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BioAssay: AID 624353

Late 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)

Name: Late 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). ..more
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 Tested Compounds
 Tested Compounds
All(25)
 
 
Active(3)
 
 
Inactive(22)
 
 
 Tested Substances
 Tested Substances
All(26)
 
 
Active(3)
 
 
Inactive(23)
 
 
AID: 624353
Data Source: The Scripps Research Institute Molecular Screening Center (IDE-CELLFREE_INH_FP_1536_3XIC50 MDCSRUN run by AP (WT FABB)..)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
Deposit Date: 2012-07-03
Hold-until Date: 2013-06-21
Modify Date: 2013-06-22

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 3
Related Experiments
Show more
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
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 insulinConfirmatorydepositor-specified cross reference: Late stage counterscreen (recombinant IDE's degradation of insulin inhibitors)
624066Late 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 artifactsConfirmatorydepositor-specified cross reference: Late stage counterscreen (fluorescent artifacts)
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 IDEConfirmatorydepositor-specified cross reference: Late stage dose response counterscreen (wild-type (WT) recombinant IDE inhibitors)
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: Late stage counterscreen (cysteine-free mutant IDE activity)
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)Othersame project related to Summary assay
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)Confirmatorysame 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: IDE-CELLFREE_INH_FP_1536_3XIC50 MDCSRUN run by AP (WT FABB) (ROUND 2)

Name: Late 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).

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.
24. Leissring, MA, Lu, A, Condron, MM, Teplow, DB, Stein, RL, Farris, W and Selkoe, DJ, Kinetics of amyloid beta-protein degradation determined by novel fluorescence- and fluorescence polarization-based assays. J Biol Chem, 2003. 278(39): p. 37314-20.

Keywords:

assay provider, counterscreen, 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, wild-type, biochemical, fluorescence polarization, FP, FABB, dose response, triplicate, 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 assay is to establish dose-response curves for compounds identified as possible IDE inhibitor probe candidates, using recombinant wild-type (WT) IDE. This biochemical assay measures shifts in the proportion of intact and cleaved forms of an IDE substrate -- the amyloid Beta-protein (ABeta) -- using a fluorescence polarization (FP)-based read-out based on a derivatized ABeta peptide [fluorescein-ABeta-(1-40)-Lys-biotin (FABetaB)] (24). Cleavage of FABetaB by IDE separates the fluoresceinated portion of the ABeta peptide from the biotinylated portion. Subsequent addition of avidin to the reaction increases the effective molecular weight of intact, biotinylated FABetaB substrate, slowing its rotation rate and reducing the degree of depolarization of plane polarized light. In contrast, cleavage of FABetaB tileds fluoresceinated fragments separated from the biotinylated portion of the molecule, the rotation of which is unaffected by the addition of avidin and which therefore tumble rapidly and strongly depolarize plane-polarized light. Thus, the relative amounts of cleaved and intact forms of the FABetaB substrate can be measured. As designed, compounds that act as IDE inhibitors will inhibit FABetaB cleavage, resulting in a population of large avidin-bound, slowly rotating FABetaB molecules. Compounds are tested in triplicate using a 10-point semi-log dilution series starting at a nominal test concentration of 100 uM (highest dose).
Protocol Summary:
10 uL of WT recombinant IDE enzyme (1 nM final, i.e. EC80) were dispensed into each well of 384-well microtiter plates. Next, 10 uL of test compound in DMSO, low control (1% DMSO final concentration), or high control (1 uM IDE inhibitor Ii1) were added to the appropriate wells. The assay was started by dispensing 10 uL of FBetaB substrate (100 nM final) in Buffer A [100 mM NaCl, 10 mM MgCl2, 50 mM HEPES, pH 7.4 supplemented with 0.1% bovine serum albumin (BSA)]. Plates were then incubated for various lengths of time at room temp (22 C), then terminated by transfer of 15 uL solution into a solution containing avidin (100 nM final) and the broad-spectrum IDE inhibitor 1,10 phenanthroline solution (2 mM final). Fluorescence polarization was read on a Molecular Devices SpectraMAX 5e multilabel plate reader (excitation = 485 nm, emission = 525 nm). FP was calculated automatically by the plate reader software (SpftMax Pro 4.0) according to the following formula:
FP = ( Raw1 - Raw2 ) / ( Raw1 + Raw2 )
Where:
Raw1 is defined as fluorescence in the S channel (same plane as polarized light source).
Raw2 is defined as the P channel (perpendicular plane to polarized light source).
For each test compound, percent inhibition was plotted against the log of the compound concentration. A four parameter variable slope equation describing a sigmoidal dose-response curve was then fitted using GraphPad Prism (GraphPad Software Inc). The software-generated IC50 values are reported.
%_Inhibition = 100 * ( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median_Low_Control ) )
Where:
Low_Control is defined as DMSO-treated wells only.
Test_Compound is defined as wells containing test compound.
High_Control is defined as wells containing reference IDE inhibitor Ii1.
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 100-80, and for inactive compounds 1-0.
List of Reagents:
IDE inhibitor Ii1(supplied by Assay Provider)
Biotinylated Abeta-peptide (supplied by Assay Provider)
Avidin (Pierce, part 21128)
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 PubChem:
Assay Format: Biochemical
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
Classification
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