5 research outputs found
Mechanistic Characterization of a 2‑Thioxanthine Myeloperoxidase Inhibitor and Selectivity Assessment Utilizing Click Chemistry–Activity-Based Protein Profiling
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-6Â(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>)
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-6Â(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