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bioRxiv · 10.1101/2025.06.13.659517

Analysis of apo and citraconate-bound hACOD1 (hIRG1) by X-ray crystallography and NMR spectroscopy: structural insights for developing chemotherapeutic agents

Monteiro, D. C. F.·Runge, B.·Fucci, I. J.
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Abstract

Human aconitate decarboxylase (ACOD1) has emerged as a therapeutic target for cancer and inflammatory diseases, with its metabolic product, itaconate, as a multifunctional metabolite shown to drive several disease states. Though extensively studied in cellulo and in vivo, this protein is biochemically and mechanistically under characterized. In this work we present a robust mechanistic study of hACOD1, with insights into its structure-activity relationships and inhibition. First true-apo and inhibited structures of this enzyme and selected point-mutants are presented, together with complementary kinetic, biochemical and biophysical analyses. We also devised a novel, low-enzyme consumption, continuous kinetic assay to support complex kinetic studies, as exemplified by the competitive inhibition assays performed. Together with molecular dynamics simulations and solution X-ray scattering experiments, all these data shed light on mechanism and dynamic behavior of the enzyme, vital insights for future drug discovery campaigns.

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Monteiro, D. C. F., Runge, B., Fucci, I. J.. 2025-06-13. Analysis of apo and citraconate-bound hACOD1 (hIRG1) by X-ray crystallography and NMR spectroscopy: structural insights for developing chemotherapeutic agents. https://doi.org/10.1101/2025.06.13.659517

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