Late 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 insulin
Name: Late 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 insulin. ..more
BioActive Compounds: 3
Depositor Specified Assays
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: INSULIN_INH_TRFRET_3XIC50 MDCSRUN run by AP
Name: Late 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 insulin.
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, insulin, insulysin, insulinase, beta amyloid, AB, A-beta, beta, inhibitors, inhibition, antagonists, inhibit, inhibitor, Alzheimer's disease, AD, diabetes, cell-free, wildtype, biochemical, HTRF, TRFRET, TR-FRET, FRET, CisBio, 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 can inhibit the ability of recombinant IDE to degrade insulin in vitro. This assay is a proximity-based immunoassay that uses two monoclonal antibodies, one labeled with Eu3+-Cryptate and one labeled with XL665 that recognize distinct epitopes on the insulin molecule. Compounds are tested in triplicate using a 10-point semi-log dilution series starting at a nominal test concentration of 100 uM (highest dose).
Details of this assay are available at the manufacturer's website: http://www.htrf.com/products/cns/insulin/.
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. To define background fluorescence (background control) a subset of wells were filled with 10 uL buffer only (no insulin). The assay was started by dispensing 10 uL of insulin (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 the reactions were terminated by addition of 5 uL of the broad-spectrum zinc-metalloprotease inhibitor 1,10 phenanthroline (2 mM final). TR-FRET was measured using a Molecular Devices SpectraMAX 5e multi-label plate reader (excitation = 337 nm, emission A = 665 nm and emission B: 620 nm, with a 400 ms delay time). Raw TR-FRET values (TRFraw) were determined from the following formula:
TRFraw = Em665 / Em620
Next, background-subtracted TR-FRET values (TRFsub) were calculated according to the following formula:
TRFsub = TRFraw - Mean_Background_Control_TRFraw
Mean_Background_Control_TRFraw is defined as the raw TR-FRET values obtained in wells containing no insulin
For each test compound, percent activity relative to Low_Control (100%) was plotted against compound concentration using Microsoft Excel, according to the following formula:
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.
IC50 values were determined by fitting a sigmoidal curve to the data set using Prism 5.0 (GraphPad Software, Inc.) Technologies Inc), then 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 was determined manually as greater than 100 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.
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 100-1, and for inactive compounds 0-0.
List of Reagents:
Recombinant wild-type IDE (supplied by Assay Provider)
IDE inhibitor Ii1 (supplied by Assay Provider)
Insulin (Sigma, part I9278)
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.
BAO: version: 1.4b1090
BAO: bioassay specification: assay stage: secondary: counter screening
BAO: bioassay specification: assay biosafety level: bsl1
BAO: assay format: biochemical format: protein format: single protein format
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: meta target: molecular target: protein target: enzyme: protease
BAO: meta target: biological process target: regulation of molecular function
BAO: meta target detail: binding reporter specification: interaction: protein-small molecule
BAO: assay design: enzyme reporter: enzyme activity: enzyme inhibition
BAO: detection technology: fluorescence: fluorescence polarization
BAO: bioassay specification: bioassay type: functional: enzyme activity
BAO: bioassay specification: assay footprint: microplate: 384 well plate
BAO: bioassay specification: assay measurement throughput quality: concentration response multiple replicates
* Activity Concentration.
Data Table (Concise)