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
BioActive Compounds: 1316
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).
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.
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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.
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.
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 )
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 ) )
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)
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.
** Test Concentration.
Data Table (Concise)