Late 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 compounds
Name: Late 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 compounds. ..more
BioActive Compound: 1
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: ARTIFACT_INH_FP_1536_3XIC50 MDCSRUN FL PROFILING
Name: Late 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 compounds.
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.
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.
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, biochemical, profiling, fluorescence, artifact, fluorescent, fluorescence polarization, FP, dose response, triplicate, HTS, high throughput screen, 1536, Scripps, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this assay is to determine whether powder samples of compounds identified as possible IDE inhibitor probe candidates are nonselective due to their optical activity or fluorescence properties. This fluorescence polarization (FP)-based assay (24) employs a derivatized Abeta- peptide [Fluorescein-Abeta-(1-40)-Lys-Biotin (FAbetaB)]. Cleavage of FAbetaB by IDE 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. Quenchers and fluorescent compounds may interfere with the fluorescence polarization readout and appear as inhibiting IDE. In order to identify such assay artifact, tests compounds are added at the end of the reaction, just before measuring fluorescence polarization, so the calculated %inhibition is a result of the optical interference by the compound rather than its impact on IDE activity. Compounds are tested in triplicate using a 10-point 1:3 dilution series starting at a nominal test concentration of 55 uM.
HEK cells were cultured in T-175 sq cm flasks at 37 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 assay, cells were suspended to 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 (0.56% DMSO 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 (150 nM final) in DMEM supplemented with 0.5% BSA. Plates were then incubated for 3.5 hours at 37 C, 95% relative humidity and 5% CO2. Next, 1 uL of 9x avidin solution (750 nM final) was added to all wells. Immediately before fluorescence polarization acquisition, 45 nL of test compound in DMSO were added to the plates. Plates were centrifuged and fluorescence polarization was read on a EnVision microplate reader (PerkinElmer, Turku, Finland) using a FITC FP filter set and a FITC dichroic mirror (excitation = 480 nm, emission = 540 nm). Fluorescence polarization was read for 10 seconds 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 )
Raw1 is defined as the S channel.
Raw2 is defined as the P channel.
The percent inhibition for each compound was calculated as follows:
%_Inhibition = 100 * ( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median_Low_Control ) )
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.
For each test compound, percent inhibition was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (Symyx Technologies Inc). The reported IC50 values were generated from fitted curves by solving for the X-intercept value at the 50% inhibition level of the Y-intercept value. In cases where the highest concentration tested (i.e. 55 uM) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 55 uM.
PubChem Activity Outcome and Score:
Compounds with an IC50 greater than 10 uM were considered inactive. Compounds with an IC50 equal to or less than 10 uM were considered active. Compounds identified as active in this assay were not pursued further, as they may interfere with the assay format or well detection.
Any compound with a percent activity value less than 50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity value greater than or equal to 50% at any test concentration was assigned an activity score greater than zero. 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 100-100 and for inactive compounds 1-0.
List of Reagents:
HEK cells (supplied by Assay Provider)
IDE inhibitor Ii1(supplied by Assay Provider)
Biotinylated Abeta-peptide (supplied by Assay Provider)
Avidin (Pierce, part 21128)
BSA (Sigma, part A9647)
DMEM (Invitrogen, 11965)
Phenol-red free DMEM (Invitrogen, part 21063)
FBS (HyClone, part SH30088.03)
Pen/Step/Neo mix (Gibco, part 15640)
1536-well plates (Greiner, part 789176)
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.
Categorized Comment - additional comments and annotations
From BioAssay Depositor:
Assay: CurveFit : Equation: =( ( [Maximal Response] * [Concentration]^[Hill Slope] ) / ( [Inflection Point Concentration]^[Hill Slope] + [Concentration]^[Hill Slope] ) ) + [Baseline Response]
Assay: CurveFit : Mask: Excluded Points
Assay: Dictionary: Version: 0.1
BAO: assay design: enzyme reporter: coupled enzyme: protease coupled enzyme
BAO: assay format: cell-based format
BAO: bioassay specification: assay biosafety level: bsl1
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: detection technology: fluorescence: fluorescence polarization
BAO: meta target: molecular target: protein target: enzyme: protease
BAO: version: 1.4b1090
Assay Format: Biochemical
* Activity Concentration. ** Test Concentration.
Data Table (Concise)