Citraconate inhibits ACOD1 (IRG1) catalysis, reduces interferon responses and oxidative stress, and modulates inflammation and cell metabolism

Fangfang Chen’s PhD project describes potentially useful properties of two previously underappreciated cousins of itaconate: mesaconate and citraconate. Probably her most important finding is that citraconate inhibits ACOD1, the enzyme that mediates itaconate synthesis during macrophage activation.
Citraconate inhibits ACOD1 (IRG1) catalysis, reduces interferon responses and oxidative stress, and modulates inflammation and cell metabolism
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Graphical abstract of the paper: shared and distinct properties of the three isomers itaconate, mesaconate, and citraconate. Only citraconate inhibits ACOD1. 

Prehistory of the project

During my clinical training in Pediatric Rheumatology at the Children's Hospital of Philadelphia (University of Pennsylvania) in the early 2000’s, I stumbled upon an interesting mRNA that was highly upregulated in a mouse model of gout. Irg1 (immune response gene 1) bore vague resemblance to a bizarre (at least to me) bacterial enzyme, 2-methylcitrate dehydratase, but otherwise its function was unknown. In 2013, Karsten Hiller’s lab (University of Luxembourg) found that Irg1 encodes cis-aconitate decarboxylase (hence the new name, ACOD1) and is responsible for itaconate synthesis during macrophage activation (1). Meanwhile my own research had switched to viral infectious diseases. Surprise, surprise, in our gene expression analyses of influenza-infected mouse lungs, we kept finding Irg1 among the most highly induced mRNAs. My PhD student Mohamed Tantawy first tested the hypothesis that itaconate could be used as antiviral substance against influenza viruses, but his funding ended and he returned to his home country. Finally (2 doctoral theses later), we published that itaconate has strong anti-interferon properties, but at the same time reduces release of influenza virus particles from cells (2). Pulmonary inflammation was a lot stronger in infected Irg1 KO mice, which agreed well with the general consensus in the field (please correct me if you disagree) that endogenous itaconate helps to limit inflammation, at least at the organismal level. This brings us to Fangfang’s project.

Solving the structure of ACOD1

When Fangfang joined the Helmholtz Center for Infection Research as M.Sc. student in 2017, it was hard to believe that the crystal structure of IRG1 (meanwhile renamed ACOD1) had not been solved. The plan to solve it actually was born when Wulf Blankenfeldt (head of the Dept. Structure and Function of Proteins) and I once chatted during lunch in the cafeteria! For her Master’s thesis, Fangfang worked in Wulf’s lab (with Konrad Büssow as mentor) and figured out how to purify active ACOD1 and make nice crystals. We were so lucky that Fangfang could continue this work as a PhD student in my lab, which resulted in the publication of the crystal structure in 2019 (3). Now that we had the structure, what was the rationale for looking for inhibitors? Meanwhile, there were first indications in the literature that ACOD1 function may not always be entirely beneficial. For instance, itaconate had been accused of promoting tumor growth, immune paralysis, and the initiation of chronic inflammatory diseases.

Searching for ACOD1 inhibitors

Looking for inhibitors, our co-author Konrad Büssow screened a variety of molecules that resembled the natural substrate of ACOD1, cis-aconitate. Of course, this included also the product (itaconate) and its naturally occurring isomers mesaconate and citraconate, which Fangfang was testing for anti-inflammatory and antiviral properties in a related project that also became part of our present paper. Did itaconate affect ACOD1 activity? No, it didn’t, which ruled out product inhibition of the enzyme. Citraconate differs from itaconate only by the arrangement of the internal double bond. We were therefore stumped when Konrad found that citraconate inhibited ACOD1 activity. But a closer look clarified that it actually is a pretty good analog of the presumed transition state between cis-aconitate and itaconate. Unfortunately, all attempts at crystallizing the enzyme/inhibitor complex failed. Enter the stage Walid A. M. Elgaher and Anna K. H. Hirsch from the Helmholtz Institute for Pharmaceutical Research Saarland (Saarbrücken, Germany). They modeled ligand-target interactions and found that citraconate fits smack into the active site -- please check out the beautiful image in Figure 3f of the paper (4)! This was gratifying to see because it agreed perfectly with results of in vitro enzyme kinetics, which had suggested competitive inhibition (i.e. the inhibitor binds to the same place as the substrate). This is shown nicely in the Lineweaver-Burk plot in Figure 3b.

Inhibiting ACOD1 with citraconate does not necessarily affect inflammatory responses in human macrophages

One surprising finding was that essentially eliminating itaconate accumulation by adding 1 mM citraconate to LPS/IFN-γ stimulated macrophages (dTHP1 cells) did not affect expression of the pro-inflammatory genes IL-6, IL-1β, and CXCL10. Remembering that the Irg1 (Acod1) KO mice had a clearly pro-inflammatory phenotype (2), this gives us some food for thought. Perhaps the full anti-inflammatory effect of itaconate requires the complex regulatory networks of an entire organ, like a lymph node or an infected lung. Also, Fangfang confirmed previous reports that itaconate levels in human macrophages are much lower than in mouse macrophages (in which most itaconate research has been done) and, perhaps, eliminating it from human cells just does not affect inflammatory responses as much. Clearly, we need to learn a lot more about function of ACOD1 and itaconate in humans.

Effects of citraconate on cells may be bimodal

As the elaborate title of our paper suggests, we did see strong immunomodulatory, anti-oxidative, and antiviral effects of citraconate. However, these appeared at much higher concentrations (10-25 mM) in the medium, and we therefore think that citraconate effects are bimodal: low doses (≤1 mM) inhibit ACOD1 activity by noncovalent binding to the active site, but effects on other potential targets are weak. In contrast, higher concentrations (≥10 mM) also exert a variety of effects by SH-alkylating a potentially broad spectrum of other targets.

Where do we go from here? 

Citraconate inhibits ACOD1 activity at micromolar concentrations but becomes toxic only at concentrations >60 mM. Nonetheless, we think this can be improved and we consider citraconate merely a scaffold for further drug optimization. In the previous paragraph, I brought up the idea that ACOD1 function in humans is, at best, underexplored but, at worst, overrated. Nonetheless, the following potential applications for ACOD1 inhibitors based on citraconate come to mind: as antineoplastics to inhibit itaconate-dependent tumors, as immune stimulants to treat hypo-inflammation of sepsis and similar scenarios, as adjuvants to enhance vaccine responses, and last but not least as research reagents to probe the physiological function of endogenous itaconate. Clearly, more indications may appear on the horizon once we understand the role of itaconate in humans better. 

References

  1. Michelucci A, Cordes T, Ghelfi J, Pailot A, Reiling N, Goldmann O, et al. Immune-responsive gene 1 protein links metabolism to immunity by catalyzing itaconic acid production. Proc Natl Acad Sci U S A. 2013;110:7820-5.
  2. Sohail A, Iqbal AA, Sahini N, Chen F, Tantawy M, Waqas F, et al. Itaconate and derivatives reduce interferon responses and inflammation in influenza A virus infection. PLoS Pathog. 2022;18:e1010219.
  3. Chen F, Lukat P, Iqbal AA, Saile K, Kaever V, van den Heuvel J, et al. Crystal structure of cis-aconitate decarboxylase reveals the impact of naturally occurring human mutations on itaconate synthesis. Proc Natl Acad Sci U S A. 2019;116:20644-54.
  4. Chen F, Elgaher WAM, Winterhoff M, Büssow K, Waqas FH, Graner E, et al. Citraconate inhibits ACOD1 (IRG1) catalysis, reduces interferon responses and oxidative stress, and modulates inflammation and cell metabolism. Nature Metabolism. 2022;4:534–546.