Cell-Based Target Engagement and Functional Assays for NLRP3 Inhibitor Profiling Help Identify Successes and Failures

Identifying Inflammasome Inhibitors: What’s Missing
The NLRP3 inflammasome is implicated in a wide range of diseases. The ability to inhibit this protein complex could provide more precise, targeted relief to inflammatory disease sufferers than current broad-spectrum anti-inflammatory compounds, potentially without side effects.

Studies of NLRP3 inflammasome inhibitors have relied on cell-free assays using purified NLRP3. But cell-free assays cannot assess physical engagement of the inhibitor and target in the cellular micro-environment. Cell-free assays cannot show if an NLRP3 inhibitor enters the cell, binds the target and how long the inhibitor binding lasts.

Cell-based assays that interrogate the physical interaction of the NLRP3 target and inhibitor inside cells are needed.

Assays Used in This Work
To demonstrate a more authentic, fundamental connection between NLRP3 and reported inhibitors, the authors of a recent Cell Chemical Biology paper demonstrated three scalable cell-based assays. These assays provide mechanistic, as well as measurable gauges of physical interaction between NLRP3 and potential inhibitors. The assays quantify target occupancy, caspase-1 activation and cytokine release.

Existing immunoassays to monitor markers of inflammasome activity, such as caspase-1 activation and
IL-1β release, include ELISAs used to measure secreted IL-1β or Western blots of cleaved IL-1β and caspase -1. These assays are performed using cell supernatants. In addition, pulldown assays have been used to demonstrate interactions of small molecules and targets like NLRP3, but these assays are not simple to run and are performed on cell lysates. Such indirect methods can’t demonstrate a relationship between inhibitors/small molecules and pathway inhibition of NLRP3.

The NLRP3 inflammasome detects stress signals, like extracellular ATP released by damaged cells. These signals activate the inflammasome and recruit caspase-1 to the inflammasome complex. Caspase-1 then cleaves and activates the proinflammatory cytokine, IL-1β.

Inflammation and Inflammasomes
Inflammation is a driver of diseases from Alzheimer’s to heart disease, arthritis to COVID-19. Environmental agents such as particulates that make up air pollution also cause inflammation. These inflammation solicitors are known as pathogen-associated molecular patterns (PAMPS) or damage-associated molecular patterns (DAMPS).

DAMPS and PAMPS result in formation of protein complexes called inflammasomes. This complex formation results in activation of caspase-1, resulting in IL-1β and IL-18 cleavage and activation, also cleavage of gasdermin D, which forms holes in cell membranes, resulting in further inflammatory compound release.

Inflammasome formation has been implicated in both chronic and acute inflammatory diseases, as well as disease due to environmental factors. In particular, the NLRP3 inflammasome, which forms in response to a wide array of insults, is of interest. A recent PubMed search on “NLRP3 inflammasome” returned over 13,400 publications.

There’s obviously tremendous interest in development of NLRP3 inhibitors. And some small molecule NLRP3 inhibitors have reached preclinical or clinical studies after demonstrating the ability to modulate the NLRP3 inflammasome. However, these inhibitors have not shown evidence of in-the-cell ability to block this target.

In their Cell Chemical Biology paper, “Interrogating direct NLRP3 engagement and functional inflammasome inhibition using cellular assays” the authors present mechanistic assays to examine direct NLRP3-compound engagement in cells and to functionally interrogate NLRP3 pathway activity. Using these assays the authors were able to rank the potency of five confirmed inhibitors. Additionally, the authors used the assays to identify two previously identified NLRP3 inhibitors that did not demonstrate direct pathway antagonism.

Interrogating Target-Inhibitor Interaction
A novel live-cell binding assay, the NanoBRET™ Target Engagement (TE) Assay, uses an energy transfer technique known as bioluminescence resonance energy transfer (BRET), to measure binding between the target protein and small molecules in live cells. This energy transfer is based on two elements: Cellular expression of the target protein fused to NanoLuc® Luciferase and a cell-permeable fluorescent NanoBRET™ tracer, which binds reversibly to the target protein.

The NanoBRET™ TE Assay can measure the ability of a drug compound to enter a cell (permeability), the amount of time the compound remains bound to the targeted molecule (residence time), provide data on the intracellular binding of the compound to proteins closely related to the target molecule (selectivity) and quantitate the intensity of compound binding to a target, as well as how much is bound (affinity and occupancy).

In the NanoBRET™ TE Assay, cells are transfected with a NanoLuc®-target protein containing vector (in this case, NLRP3). Then a cell-permeable fluorescent tracer that can bind to the target is added. When the fluorescent tracer binds to the NanoLuc®-target fusion, a BRET signal results. This tracer binding to the target is reversible, so that if other target-binding molecules (e.g., NLRP3 inhibitors) are present, the competition results in a decrease in tracer binding and a loss of the BRET signal.

Two additional functional bioluminescent immunoassays were used to identify caspase-1 activation and IL-1β release in cell culture medium, as downstream measures of NLRP3 inflammasome activity. The Lumit® immunoassay for IL-1β uses NanoBiT® split-luciferase technology. Antibodies are chemically-labeled with two NanoBiT® subunits—small bit (SmBiT) and large bit (LgBiT). When the antibodies bind to the target, SmBiT and LgBiT are brought together, generating a luminescent signal. The functional assay used to monitor caspase-1 activity, Caspase-Glo® 1 Inflammasome Assay, is a bioluminescent assay with a coupled-enzyme system using Z-WEHD-aminoluciferin substrate and Ultra-Glo™ luciferase.

Because caspase-1 activation and IL-1β release are both steps in the cellular response to NLRP3 inflammasome development, NLRP3 inhibition produced similar results in both assays. Inhibitor MCC950 was used in these studies to confirm inhibition of caspase-1 activity and IL-1β release from LPS-primed, nigericin-treated THP-1 cells due to NLRP3 blocking.

These assays for biophysical interaction and pathway analysis, were combined to examine seven previously identified small molecule inflammasome inhibitors, in order to evaluate the inhibitors for interactions with NLRP3. These molecules were MCC950 (a potent NLRP3 inhibitor that blocks ATP hydrolysis), oridonin, NBC6, NBC19, CY-09, OXSI-2 and OLT1177.

Study Results and Conclusions
To determine and quantify intracellular engagement of NLRP3 inhibitors, the NLRP3 target engagement assay was run using HEK293 cells and consistent fluorescent tracer concentrations. The authors reported NLRP3 engagement for oridonin, NBC9, NBC19 and CY-09, with complete inhibition by these compounds except CY-09, which demonstrated partial inhibition. Two functional assays confirmed these results by directly measuring caspase-1 and IL-1β.

Surprisingly, the results showed no direct engagement nor pathway inhibition for two previously reported NLRP3 inhibitors, OXSI-2 and OLT1177. OLT1177 is currently being tested in phase 2 clinical trials.

Details on the work done by Teske et al. can be found in the paper cited below.

To summarize, the methods used here to investigate inflammasome activation were able to determine intracellular engagement by several inhibitors of NLRP3. Use of these inhibitors resulted in a coincident decrease of caspase-1 activity and IL-1β release in functional assays.

Importantly, these assays showed that two compounds, OXSI-2 and OLT1177, may be incorrectly classified as NLRP3 inhibitors. These compounds require further study to determine their cellular mode of action.

A researcher at the “Inflammasome Therapeutics Summit”, December 2023, where this work was presented, noted to these authors the importance of thorough characterization of drug candidates such as NLRP3 inhibitors, to ensure they reach their potential as therapeutic drugs in inflammatory diseases.


Teske, K.A. et al. (2023) Interrogating direct NLRP3 engagement and functional inflammasome inhibition using cellular assays. Cell Chem. Biol. Accessed 19-Dec-23.

Learn More

Inflammasome Activation web page has details on inflammasomes, a link to this paper featured in this blog and a video presented by author Martha O’Brien on this work.

NanoBRET™ Target Engagement web page includes video and diagrams, as well as product offerings, to explain this technology.

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Kari Kenefick

Kari Kenefick

Kari has been a science writer/editor for Promega since 1996. Prior to that she enjoyed working in veterinary microbiology/immunology, and has an M.S. in Bacteriology, U of WI-Madison. Favorite topics include infectious disease, inflammation, aging, exercise, nutrition and personality traits. When not writing, she enjoys training her dogs in agility and obedience. About the practice of writing, as we say for cell-based assays, "add-mix-measure".

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