckman, EA, Sambamurti, K, Dobbs, N, Chow, KM, Eckman, CB, Hersh, LB and Thiele, DL, Amyloid-beta peptide levels in brain are inversely correlated with insulysin activity levels in vivo. Proc Natl Acad Sci U S A, 2003. 100(10): p. 6221-6.
16. Zhao, J, Li, L and Leissring, MA, Insulin-degrading enzyme is exported via an unconventional protein secretion pathway. Mol Neurodegener, 2009. 4: p. 4.
17. Perez, A, Morelli, L, Cresto, JC and Castano, EM, Degradation of soluble amyloid beta-peptides 1-40, 1-42, and the Dutch variant 1-40Q by insulin degrading enzyme from Alzheimer disease and control brains. Neurochem Res, 2000. 25(2): p. 247-55.
18. Zhao, Z, Xiang, Z, Haroutunian, V, Buxbaum, JD, Stetka, B and Pasinetti, GM, Insulin degrading enzyme activity selectively decreases in the hippocampal formation of cases at high risk to develop Alzheimer's disease. Neurobiol Aging, 2007. 28(6): p. 824-30.
19. Abraham, R, Myers, A, Wavrant-DeVrieze, F, Hamshere, ML, Thomas, HV, Marshall, H, Compton, D, Spurlock, G, Turic, D, Hoogendoorn, B, Kwon, JM, Petersen, RC, Tangalos, E, Norton, J, Morris, JC, Bullock, R, Liolitsa, D, Lovestone, S, Hardy, J, Goate, A, O'Donovan, M, Williams, J, Owen, MJ and Jones, L, Substantial linkage disequilibrium across the insulin-degrading enzyme locus but no association with late-onset Alzheimer's disease. Hum Genet, 2001. 109(6): p. 646-52.
20. Prince, JA, Feuk, L, Gu, HF, Johansson, B, Gatz, M, Blennow, K and Brookes, AJ, Genetic variation in a haplotype block spanning IDE influences Alzheimer disease. Hum Mutat, 2003. 22(5): p. 363-71.
21. Ertekin-Taner, N, Allen, M, Fadale, D, Scanlin, L, Younkin, L, Petersen, RC, Graff-Radford, N and Younkin, SG, Genetic variants in a haplotype block spanning IDE are significantly associated with plasma Abeta42 levels and risk for Alzheimer disease. Hum Mutat, 2004. 23(4): p. 334-42.
22. Bian, L, Yang, JD, Guo, TW, Sun, Y, Duan, SW, Chen, WY, Pan, YX, Feng, GY and He, L, Insulin-degrading enzyme and Alzheimer disease: a genetic association study in the Han Chinese. Neurology, 2004. 63(2): p. 241-5.
23. Vepsalainen, S, Parkinson, M, Helisalmi, S, Mannermaa, A, Soininen, H, Tanzi, RE, Bertram, L and Hiltunen, M, Insulin-degrading enzyme is genetically associated with Alzheimer's disease in the Finnish population. J Med Genet, 2007. 44(9): p. 606-8.

Keywords:

late stage, powders, synthesized, purchased, Insulin degrading enzyme, IDE, insulysin, insulinase, beta amyloid, AB, A-beta, beta, inhibitors, inhibition, antagonists, inhibit, inhibitor, Alzheimer's disease, AD, diabetes, cell-free, wildtype, biochemical,FRET, FRET1, QFRET, triplicate, assay provider, Scripps, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
Protocol
Assay Overview:
The purpose of this biochemical assay is to determine whether powder samples of compounds identified as possible IDE inhibitor probe candidates are artifacts due to their interference with the FP assay format. This IDE activity assay differs from the primary FP-based HTS assay (AID 434962). In this assay, a fluorogenic IDE substrate (FRET1) is incubated with wild-type (WT) recombinant IDE in the presence of compounds in reaction buffer. The reaction is monitored by quantification of fluorescence intensity as a function of time. Percent activity is calculated relative to controls containing no experimental compound (100%) or no enzyme (0%). Compounds are tested in triplicate using a 10-point, semilog dilution series starting at a nominal test concentration of 100 uM.
Protocol Summary:
10 uL of wildtype (WT) recombinant IDE enzyme (1 nM final, i.e., EC80) were dispensed into each well of 384-well microtiter plates, together with control wells loaded with buffer only (Low_Control). Next, 10 uL of test compounds in DMSO (1% final concentration) or DMSO only (High_Control) were added to the appropriate wells. The assay was started by dispensing 10 uL FRET1 substrate (1 uM final) in Buffer A [100 mM NaCl, 10 mM MgCl2, 50 mM HEPES, pH 7.4 supplemented with 0.1% bovine serum albumin (BSA)]. Activity was read continuously at 2 min intervals for 2 h at room temperature (22 C) using a Molecular Devices SpectraMAX 5e multilabel plate reader (excitation = 335 nm, emission = 385 nm).
For each test compound activity was determined from the rate of increase of fluorescence as a function of time relative to controls, using the linear portion of the progress curves (i.e., 0-30% of complete hydrolysis).
For each test compound, percent activity relative to High_Control (100%) was plotted against compound concentration using Microsoft Excel, according to the following formula:
%_Activity = 100 * ( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median_Low_Control ) )
Where:
Test_Compound indicates wells containing test compound
Low_Control indicates wells containing Buffer only
High_Control indicates wells containing DMSO only
IC50 values were determined by fitting a sigmoidal curve to the data set using Prism 5.0 (GraphPad Software, Inc.) Technologies Inc), solving for the concentration corresponding to 50% activity. In cases where the highest concentration tested (i.e. 100 uM) did not result in greater than 50% inhibition, the IC50 value was determined manually as greater than 100 uM.
PubChem Activity Outcome and Score:
Compounds with an IC50 value greater than 10 uM were considered inactive. Compounds with an IC50 value equal to or less than 10 uM were considered active.
Activity score was then ranked by the potency, with the most potent compounds assigned the highest activity scores.
The PubChem Activity Score range for active compounds is 100-81 and for inactive compounds 56-0.
List of Reagents:
Recombinant wild-type IDE (supplied by Assay Provider)
Fluorescence dequenching substrate (FRET1) (supplied by Assay Provider)
BSA (Sigma, part A9647)
NaCl (Sigma, part S9888)
MgCl2 (Sigma, part M9272)
HEPES (Sigma, part H3375)
384-well NBS, low-volume, black plates (Corning, part 3676)
Comment
This assay was run by the assay provider. This assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this case the results of each separate campaign were assigned "Active/Inactive" status based upon that campaign's specific compound activity cutoff value. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that quench or emit fluorescence within the well. All test compound concentrations reported are nominal; the specific concentration for a particular test compound may vary based upon the actual sample provided.
Categorized Comment - additional comments and annotations
From BioAssay Depositor:
BAO: assay design: enzyme reporter: enzyme activity: enzyme inhibition
BAO: assay format: biochemical format: protein format: single protein format
BAO: bioassay specification: assay biosafety level: bsl1
BAO: bioassay specification: assay footprint: microplate: 384 well plate
BAO: bioassay specification: assay measurement throughput quality: concentration response multiple replicates
BAO: bioassay specification: assay measurement type: endpoint assay
BAO: bioassay specification: assay readout content: assay readout method: regular screening
BAO: bioassay specification: assay readout content: content readout type: single readout
BAO: bioassay specification: assay stage: secondary: counter screening
BAO: bioassay specification: bioassay type: functional: enzyme activity
BAO: detection technology: fluorescence: fluorescence polarization
BAO: meta target detail: binding reporter specification: interaction: protein-small molecule
BAO: meta target: biological process target: regulation of molecular function
BAO: meta target: molecular target: protein target: enzyme: protease
BAO: version: 1.4b1090
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1QualifierActivity Qualifier identifies if the resultant data IC50 came from a fitted curve or was determined manually to be less than or greater than its listed IC50 concentration.String
2IC50*The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar.FloatμM

* Activity Concentration.
Additional Information
Grant Number: 1 R03 DA024888-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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