Single concentration confirmation of uHTS for the identification of inhibitors of NALP3 in yeast using a luminescent assay
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 more ..
BioActive Compounds: 1089
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 NLRP3 (NALP3), 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 b-galactosidase. Inhibition of any of the activities of the upstream components such as the oligimerization of NAPL3 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 "uHTS Luminescent assay for identification of inhibitors of NALP3 in yeast" (AID 2825)
1. Lopez-Otin C, Overall CM. Protease degradomics: a new challenge for proteomics. Nat Rev Mol Cell Biol 2002;3(7):509-19.
2. Reed JC. Caspases and cytokines: roles in inflammation and autoimmunity. Adv Immunol 1999;73:265-99.
3. Talanian R, Quinlan C, Trautz S, et al. Substrate specificities of caspase family proteases. J Biol Chem 1997;272:9677-82.
4. Timmer JC, Salvesen GS. Caspase substrates. Cell Death Differ 2007;14(1):66-72.
5. Salvesen GS, Renatus M. Apoptosome: The seven-spoked death machine. Develop Cell 2002;2:256-7.
6. Martinon F, Burns K, Tschopp J. The Inflammasome: A molecular platform triggering activation of inflammatory caspases and processing of proIL-1b. Mol Cell 2002;10:417-26.
7. Faustin B, Lartigue L, Bruey JM, et al. Reconstituted NALP1 inflammasome reveals two-step mechanism of Caspase-1 activation. Molecular Cell 2007;25(5):713-24.
8. Stehlik C, Reed JC. The PYRIN connection: novel players in innate immunity and inflammation. J Exp Med 2004;200:551-8.
9. Martinon F, Tschopp J. NLRs join TLRs as innate sensors of pathogens. Trends Immunol 2005;26:447-54.
10. Church L, Cook, G, McDermott, M. Primer: inflammasomes and interleukin 1beta in inflammatory disorders. Nat Clin Pract Rheumatol 2008;4(1):34-42
11. Slee E, Harte M, Kluck R, et al. Ordering the cytochrome c-initiated caspase cascade: Hierarchic activation of caspases -2,-3,-6,-7,-8 and -10 in a caspase-9-dependent manner. J Cell Biol 1999;144:281-92.
12. Bruey JM, Bruey-Sedano N, Luciano F, et al. Bcl-2 and Bcl-XL regulate proinflammatory caspase-1 activation by interaction with NALP1. Cell. 2007; 6;129(1):45-56.
Yeast Strain: ASC/NALP3/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 b-Galactosidase Reporter Gene Assay System for Yeast or Mammalian Cells (Applied Biosystems Group, T1030).
I. Compound Addition:
1. Using LabCyte Echo, transfer 15 nL from a 2 mM Echo qualified plate containing test compounds into assay plate columns 3 - 48 (final concentration of test compounds is 10 uM, 0.5 % DMSO). Transfer 15 nL of DMSO to positive and negative control wells in columns 1 - 4.
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 oC 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 3 ul 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 3 ul 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 oC, 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. 3 ul 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 % Activity >= 50% for either replicate are considered "active" in this assay.
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 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. The score is correlated with % activity in the assay:
a. If outcome of the primary screen is inactive, then the assigned score is 0
b. If outcome of the primary screen is inconclusive, then the assigned score is 10
c. If outcome of the primary screen is active, then the assigned score is 20
Scoring for Single concentration confirmation screening is not applicable to this assay.
d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21
e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25
f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30
This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.
2) Second tier (41-80 range) is reserved for dose-response confirmation data
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues
** Test Concentration.
Data Table (Concise)