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
BioActive Compounds: 9
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.
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.
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.
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.
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 ) )
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)
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
* Activity Concentration.
Data Table (Concise)