Runge, B. | bioRxiv Science

bioRxiv2025

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.

biochemistry↗

bioRxiv2020

Permeability of the HIV-1 capsid to metabolites modulates viral DNA synthesis

Reverse transcription, an essential event in the HIV-1 lifecycle, requires deoxynucleotide triphosphates (dNTPs) to fuel DNA synthesis, thus requiring penetration of dNTPs into the viral core. The central cavity of the capsid protein (CA) hexamer reveals itself as a plausible channel that allows the passage of dNTPs into assembled capsids. Nevertheless, the molecular mechanism of nucleotide import into the capsid remains unknown. Employing all-atom molecular dynamics simulations, we established that cooperative binding between nucleotides inside a CA hexamer cavity results in energetically-favorable conditions for passive translocation of dNTPs into the HIV-1 capsid. Furthermore, binding of the host cell metabolite inositol hexakisphosphate (IP6) enhances dNTP import, while binding of synthesized molecules like benzenehexacarboxylic acid (BHC) inhibits it. The enhancing effect on reverse transcription by IP6 and the consequences of interactions between CA and nucleotides were corroborated using atomic force microscopy, transmission electron microscopy, and virological assays. Collectively, our results provide an atomistic description of the permeability of the HIV-1 capsid to small molecules and reveal a novel mechanism for the involvement of metabolites in HIV-1 capsid stabilization, nucleotide import and reverse transcription.

biophysics↗