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-1H-purin-6(9H)-one (A), has recently been reported to inhibit MPO by covalently modifying the heme prosthetic group. Here we report a detailed mechanistic characterization demonstrating that A possesses all the distinguishing features of a mechanism-based inactivator. A is a time-dependent MPO inhibitor and displays saturable inactivation kinetics consistent with a two-step mechanism of inactivation and a potency (k(inact)/K(I) ratio) of 8450 ± 780 M⁻¹ s⁻¹. MPO inactivation by A is dependent on MPO catalysis and is protected by substrate. A reduces MPO compound I to compound II with a second-order rate constant of (0.801 ± 0.056) × 106 M⁻¹ s⁻¹, 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 A labels multiple other protein targets in the presence of MPO. By synthesizing an alkyne analogue of A 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 A, which we determined to be 15.6. This study highlights critical methods that can guide the discovery and development of next-generation MPO inhibitors.

Three-dimensional model of a selective theophylline-binding RNA molecule.

A three-dimensional (3D) model for an RNA molecule that selectively binds theophylline but not caffeine is proposed. This RNA, which was found using SELEX (Jenison et al., 1994), is 10,000 times more specific for theophylline (Kn = 320 nM) than for caffeine (KD = 3.5 mM), although the two ligands are identical except for a methyl group substituted at N7 (present only in caffeine). The binding affinity for ten xanthine-based ligands was used to derive a comparative molecular field analysis model (R2 = 0.93 for three components, with cross-validated R2 of 0.73), using the SYBYL and GOLPE programs. A pharmacophoric map was generated to locate steric and electrostatic interactions between theophylline and the RNA binding site. This information was used to identify putative functional groups of the binding pocket and to generate distance constraints. On the basis of a model for the secondary structure (Jenison et al., 1994), the 3D structure of this RNA was then generated using the following method: each helical region of the RNA molecule was treated as a rigid body; single-stranded loops with specific end-to-end distances were generated. The structures of RNA-xanthine complexes were studied using a modified Monte Carlo algorithm. The detailed structure of an RNA-ligand complex model, as well as possible explanations for the theophylline selectivity are discussed.

Assessment of the enantiomeric purity of R- and S-N6-phenylisopropyladenosine (PIA): implications for adenosine receptor subclassification.

A chiral column high-performance liquid chromatographic method was developed for the assessment of the enantiomeric purity of the stereoisomers of N6-phenylisopropyladenosine (PIA). The observed chiral purity of R-PIA was greater than 99.9%, whereas S-PIA was found to contain 4.4% of the R-enantiomer. In radioligand binding studies, the observed affinity of S-PIA for the adenosine A1 receptor (IC50 240 nM) could entirely be attributed to its content of R-PIA (IC50 7.8 nM). Calculation of a theoretical IC50 of pure S-PIA for the A2 receptor yielded a value of 6700 nM, which was 35-fold higher than for R-PIA (190 nM). Concludingly, the utilization of enantiomeric impure S-PIA in the definition of adenosine receptor subclasses is questionable.

Urid acid accumulated in isolated kidney grafts: an assessment of agonal ischaemia.

Hypoxanthine-xanthine washed out from kidney tissue during preservation has previously been found to be a reliable measure of the in vitro assessment of the deleterious effect of ischaemia on the functional regenerative ability of the graft. We have now studied the question: can uric acid accumulation in the isolated kidney graft be employed as a retrospective measure of the agonal ischaemia? We have found that uric acid alone accumulates in renal tissue during the agonal phase, in an amount that remains unchanged during the subsequent in vitro ischaemia. The determination of hypoxanthine-xanthine and uric acid in kidney perfusate samples during preservation must thus be presumed to be an optimal clinical-chemical method of assessing the graft ischaemia.