Inhibition of NF-κB Signaling by the Reactive Glycolytic Metabolite Methylglyoxal

Caroline Stanton, Woojin Choi, R. Luke Wiseman, Michael J. Bollong

Posted on: 10 November 2025

Preprint posted on 16 May 2025

This research uncovers how tinkering with a basic energy pathway in cells can unexpectedly switch off key immune signals—pointing to new ways of controlling inflammation and even cancer.

Selected by Yan Aveiro dos Reis, Marcus Oliveira

Categories: biochemistry, pharmacology and toxicology

Why this preprint is important?

The study importantly describes how the molecule CBR-470-2 modulates immune response via NF-κB activity, proposing a novel pharmacological mechanism that inhibits glycolysis to regulate immune function, with insights from both monocyte and lung adenocarcinoma cell models suggesting its potential as a pharmacological approach for cancer treatment and understanding its effects on normal immune cells.

Background

Activation of the transcription factor NF-κB requires the formation of dimers that translocate from the cytosol to the nucleus to initiate gene transcription (1). Under basal conditions, NF-κB dimmers are maintained in the cytosol through their interaction with the IκB family of inhibitory proteins. In response to inflammatory stimuli, the IκB kinase (IKK) complex phosphorylates IκB proteins, targeting them for degradation and allowing NF-κB dimers to enter the nucleus (1). This signaling cascade can be triggered by reactive oxygen species (ROS) and electrophilic molecules, leading to the expression of pro-inflammatory cytokines (2). In contrast, the transcription factor NRF2 functions as a key regulator of cellular redox status and promotes the resolution of inflammation. NRF2 is activated when its cytoplasmic repressor, KEAP1, senses ROS or other electrophilic chemicals (3). Once activated, NRF2 dampens innate immune signaling and can inhibit the NF-κB pathway. Previous studies have shown that specific inhibition of phosphoglycerate kinase 1 (PGK1) by CBR-470-2 increases intracellular levels of the reactive electrophile methylglyoxal (MGO), which is known to inhibit KEAP1 (4). Based on this mechanism, the authors hypothesize that the accumulation of MGO following PGK1 inhibition will modulate NF-κB signaling.

Key findings

PGK1 inhibition reduces the transcriptional output of NF-κB.

In immunologically stimulated THP-1 cells (monocytes), it was observed that CBR-470-2 inhibited NF-κB activity (Fig. 1B-E) and nuclear translocation (Fig. 1F-G). Further evidence of PGK1 inhibition with siRNA recapitulated the decrease in NF-κB transcriptional activity to a certain degree (Fig. 2D-F). These findings suggest a crosstalk between glycolysis and NF-κB mediated by PGK1 activity

CBR-470-2 inhibits NF-κB in a NFR2 independent way.

Pharmacological inhibition of NRF2 did not change the dose-dependent inhibition of NF-κB by CBR-470-2 in immunostimulated monocytes (Fig. 2A). The authors suggested that NRF2 activation is not relevant to the observed inhibitory mechanism. On the other hand, treating these stimulated cells with GSH made them more resistant to PGK1 inhibition (Fig. 2B). The authors suggested that CBR-470-2 is driving the accumulation of electrophilic molecules.

MGO treatment reproduced the effect of CBR-470-2 inhibition

Given that MGO is a byproduct of glycolysis, the authors sought to analyze the specific relevance of increased MGO levels to NF-κB modulation. By treating either immunostimulated monocytes or A549 (lung adenocarcinoma) cells with MGO, the authors were able to replicate the NF-κB inhibition (Fig. 5) previously observed during PGK1 inhibition. In the lung adenocarcinoma cells, no difference was observed in sensitivity to MGO during NRF2 inhibition; therefore, the group suggested that the MGO-mediated inhibition of NF-κB is independent of the canonical NRF2 pathway.

Comments

C1: Glycolysis is a major pathway responsible for many cellular processes, such as ATP synthesis and NADH/NAD+ redox balance. It would enrich this preprint’s discussion to consider reduced ATP synthesis and altered NADH/NAD+ levels as factors that could interfere with NF-κB activity.

C2: Inhibition of glycolysis can lead to an increased flux through other metabolic pathways, such as lipid and ketone body catabolism, which can also contribute to MGO accumulation (5,6). It would enrich this pre-print’s discussion to consider these other metabolic pathways as possible generators of MGO.

C3: Reactions between GSH and MGO can lead to D-lactate production (7,8). It would enrich this preprint’s discussion to consider a possible accumulation of D-lactate and its potential roles as a regulator of the immune response.

C4: In these NRF2-inhibited cells (Fig 2A), it is possible to observe an approximate 80% decrease in NF-κB activity. This contrasts with the results in Figure 1A, where cells with functional NRF2 showed a complete depletion of the NF-κB signal. Could the authors pinpoint the cause of this remaining ~20% of NF-κB activity in the NRF2-inhibited cells?

C5: Could the authors provide additional details on the settings used to quantify NF-κB localization with the Cell Insight CX5 HCS Platform for the experiments shown in Figures 2F and 3E? For example, what specific parameters were used for image acquisition and analysis?

Disclaimer

Google Gemini was used for the grammatical review for this report.

References

Liu T, Zhang L, Joo D, Sun SC. NF-κB signaling in inflammation. Signal Transduct Target Ther. 14 de julho de 2017;2(1):17023.
Morgan MJ, Liu Z gang. Crosstalk of reactive oxygen species and NF-κB signaling. Cell Res. janeiro de 2011;21(1):103–15.
Baird L, Yamamoto M. The Molecular Mechanisms Regulating the KEAP1-NRF2 Pathway. Mol Cell Biol. 15 de junho de 2020;40(13):e00099-20.
Bollong MJ, Lee G, Coukos JS, Yun H, Zambaldo C, Chang JW, et al. A metabolite-derived protein modification integrates glycolysis with KEAP1–NRF2 signalling. Nature. outubro de 2018;562(7728):600–4.
Salomón T, Sibbersen C, Hansen J, Britz D, Svart MV, Voss TS, et al. Ketone Body Acetoacetate Buffers Methylglyoxal via a Non-enzymatic Conversion during Diabetic and Dietary Ketosis. Cell Chem Biol. agosto de 2017;24(8):935-943.e7.
Berdowska I, Matusiewicz M, Fecka I. A Comprehensive Review of Metabolic Dysfunction-Associated Steatotic Liver Disease: Its Mechanistic Development Focusing on Methylglyoxal and Counterbalancing Treatment Strategies. Int J Mol Sci. 7 de março de 2025;26(6):2394.
Cai M, Wan J, Cai K, Song H, Wang Y, Sun W, et al. Understanding the Contribution of Lactate Metabolism in Cancer Progress: A Perspective from Isomers. Cancers. 23 de dezembro de 2022;15(1):87.
Li J, Ma P, Liu Z, Xie J. L- and D-Lactate: unveiling their hidden functions in disease and health. Cell Commun Signal. 12 de março de 2025;23(1):134.

Tags: disease, inflammation, nf-κb, pathology, pharmacology

doi: https://doi.org/10.1242/prelights.41998

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