Cancer Drug GSK461364 Shuts Down NLRP3 Inflammasome Inflammation in Mice
A PLK1-inhibiting cancer compound blocks NLRP3 inflammasome assembly by preventing NEK7 phosphorylation, easing endotoxemia and colitis in mice.
Summary
Overactive NLRP3 inflammasomes drive many inflammatory diseases, yet most NLRP3-targeting drugs have stalled in clinical development. Researchers screened 429 kinase inhibitors in mouse macrophages and found GSK461364, a PLK1 inhibitor that was already tested in cancer trials, strongly blocked NLRP3-dependent IL-1β release. It did not affect AIM2 or NLRC4 inflammasomes. In mice, it reduced inflammation in LPS-induced endotoxemia and DSS-induced colitis without visible toxicity over 9 days. Mechanistically, PLK1 appears to phosphorylate NEK7, likely at Ser221 and Ser260, which strengthens NEK7-NLRP3 binding needed for inflammasome assembly. Blocking PLK1 reduces this phosphorylation and inflammasome activation. The work is preclinical, but it suggests repurposing PLK1 inhibitors for NLRP3-driven inflammation.
Detailed Summary
The NLRP3 inflammasome is a protein complex that triggers caspase-1 activation, IL-1β release and pyroptotic cell death. Its overactivation is linked to colitis, gout, atherosclerosis, sepsis and type 2 diabetes, all relevant to inflammaging. Many direct NLRP3 inhibitors, such as MCC950, have failed clinically, so new targets and drugs are needed.
The team screened a 429-compound kinase inhibitor library in LPS-primed mouse peritoneal macrophages stimulated with nigericin, adding compounds after priming to avoid effects on the priming step. Six compounds cut IL-1β without lowering TNF-α, including four with previously reported NLRP3-suppressing activity, which validated the screen. The sixth, GSK461364, a PLK1 inhibitor, was new. At 10 µM it blocked IL-1β release, LDH release (cell death), caspase-1 cleavage, and ASC oligomerization and speck formation after ATP, nigericin or MSU. It had little effect on AIM2 or NLRC4 inflammasomes, which points to NLRP3 selectivity.
In vivo, intraperitoneal GSK461364 (10 and 25 mg/kg) lowered serum IL-1β, but not TNF-α or IL-6, in LPS endotoxemia. It also reduced lung inflammation and cleavage of GSDMD and caspase-1. In DSS colitis, it reduced weight loss, disease activity scores, colon shortening, tissue damage and colonic ASC oligomerization. A 25 mg/kg dose over 9 days produced no changes in body weight, liver or kidney markers, or organ histology, and no cytotoxicity appeared in cultured cells at 1–20 µM.
The drug did not alter mitochondrial damage, mitochondrial ROS, potassium or chloride efflux, or expression of inflammasome components. GSK461364 bound PLK1 (SPR KD about 5.21 µM), PLK1 silencing mimicked its effects, and the drug lost activity in PLK1-silenced cells. PLK1 interacted with NEK7 but not NLRP3. PLK1 knockdown or inhibition reduced nigericin-induced NEK7 Ser/Thr phosphorylation and weakened NEK7-NLRP3 association. In vitro kinase assays (Phos-tag, ADP-Glo) showed direct PLK1 phosphorylation of NEK7, which GSK461364 reduced. NEK7 S221A and S260A mutants impaired IL-1β production in reconstituted HEK293T cells.
The findings outline a PLK1/NEK7/NLRP3 axis and support repurposing PLK1 inhibitors for inflammation. Caveats are important: the data are from mice and cell models, phosphorylation sites are described as 'likely' (mutational and predicted rather than mass-spectrometry-confirmed), and PLK1 inhibition carries antimitotic effects that could limit chronic use. The work also conflicts with other reports on PLK1 and PLK4 in NLRP3 regulation.
Key Findings
- Screening 429 kinase inhibitors identified GSK461364, a PLK1 inhibitor, as a selective NLRP3 inflammasome blocker with little effect on AIM2 or NLRC4.
- GSK461364 reduced serum IL-1β, lung inflammation and GSDMD/caspase-1 cleavage in LPS endotoxemia, and eased DSS colitis in mice.
- PLK1 phosphorylates NEK7, likely at Ser221 and Ser260, enhancing NEK7-NLRP3 binding; GSK461364 blocks this phosphorylation.
- The drug lost its effect in PLK1-silenced macrophages, supporting PLK1 as its functional target for NLRP3 inhibition.
- No toxicity was seen in cells (1–20 µM) or in mice given 25 mg/kg over 9 days.
Methodology
A 429-compound kinase inhibitor library was screened by IL-1β ELISA in LPS-primed mouse peritoneal macrophages stimulated with nigericin. Hits were validated with multiple inflammasome agonists, ASC oligomerization and speck assays, and PLK1 siRNA knockdown. Target engagement was shown by CETSA and SPR. NEK7 phosphorylation was assessed by co-IP, Phos-tag, ADP-Glo and site mutants, with efficacy tested in mouse LPS endotoxemia (n=6) and DSS colitis models.
Study Limitations
All evidence comes from mouse and cell models with short-term dosing, and the toxicity assessment covered only 9 days. Ser221/Ser260 as phosphorylation sites rest on prediction and mutant functional assays rather than direct site mapping, and the findings conflict with reports of PLK1-NLRP3 binding and PLK4-mediated NEK7 effects. Because PLK1 is a mitotic kinase, chronic anti-inflammatory use raises off-target and antiproliferative concerns that were not tested, and the moderate binding affinity (KD about 5 µM) leaves room for off-target effects.
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