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

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.