5 research outputs found

    Mechanistic Characterization of a 2‑Thioxanthine Myeloperoxidase Inhibitor and Selectivity Assessment Utilizing Click Chemistry–Activity-Based Protein Profiling

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    Myeloperoxidase (MPO) is a heme peroxidase that catalyzes the production of hypochlorous acid. Despite a high level of interest in MPO as a therapeutic target, there have been limited reports about MPO inhibitors that are suitable for evaluating MPO in pharmacological studies. 2-Thioxanthine, 3-(2-ethoxypropyl)-2-thioxo-2,3-dihydro-1<i>H</i>-purin-6­(9<i>H</i>)-one (<b>A</b>), has recently been reported to inhibit MPO by covalently modifying the heme prosthetic group. Here we report a detailed mechanistic characterization demonstrating that <b>A</b> possesses all the distinguishing features of a mechanism-based inactivator. <b>A</b> is a time-dependent MPO inhibitor and displays saturable inactivation kinetics consistent with a two-step mechanism of inactivation and a potency (<i>k</i><sub>inact</sub>/<i>K</i><sub>I</sub> ratio) of 8450 ± 780 M<sup>–1</sup> s<sup>–1</sup>. MPO inactivation by <b>A</b> is dependent on MPO catalysis and is protected by substrate. <b>A</b> reduces MPO compound I to compound II with a second-order rate constant of (0.801 ± 0.056) × 10<sup>6</sup> M<sup>–1</sup> s<sup>–1</sup>, and its irreversible inactivation of MPO occurs prior to release of the activated inhibitory species. Despite its relatively high selectivity against a broad panel of more than 100 individual targets, including enzymes, receptors, transporters, and ion channels, we demonstrate that <b>A</b> labels multiple other protein targets in the presence of MPO. By synthesizing an alkyne analogue of <b>A</b> and utilizing click chemistry–activity-based protein profiling, we present that the MPO-activated inhibitory species can diffuse away to covalently modify other proteins, as reflected by the relatively high partition ratio of <b>A</b>, which we determined to be 15.6. This study highlights critical methods that can guide the discovery and development of next-generation MPO inhibitors

    Deconstruction of Activity-Dependent Covalent Modification of Heme in Human Neutrophil Myeloperoxidase by Multistage Mass Spectrometry (MS<sup>4</sup>)

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    Myeloperoxidase (MPO) is known to be inactivated and covalently modified by treatment with hydrogen peroxide and agents similar to 3-(2-ethoxypropyl)-2-thioxo-2,3-dihydro-1<i>H</i>-purin-6­(9<i>H</i>)-one (<b>1</b>), a 254.08 Da derivative of 2-thioxanthine. Peptide mapping by liquid chromatography and mass spectrometry detected modification by <b>1</b> in a labile peptide–heme–peptide fragment of the enzyme, accompanied by a mass increase of 252.08 Da. The loss of two hydrogen atoms was consistent with mechanism-based oxidative coupling. Multistage mass spectrometry (MS<sup>4</sup>) of the modified fragment in an ion trap/Orbitrap spectrometer demonstrated that <b>1</b> was coupled directly to heme. Use of a 10 amu window delivered the full isotopic envelope of each precursor ion to collision-induced dissociation, preserving definitive isotopic profiles for iron-containing fragments through successive steps of multistage mass spectrometry. Iron isotope signatures and accurate mass measurements supported the structural assignments. Crystallographic analysis confirmed linkage between the methyl substituent of the heme pyrrole D ring and the sulfur atom of <b>1</b>. The final orientation of <b>1</b> perpendicular to the plane of the heme ring suggested a mechanism consisting of two consecutive one-electron oxidations of <b>1</b> by MPO. Multistage mass spectrometry using stage-specific collision energies permits stepwise deconstruction of modifications of heme enzymes containing covalent links between the heme group and the polypeptide chain
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