Plant Compound Calein C Blocks Key Bacterial Antioxidant Enzyme, Study Finds

A small molecule extracted from a Brazilian wildflower has emerged as a surprising new weapon in the fight against antibiotic resistance. In a study published in Applied Microbiology and Biotechnology, researchers report that calein C, a sesquiterpene lactone isolated from the plant Calea pinnatifida, irreversibly inhibits AhpC, a 2-Cys peroxiredoxin from Escherichia coli that serves as a critical antioxidant shield for many pathogenic bacteria. The finding is significant because AhpC has been identified as a virulence factor in a range of bacterial species, yet inhibitors of this enzyme remain poorly characterized. By combining computational modeling, purified-enzyme biochemistry, and bacterial growth assays, the team, led by Sabrina Vargas and Marcos A. de Oliveira of São Paulo State University together with collaborators at the University of São Paulo, the Federal University of São Paulo, and the Federal University of ABC, has delivered the first characterization of a sesquiterpene lactone capable of disabling a bacterial two-cysteine peroxiredoxin.

To appreciate why this matters, it helps to understand what AhpC does inside a bacterial cell. When the immune system attacks an infection, or when antibiotics such as bactericidal drugs go to work, they generate reactive oxygen species, including hydrogen peroxide and organic hydroperoxides. These molecules damage DNA, proteins, and membranes, and they are a central part of how host defenses and many antibacterial therapies kill microbes. Alkyl hydroperoxide reductase C, or AhpC, is one of the frontline enzymes bacteria deploy to neutralize these oxidants. As a typical 2-Cys peroxiredoxin, it relies on a highly reactive peroxidatic cysteine residue at its active site. This cysteine attacks the hydroperoxide substrate, becoming oxidized in the process, and is then regenerated through a catalytic cycle involving a resolving cysteine and the thioredoxin–thioredoxin reductase system. Knock out this cycle, and the bacterium becomes far more vulnerable to the oxidative onslaught it faces inside a host.

The research team began with calein C, a sesquiterpene lactone obtained from Calea pinnatifida, a plant native to the Brazilian Cerrado. Sesquiterpene lactones are a large family of plant secondary metabolites built from three isoprene units and characterized by an alpha-methylene-gamma-lactone ring, a structural motif well known for reacting with cysteine residues in proteins. This inherent electrophilicity made the compound an attractive candidate for targeting the reactive cysteine at the heart of AhpC catalysis. Using molecular docking simulations, the investigators showed that calein C binds stably within the active site pocket of E. coli AhpC, positioning its lactone moiety close to the peroxidatic cysteine and adopting a conformation consistent with covalent attack on the enzyme’s catalytic machinery.

Biochemical experiments with purified EcAhpC confirmed the computational predictions and quantified the potency of the interaction. Calein C inhibited the peroxidase activity of the enzyme with an IC50 of approximately 1.5 micromolar, a value the authors describe as indicating a potent inhibitor for this target class. Detailed kinetic analysis pointed to irreversible inhibition, consistent with a mechanism in which the compound forms a covalent adduct with the essential cysteine and permanently inactivates the enzyme rather than merely competing with substrate. Irreversible inhibition of a virulence-associated antioxidant enzyme is a particularly attractive pharmacological strategy, because a permanently disabled enzyme cannot be rescued by dilution or simple competition, and lower sustained concentrations of the inhibitor may be required to suppress bacterial antioxidant defenses during an infection.

Translating the enzyme result to living bacteria revealed a more nuanced picture, but also one of genuine therapeutic promise. On its own, calein C effectively inhibited the growth of Gram-positive bacteria, whose single membrane leaves the periplasmic and cytoplasmic targets more accessible. Against Gram-negative strains, however, the compound showed no intrinsic antibacterial activity, a result the authors attribute largely to the formidable outer membrane barrier and the activity of multidrug efflux pumps that characterize organisms such as E. coli. Rather than abandoning the Gram-negative experiments, the team turned to a combination strategy that has become one of the most actively pursued approaches in modern antimicrobial research: adjuvant therapy, in which a compound with modest standalone activity is paired with an established antibiotic or a permeabilizing agent to produce an effect greater than either drug alone.

The combination assays delivered the study’s most striking results. When calein C was combined with gentamicin, an aminoglycoside antibiotic whose bactericidal activity is tightly linked to the production of reactive oxygen species, growth inhibition of Gram-positive bacteria was significantly enhanced. The logic is compelling: gentamicin stresses the cell and stimulates oxidant formation, while calein C simultaneously removes the bacterium’s capacity to detoxify those oxidants, creating a lethal synergy. For Gram-negative bacteria, the researchers paired calein C with polymyxin B nonapeptide, or PMBN, a derivative of polymyxin B that permeabilizes the outer membrane without itself being strongly bactericidal. With the membrane barrier weakened, calein C could gain access to intracellular AhpC, and the combination produced enhanced growth inhibition of Gram-negative strains that the lactone could not touch on its own.

These findings position AhpC as a promising target for overcoming antibiotic resistance and calein C as a prototype for a new chemical class of anti-virulence agents. The concept of disarming bacterial antioxidant defenses rather than directly killing the cells offers several advantages. Anti-virulence strategies may impose weaker selective pressure for classical resistance mutations, and sensitizing bacteria to oxidative stress could rejuvenate existing antibiotics whose clinical utility is eroding. The work also expands the known chemical space of peroxiredoxin inhibitors. Although peroxiredoxins have long been studied in the context of parasite and cancer biology, validated inhibitors of bacterial 2-Cys peroxiredoxins have been scarce, and this is the first report of a sesquiterpene lactone with this activity, opening a natural-product scaffold that medicinal chemists can now optimize.

The study is a product of a broad Brazilian research collaboration supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo through the CEPID REDOXOMA program and by the Conselho Nacional de Desenvolvimento Científico e Tecnológico. Natural products from Brazilian biodiversity have historically yielded pharmacologically important molecules, and Calea pinnatifida, used in traditional medicine, continues to attract phytochemical interest. By coupling natural products chemistry with redox biology and microbiology, the team illustrates how systematically pairing plant-derived electrophiles with vulnerable bacterial enzymes can generate candidate adjuvants for the antibiotic pipeline, a pipeline that global health authorities have repeatedly described as dangerously thin in the face of rising multidrug resistance.

Considerable work remains before calein C or its derivatives could approach the clinic. The researchers will need to confirm the covalent modification site on AhpC at atomic resolution, evaluate selectivity against human peroxiredoxins, optimize compound stability and delivery, and test efficacy in infection models. The requirement for permeabilizing partners in Gram-negative organisms adds a layer of formulation complexity, although combination regimens of this kind are already established in practice. Even so, the study’s central message stands out clearly in a field hungry for new ideas: an old enzyme that bacteria depend on to survive oxidative attack can be permanently shut down by a molecule from a Brazilian shrub, and doing so makes existing antibiotics work better. As resistance continues to outpace the discovery of entirely new drug classes, strategies that strip pathogens of their biochemical armor may prove to be among the most valuable additions to the antimicrobial arsenal.

Subject of Research: Inhibition of the bacterial 2-Cys peroxiredoxin AhpC by the plant-derived sesquiterpene lactone calein C as a strategy against antibiotic resistance

Article Title: The sesquiterpene lactone Calein C irreversibly inhibits AhpC: a 2-Cys peroxiredoxin from E. coli

Article References: Vargas, S., Cabrera, V. I. M., dos Santos, M. C., Tairum, C. A., Caldas, L. A., Sartorelli, P., Soares, S. R., Toyama, M. H., Netto, L. E. S., Lago, J. H. G., & de Oliveira, M. A. (2026). The sesquiterpene lactone Calein C irreversibly inhibits AhpC: a 2-Cys peroxiredoxin from E. coli. Applied Microbiology and Biotechnology. https://doi.org/10.1007/s00253-026-14028-0

Image Credits: AI Generated

DOI: 10.1007/s00253-026-14028-0

Keywords: AhpC, calein C, sesquiterpene lactone, Escherichia coli, peroxiredoxin, antimicrobial resistance, antioxidant enzymes, natural products, antibiotic adjuvants, reactive oxygen species, Calea pinnatifida, oxidative stress