MLPCN ERAP1 Measured in Biochemical System Using Plate Reader - 7016-01_Inhibitor_Dose_CherryPick_Activity_Set2
Keywords: ERAP-1, aminopeptidase major histocompatibility complex (MHC),L-leucine-7-amido-4-methylcoumarin, L-Leucinethiol, Envision, Genedata, ..more
BioActive Compounds: 95
Keywords: ERAP-1, aminopeptidase major histocompatibility complex (MHC),L-leucine-7-amido-4-methylcoumarin, L-Leucinethiol, Envision, Genedata,
ankylosing spondylitis, autoimmune diseases,genome-wide association studies,endoplasmic reticulum, arthritis, psoriasis
ERAP1 is an aminopeptidase involved in processing peptide antigens for presentation by MHC proteins. ERAP1 is required for trimming certain antigenic precursors after transport to the endoplasmic reticulum so that they can be bound by major histocompatibility complex (MHC) proteins and presented to cells of the immune system. Recently ERAP1 has been implicated in genome-wide association studies with ankylosing spondylitis and other autoimmune diseases. Chemical probes are necessary for understanding ERAP1###s role in antigen selection and development of autoimmune disease. However, progress in this area currently is constrained by the lack of any chemical probes specific for ERAP1. Such probes would be useful in identifying the peptides targeted by the autoimmune T cells, for potential antigen-based tolerization therapy, for understanding the etiology of autoimmune disease, and could provide prototypes for potential therapeutic intervention based on inhibition of disease-specific antigen processing pathways.
The goal of this project is to find selective inhibitors of ERAP1. This assay is based on hydrolysis of the L-AMC amide bond liberating fluorescent 7-aminomethylcomarin (AMC). Inhibitors preventing this hydrolysis will result in a lower signal outcome. Related enzymes IRAP and ERAP2 will be used to test for selectivity in secondary assays. Autofluorescent compounds will be identified and filtered as necessary by taking T=0 and T= 60 minute reads.
Expected Outcome: There will be a loss of signal seen with successful inhibitors of ERAP1. Per the protocol, fluorescence measurements are taken at time=0 and time=60 minutes. The difference (T60-T0) is used as the calculated layer in Genedata to which normalization and correction algorithms are applied.
Add the following to a 1536 Corning black plate (or equivalent)
3.0 ul of 2.33ng/ul ERAP1 enzyme from Stern lab at (2.33X) prep in 20mM TRIS-HCl-100mM NaCl pH 7.5 ERAP buffer
3.0uL of 583uM L-leucine-7-amido-4-methylcoumarin (L-AMC, Sigma L-2145)) substrate (2.33X) prep in ERAP buffer plus 0.023% BSA
1.0 ul L-Leucinethiol poscon (Sigma L-8397) @ 700uM (7x) in buffer or 1ul ERAP buffer to non-poscon wells
Read Time 0 fluorescence at 380/450nm Using Perkin Elmer Envision reader
Incubate 1 hour at room temp.
Read Time 60 minute fluorescence at 380/450nm
Discard plates. Process data in Genedata
PRESENCE OF CONTROLS: Neutral control wells (NC; n=128) and positive control wells (PC; n=128) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
The compounds were assayed in multiple independent instances using an identical protocol; each instance is called a 'test'. For each test, the following analysis was applied:
ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (2)(Max_Concentration).
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v10.0.2):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.
PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.10.0.2) was applied.
MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): AC50
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
pAC was set to equal -1*log10(AC)
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, or
b) The fit was deemed not valid due to poor fit quality.
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.
Once the data for each test was processed, the test number was appended to all column headers in that test's data set. The individual test results were then aggregated as follows:
1. The final PUBCHEM_ACTIVITY_OUTCOME was set to:
a. '2' (active) when all test outcomes were '2' (active), or
b. '1' (inactive) when all test outcomes were '1' (inactive), or
c. '3' (inconclusive) when the test outcomes were mixed.
2. The final ACTIVE_CONCENTRATION (AC) was set as follows:
a. If the final PUBCHEM_ACTIVITY_OUTCOME = 2, AC was set as the mean of the constituent test active concentrations;
b. If the final PUBCHEM_ACTIVITY_OUTCOME = 1 or 3, AC was left empty.
3. The final PUBCHEM_ACTIVITY_SCORE was calculated based on the aggregated ACTIVE_CONCENTRATION, using the same logic described above for individual test scores.
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)