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Innate Immunity: Mechanisms and Modulation | EK39


EK39-EPOSTER-OBRIEN-MARTHA - Cell-Based Methods to Quantify Inflammasome Function and Target Engagement


Apr 12, 2021 12:00am ‐ Apr 12, 2021 12:00am

Description

Cell-Based Methods to Quantify Inflammasome Function and Target Engagement Martha O’Brien1, Matt Robers1, Cesear Corona2, Jim Vasta1, Jennifer Wilkinson1, Ngan Lam1, Kaitlin Dunn1, James Cali1, and Dan Lazar1 1Promega Corporation, 5430 E. Cheryl Parkway, Madison, WI 53711, 2Promega Biosciences, 277 Granada Drive, San Luis Obispo, CA 93401 Inflammasomes are implicated in numerous diseases related to chronic and acute inflammation. A mechanistic understanding of inflammasomes is critical for the development of modulatory drugs, especially for the NLRP3 inflammasome that responds to a wide array of stimuli. We have developed three assays for quantifying inflammasome function. Two are bioluminescent assays and monitor the inflammasome outputs of caspase-1 activity and IL-1β release. The third is a NanoBRET™ NLRP3-specific target engagement assay. The caspase-1 activity assay is a single-step, bioluminescent format combining the Z-WEHD-aminoluciferin substrate with a thermostable luciferase in an optimized reagent. Specificity for caspase-1 is dialed in with inhibitors that are combined in a lytic reagent. To enable direct measurement of released IL-1β from cells with no transfer or wash steps, we applied our NanoBiT® technology for development of human and mouse IL-1β immunoassays. These IL-1β immunoassays are direct in-solution assays with just two additions, a mixture of LgBiT and SmBiT-labeled anti-IL-1β antibodies followed by a detection reagent. The entire assay time is ~70 min and uses a standard luminometer for recording luminescence. We show IL-1β release from human THP-1 cells, PBMCs, and murine J774A.1 macrophages. IL-1β release was not detected from murine RAW264.7 ASC-deficient macrophages, supporting specificity of the mouse IL-1β immunoassay for mature IL-1β. Both the caspase-1 and IL-1β assays can be run directly with cells in culture or with transferred culture medium, enabling application of both luminescent assays to the same cell wells via split-sample analysis. Both these assays are easily scalable, have been tested in 384-well plates, and are suitable for high throughput screening. For the NLRP3 target engagement assay, cells are transfected with a NLRP3-Nanoluc construct and incubated with a cell-permeable, fluorescent NLRP3 binder (tracer), generating a bioluminescent resonance energy transfer (BRET) signal. This system enables quantitation of engagement for NLRP3 NACHT domain inhibitors in live, intact cells. Using NanoBRET, target occupancy can be observed for the Walker B motif binder MCC950, as well as CY-09 that utilizes a distinct engagement mechanism. MCC950 inhibited caspase-1 activity, IL-1β release, and the BRET signal when tested in each of the assays and all three inhibition curves demonstrated excellent correspondence with comparable IC50s. In total, these three assays significantly expand the toolbox for probing inflammasome function and rapidly screening for inflammasome modulators.

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