qHTS for Inhibitors of Inflammasome Signaling: IL-1-beta AlphaLISA Primary Screen
Inflammasomes are a set of intracellular multi-protein complexes that enable autocatalytic activation of inflammatory caspases and drive the innate immune response to counter harmful agents. Inflammasomes have been shown to contribute to the pathology of multiple autoinflammatory and autoimmune diseases, such as the Cryopryrin-Associated Periodic Syndrome (CAPS) and Chronic Obstructive Pulmonary more ..
BioActive Compounds: 17186
Inflammasomes are a set of intracellular multi-protein complexes that enable autocatalytic activation of inflammatory caspases and drive the innate immune response to counter harmful agents. Inflammasomes have been shown to contribute to the pathology of multiple autoinflammatory and autoimmune diseases, such as the Cryopryrin-Associated Periodic Syndrome (CAPS) and Chronic Obstructive Pulmonary Disease (COPD). Patients with CAPS exhibit excessive production of IL-1B and IL-18 due to a gain-of-function mutation in NLRP3 , while COPD patients have elevated levels of IL-1B as a downstream consequence of inflammasome activation . Anakinra has become the standard therapy for treating CAPS, but this drug has been shown to be inefficient because it relies on the stoichiometric scavenging of IL-1B rather than blocking inflammasome activation. Hence, the development of novel anti-inflammatory therapies for these chronic inflammatory diseases are of critical need.
A qHTS assay, based on AlphaLISA technology, was developed to screen the Molecular Libraries Small Molecule Repository (MLSMR) to look for novel inflammasome inhibitors. The assay detects IL-1B using AlphaLISA anti-IL-1B acceptor and donor beads. A decreased emission signal, indicating reduced IL-1B secretion, is the desired outcome for this assay.
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]
MLPCN Grant: MH095513
Assay Provider: Shyam Biswal Ph.D., Johns Hopkins University
 Immunity. 2012 Jul 27;37(1):85-95. PMID: 22819042
 Curr Pharm Des. 2012;18(16):2320-8. PMID: 22390695
Three microliters containing 3000 THP-1 cells were dispensed into the wells of solid, white, tissue culture treated Greiner 1,536-well plates. Library compounds and positive control, Glyburide, were pin-transferred (23 nL) by a Kalypsys pin-tool equipped with a 1,536-pin array, resulting in final compound concentration ranges of 2.5 nM to 57.5 uM. After 1 hr of cell culture incubation (37o C, 5% CO2), 1 microL of 1 microg/mL lipopolysaccharide (250 ng/mL, final) and 5 mM ATP (1.25 mM, final) were added to stimulate the cells. After 16 hr of cell culture incubation with the compounds, 1.5 uL of anti-IL-1B acceptor beads (6 ug/mL, final) and biotinylated antibody (0.6 nM, final) were added to the plate, followed by a 1 hr incubation at ambient temperature. To detect the secreted IL-1B, 1.5 uL of AlphaLISA SA-Donor beads (24 ug/mL, final) were added, followed by 30 min of incubation in the dark at ambient temperature. The AlphaLISA signal was read by a PerkinElmer EnVision plate reader with an ultrasensitive luminescence detector and 1536 Plate HTS AlphaScreen aperture (excitation of 680nm and emission of 615nm).
1. The screen was ran at two concentrations (11.50uM and 57.50 uM). Maximum inhibition response is obtained and active compound is defined by a max response <= -50%; inactive compound is defined by a max response > -30%; inconclusive compound is defined by a max response in between -30% and -50%
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, PUBCHEM_ACTIVITY_SCORE is 95. For all inconclusive compounds, PUBCHEM_ACTIVITY_SCORE is 50.
3. Active compounds were reacquired for dose response validation.
Data Table (Concise)