Cancer cells make extensive use of the folate cycle to sustain increased anabolic metabolism. Multiple chemotherapeutic drugs interfere with the folate cycle, including methotrexate and 5-fluorouracil that are commonly applied for the treatment of leukemia and colorectal cancer (CRC), respectively. Despite high success rates, therapy-induced resistance causes relapse at later disease stages. Depletion of folylpolyglutamate synthase (FPGS), which normally promotes intracellular accumulation and activity of both natural folates and methotrexate, is linked to methotrexate and 5-fluorouracil resistance and its association with relapse illustrates the need for improved intervention strategies. In this study, we characterize a novel antifolate (C1) that, like methotrexate, potently inhibits dihydrofolate reductase (DHFR) and downstream one-carbon metabolism. Contrary to methotrexate, however, C1 displays optimal efficacy in FPGS-deficient contexts, due to decreased competition with intracellular folate concentrations for interaction with DHFR. Indeed, we show that FPGS-deficient patient-derived CRC organoids display enhanced sensitivity to C1-induced growth inhibition, while FPGS-high CRC organoids are more sensitive to methotrexate. Our results thus argue that polyglutamylation-independent antifolates can be applied to exert selective pressure on FPGS-deficient cells during chemotherapy, employing a vulnerability created by polyglutamylation deficiency.

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Peptide antibiotics have gathered attention given the urgent need to discover antimicrobials with new mechanisms of action. Their extended role as immunomodulators makes them interesting candidates for the development of compounds with dual mode of action. The objective of this study was to test the antiinflammatory capacity of a recently reported chimeric peptidomimetic antibiotic (CPA) composed of polymyxin B nonapeptide (PMBN) and a macrocyclic β-hairpin motif (MHM). We investigated the potential of CPA to inhibit lipopolysaccharide (LPS)-induced activation of RAW264.7 macrophages. In addition, we elucidated which structural motif was responsible for this activity by testing CPA, its building blocks, and their parent compounds separately. CPA showed excellent LPS neutralizing activity for both smooth and rough LPSs. At nanomolar concentrations, CPA completely inhibited LPS-induced nitric oxide, TNF-α, and IL-10 secretion. Murepavadin, MHM, and PMBN were incapable of neutralizing LPS in this assay, while PMB was less active compared to CPA. Isothermal titration calorimetry showed strong binding between the CPA and LPS with similar binding characteristics also found for the other compounds, indicating that binding does not necessarily correlate with neutralization of LPS. Finally, we showed that CPA-killed bacteria caused significantly less macrophage activation than bacteria killed with gentamicin, heat, or any of the other compounds. This indicates that the combined killing activity and LPS neutralization of CPA can prevent unwanted inflammation, which could be a major advantage over conventional antibiotics. Our data suggests that immunomodulatory activity can further strengthen the therapeutic potential of peptide antibiotics and should be included in the characterization of novel compounds.