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BioAssay: AID 434962

Fluorescence polarization-based cell-based primary high throughput screening assay to identify inhibitors of insulin-degrading enzyme (IDE)

Name: Fluorescence polarization-based cell-based primary high throughput screening assay to identify inhibitors of insulin-degrading enzyme (IDE). ..more
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 Tested Compounds
 Tested Compounds
All(324751)
 
 
Active(1316)
 
 
Inactive(323436)
 
 
 Tested Substances
 Tested Substances
All(324858)
 
 
Active(1316)
 
 
Inactive(323542)
 
 
AID: 434962
Data Source: The Scripps Research Institute Molecular Screening Center (IDE_INH_FP_1536_1X%INH PRUN)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-06-03
Modify Date: 2010-06-07

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 1316
Depositor Specified Assays
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AIDNameTypeComment
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 IDEconfirmatory
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 insulinconfirmatory
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)other
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 IDEconfirmatory
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)confirmatory
463220Dose Response: Fluorescence polarization-based cell-based high throughput dose response assay for inhibitors of insulin-degrading enzyme (IDE)confirmatory
434984Summary of probe development efforts to identify inhibitors of insulin-degrading enzyme (IDE)summary
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)other
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 cellsconfirmatory
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 IDEconfirmatory
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 compoundsconfirmatory
449730Counterscreen for IDE inhibitors: Luminescence-based cell-based high throughput assay to identify cytotoxic compounds using HEK cellsscreening
463221Counterscreen for IDE inhibitors: Luminescence-based cell-based high throughput dose response assay to identify compounds that are cytotoxic to HEK cellsconfirmatory
435028Fluorescence polarization-based cell-based high throughput confirmation assay for inhibitors of insulin-degrading enzyme (IDE)screening
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 artifactsconfirmatory
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)confirmatory
Description:
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Malcolm Leissring, Mayo Clinic College of Medicine
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R03 DA024888-01
Grant Proposal PI: Malcolm Leissring
External Assay ID: IDE_INH_FP_1536_1X%INH PRUN

Name: Fluorescence polarization-based cell-based primary high throughput screening assay to identify inhibitors of insulin-degrading enzyme (IDE).

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:

Insulin degrading enzyme, IDE, insulysin, insulinase, beta amyloid, AB, A-beta, beta, inhibitors, inhibition, antagonists, inhibit, inhibitor, Alzheimer's disease, AD, diabetes, HEK cells, fluorescence polarization, FP, primary, primary screen, HTS, high throughput screen, 1536, 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 determine the ability of test compounds to inhibit endogenous cellular IDE activity by measuring shifts in the proportion of intact and cleaved forms of the IDE substrate Abeta. This fluorescence polarization (FP)-based assay (24) employs a derivatized Abeta- peptide [Fluorescein-Abeta-(1-40)-Lys-Biotin (FAbetaB)]. Cleavage of FAbetaB by IDE (provided by live HEK cells in the reaction) separates the Fluorescein moiety from the biotinylated moiety. Subsequent addition of avidin to the reaction increases the effective molecular weight of intact, biotinylated FAbetaB substrate, slowing their rotation rate and reducing the degree of depolarization of plane polarized light. In contrast, cleaved FAbetaB substrate species have their Fluorescein moiety separated from the rest of the molecule, which has a low molecular weight, hence rotating rapidly and causing strong depolarization. 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 singlicate at a nominal concentration of 5.59 uM.

Protocol Summary:

HEK cells were routinely cultured in T-175 sq cm flasks at 37 degrees C and 95% relative humidity (RH). The growth media consisted of Dulbecco's Modified Eagle's Media (DMEM) containing 10% v/v fetal bovine serum, 1X antibiotic mix (penicillin, streptomycin, and neomycin). Prior to the start of the assay, cells were resuspended at a concentration of 1 million cells/ml in phenol red-free DMEM supplemented with 0.1% BSA. Four uL of HEK cell suspension were then dispensed into each well of 1536-well microtiter plates (4,000 cells per well). Next, 45 nL of test compound in DMSO, low control (DMSO alone, 0.56% final concentration) or high control (60 uM of IDE reference inhibitor Ii1) were added to the appropriate wells.

The assay was started by dispensing 4 uL of 2X FAbetaB substrate solution (125 nM final) in DMEM supplemented with 0.5% BSA. Plates were then incubated for 3.5 hours at 37 degrees Celsius, 95% relative humidity and 5% CO2. Next, 1 uL of 9X avidin solution (625 nM final) was added to all wells. Plates were centrifuged and fluorescence polarization was measured on a EnVision microplate reader (PerkinElmer, Turku, Finland) using a FITC dichroic mirror and a FITC FP filter set (excitation = 480 nm, emission = 540 nm). Fluorescence polarization was read using 30 flashes/well for each polarization plane (parallel and perpendicular).

Prior to further calculations, the following formula was used to calculate fluorescence polarization (FP):

FP = ( Raw1 - Raw2 ) / ( Raw1 + Raw2 )

Where:

Raw1 is defined as the S channel.
Raw2 is defined as the P channel.

The percent inhibition for each compound was calculated as follows:

100 * ( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median_Low_Control ) )

Where:

Low_Control is defined as wells containing DMSO.
Test_Compound is defined as wells containing test compound.
High_Control is defined as wells containing reference IDE inhibitor Ii1.

A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition than the cutoff parameter was declared active.

PubChem Activity Outcome and Score:

The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. % Inhibition values of greater than or equal to 100 are reported as activity score 100. Negative % inhibition values are reported as activity score zero.

The PubChem Activity Score range for active compounds is 100-8, and for inactive compounds 8-0.

List of Reagents:

HEK cells (supplied by Assay Provider)
Biotinylated Abeta-peptide (supplied by Assay Provider)
Avidin (Pierce, 21128)
BSA (Sigma, A9647)
DMEM (Invitrogen, 11965)
Phenol-red free DMEM (Invitrogen, 21063)
FBS (Hyclone, SH30088.03)
Pen/Step/Neo mix (Gibco, 15640)
1536-well plates (Greiner, part 789176)
Comment
Due to the increasing size of the MLPCN compound library, 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. All data reported were normalized on a per-plate basis. 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 by the MLSMR.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Inhibition (5.59μM**)Normalized percent inhibition of the primary screen at a compound concentration of 5.59 micromolar.Float%

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

Data Table (Concise)
Classification
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