Dose Response confirmation of inhibitors of NALP1 in yeast using a Caspase-1-ASC counter screen.
- Deposit:2010-10-21
- Modify:2010-10-21
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Production Centers Network (MLPCN)
Grant Number: 1 U01 AI078048
Assay Provider: Dr. John C. Reed, Sanford-Burnham Medical Research Institute, San Diego, CA
NLR family proteins are an important component of the innate immune system of vertebrates. These proteins possess a nucleotide-binding oligomerization domain, called NACHT, in combination with variable numbers of Leucine-Rich Repeat (LRR) domains that bind molecules produced by pathogens and probably also products of tissue injury. Among the effector mechanisms of NLR family proteins is activation of Caspase-1, which cleaves and activates pro-inflammatory cytokines. We present here a unique primary assay, in which we have reconstituted the mammalian Caspase mediated IL-1 activation pathway consisting of NLRP1 (NALP1), ASC, and Caspase-1 in Saccharomyces cerevisiae. The assay, conducted in liquid culture and formatted into microtiter plates, utilizes a cleavable chimeric transcription factor initially inserted into the cell membrane that upon specific proteolysis by activated Caspase-1 separates from the cell membrane and enters the nucleus to drive the expression of selectable marker genes and expression of beta-galactosidase. Inhibition of any of the activities of the upstream components such as the oligimerization of NAPL1 and ASC to form a complex with Caspase-1 or the direct inhibition of the Caspase-1 will result in a positive phenotype.
The goal of this assay is to confirm hits in "Colorimetric Assay for Inhibitors for NALP1" (AID 2071).
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Assay Materials:
Yeast Strain: ASC/Caspase1
SD-Broth Growth Media: 6.8 g Yeast Nitrogen Base w/o amino acids, 8 mg arginine, 20 mg threonine, 12 mg isoleucine, 24 mg phenylalanine, 8 mg valine, supplemented with 2% alpha- D-glucose and 0.05 % leucine (5 mg/per liter).
SD Broth Selection Media: 6.8 g Yeast Nitrogen Base w/o amino acids, 8 mg arginine, 20 mg threonine, 12 mg isoleucine, 24 mg phenylalanine, 8 mg valine, supplemented with 1 % galactose and 0.2% raffinose
SD-media Agar: SD Broth Growth Media with 1.7 % bacto-agar.
Assay Plate: Corning 1536 Well White Plate (3725)
Detection Reagent: Gal-Screen ss-Galactosidase Reporter Gene Assay System for Yeast or Mammalian Cells (Applied Biosystems Group, T1030).
I. Compound Addition:
1. Using a Labcyte Echo, DMSO and test compounds are transferred in to assay plates. DMSO only is transferred to columns 1-4 (Control wells), while varying volumes of test compounds are transferred to columns 4-44 to achieve the desired test concentrations. Compounds are transferred from a 10 mM stock to give the stated final concentration. Test compound wells in the assay plate are back-filled with DMSO to equalize final assay concentrations.
2. Centrifuge plates at 1000 rpm for 1 min.
II. Set up of NALP assay:
The day before the screen, frozen culture was thawed at room temperature and resuspended in growth medium at a cell density of 6x10;7/ml in approx. 100 ml. The culture was grown overnight at 30 degrees C with shaking (225 rpm).
3. In the morning of the Set-Up day prepare sufficient amount of SD/Gal/Raff selection media for negative control and compound wells and SD medium (Yeast Nitrogen Base w/o Amino Acids) for positive control wells in order to obtain enough yeast cell culture for plating the desired number of 1536 well plates with 2.5 microl yeast cell suspension / well.
4. Count the yeast cells from the overnight Growth Media culture.
5. Dilute yeast to a final concentration of 3.33x10;7 cells/ml in Selection Media.
6. Pellet at 2400 rpm for 5 min at RT. Aspirate off supernatant.
7. Add 50 ml of sterile Water. Re-suspend cells by gently shaking. Pellet again at the same conditions, and wash the yeast cells in 50ml sterile water a second time.
8. Re-suspend in Selection Media.
For negative control and compound wells as described in the plate map:
9. For positive control wells, follow steps 6 and 7 and use SD medium.
10. Add 2.5 microl yeast cells per well using combi and cover each plate with plastic lid.
11. Leave the plates at RT to equilibrate without stacking for about 30 min.
12. Spin the plates at 1000 rpm for 1 min, incubate at 30 degrees C, inverted in a stack of 4, wrapped in saran wrap for 22-24 hours.
IV. Reading plates:
13. After 22-24 hours of incubation, allow plates to equilibrate at RT for about 30 min.
14. 3microl of substrate solution (should be at RT) is added to all the wells of each plate using combi.
15. Plates are spun again at 1000 rpm, and left at RT for 60 min.
16. Read plates using a Perkin Elmer ViewLux using a luminescence protocol.
Compounds with an IC50_Mean < 100 uM are marked as active.
To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented. Its utilization for the assay is described below.
Activity Scoring
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data.
2) Second tier (41-80 range) is reserved for dose-response confirmation data
a. Inactive compounds of the confirmatory stage are assigned a score value equal 41.
b. The score is linearly correlated with a compound's potency and, in addition, provides a measure of the likelihood that the compound is not an artifact based on the available information.
c. The Hill coefficient is taken as a measure of compound behavior in the assay via an additional scaling factor QC:
QC = 2.6*[exp(-0.5*nH^2) - exp(-1.5*nH^2)]
This empirical factor prorates the likelihood of target- or pathway-specific compound effect vs. its non-specific behavior in the assay. This factor is based on expectation that a compound with a single mode of action that achieved equilibrium in the assay demonstrates the Hill coefficient value of 1. Compounds deviating from that behavior are penalized proportionally to the degree of their deviation.
d. Summary equation that takes into account all the items discussed above is
Score = 44 + 6*(pIC50-3)*QC,
Where pIC50 is a negative log(10) of the IC50 value expressed in mole/L concentration units. This equation results in the Score values above 50 for compounds that demonstrate high potency and predictable behavior. Compounds that are inactive in the assay or whose concentration-dependent behavior are likely to be an artifact of that assay will generally have lower Score values.
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues
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