MLPCN ERAP1 Measured in Biochemical System Using Plate Reader - 7016-01_Inhibitor_SinglePoint_HTS_Activity
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 more ..
BioActive Compounds: 499
Keywords: aminopeptidase major histocompatibility complex (MHC)
ankylosing spondylitis, autoimmune diseases
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.
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) and positive control wells (PC) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
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.
All well activities were then multiplied by -1 to create a positive activity readout value range, to match Pubchem convention.
PATTERN CORRECTION: A Runwise Median pattern correction algorithm from Genedata Assay Analyzer (v.10.0.2) was applied to account for a liquid dispensing effect noted in an edge row of some of the plates.
This was set as equal to the mean of the normalized sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at -30%.
PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 100.
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T
Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T
Samples passing AT only were assigned an outcome of 2 (active) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T
Samples passing PAR_T only were assigned an outcome of 2 (active) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T
** Test Concentration.
Data Table (Concise)