Potent reversible inhibition of myeloperoxidase by aromatic hydroxamates

Abstract

The neutrophil enzyme myeloperoxidase (MPO) promotes oxidative stress in numerous inflammatory pathologies by producing hypohalous acids. Its inadvertent activity is a prime target for pharmacological control. Previously, salicylhydroxamic acid was reported to be a weak reversible inhibitor of MPO. We aimed to identify related hydroxamates that are good inhibitors of the enzyme. We report on three hydroxamates as the first potent reversible inhibitors of MPO. The chlorination activity of purified MPO was inhibited by 50% by a 5 nm concentration of a trifluoromethyl-substituted aromatic hydroxamate, HX1. The hydroxamates were specific for MPO in neutrophils and more potent toward MPO compared with a broad range of redox enzymes and alternative targets. Surface plasmon resonance measurements showed that the strength of binding of hydroxamates to MPO correlated with the degree of enzyme inhibition. The crystal structure of MPO-HX1 revealed that the inhibitor was bound within the active site cavity above the heme and blocked the substrate channel. HX1 was a mixed-type inhibitor of the halogenation activity of MPO with respect to both hydrogen peroxide and halide. Spectral analyses demonstrated that hydroxamates can act variably as substrates for MPO and convert the enzyme to a nitrosyl ferrous intermediate. This property was unrelated to their ability to inhibit MPO. We propose that aromatic hydroxamates bind tightly to the active site of MPO and prevent it from producing hypohalous acids. This mode of reversible inhibition has potential for blocking the activity of MPO and limiting oxidative stress during inflammation.

Keywords: Crystal Structure; Enzyme Inhibitors; Hydroxamate; Myeloperoxidase; Neutrophil; Oxidative Stress; Reversible Inhibition; Surface Plasmon Resonance (SPR).

FIGURE 1.

Normal catalytic cycling of myeloperoxidase. The native state of the enzyme, ferric MPO, reacts with hydrogen peroxide to form the redox intermediate Compound I. Compound I either oxidizes chloride to regenerate ferric MPO via the halogenation cycle (a) or will oxidize an organic substrate (RH) to a free radical (R^•), forming the redox intermediate Compound II, which can be reduced back to the native state via the peroxidation cycle (b).

FIGURE 2.

Chemical structures of aromatic hydroxamates (RC(O)NHOH) that inhibit MPO. The structures are shown for SHA, 2-(3,5-bistrifluoromethylbenzylamino)-6-oxo-1H-pyrimidine-5-carbohydroxamic acid (HX1), 4-benzyl-2-hydroxybenzenecarbohydroxamic acid (HX2), and 2-(benzylamino)-6-oxo-3H-pyrimidine-5-carbohydroxamic acid (HX3).

FIGURE 3.

Inhibition of HOCl production by aromatic hydroxamates. A, MPO (2 nm) was preincubated with either SHA, HX1, HX2, or HX3 for 15 min prior to the addition of H₂O₂ (10 μm). HOCl production was determined after 1 min by the taurine chloramine assay in the presence of 1 mm tyrosine. Data are presented as percentage of control HOCl production determined in the absence of inhibitor and represent the mean ± S.E. (error bars) of 3–51 independent experiments. B, MPO (5 nm) was incubated at room temperature in 50 mm phosphate buffer, pH 7.4 containing 140 mm NaCl, 200 μm urate, 50 μm tyrosine, 50 μm tryptophan, 1 mg/ml albumin,1 mm methionine with or without inhibitor HX1 (●) or TX1 (○). Reactions were started by adding 20 μm H₂O₂, and the consumption of H₂O₂ was measured after 15 min. Inhibition of H₂O₂ consumption was measured relative to the full system lacking added inhibitor in which approximately 10 μm H₂O₂ was consumed. Data are means ± range (error bars) of duplicates and are representative of two to three separate experiments. C, MPO was immobilized on protein immobilizer plates (Exiqon) and incubated with either SHA (■) or HX1 (●) for 15 min prior to the addition of 10 μm H₂O₂ substrate. HOCl production was determined after 1 min by the taurine chloramine assay. After extensive washing with assay buffer, a further 10 μm H₂O₂ was added, and HOCl production was redetermined (postwash SHA (□) and HX1 (○)). Data are presented as percentage of control HOCl production determined in the absence of compounds and represent the mean ± S.E. (error bars) of three independent experiments.

FIGURE 4.

Chlorination of tyrosine residues by stimulated neutrophils and inhibition by HX1. Neutrophils were incubated with human serum albumin and stimulated with PMA. The proteins were digested with Pronase and then analyzed for their content of 3-chlorotyrosine. A, a typical chromatogram of 3-chlorotyrosine showing the characteristic 3:1 isotopic ratios for the internal standard (Cl-Y¹³C₉) and chlorinated tyrosine (Cl-Y¹²C) produced by neutrophils. B, inset, the 3-chlorotyrosine content of proteins from the supernatant of neutrophils stimulated with PMA in the absence or presence of 1 μm HX1. Data are means ± S.E. (error bars) of three to seven measurements taken over three separate experiments. B, inhibition of 3-chlorotyrosine formation by PMA-stimulated neutrophils with increasing concentrations of HX1 relative to the full system without HX1. Data are plotted as mean ± range of duplicates and are representative of three experiments. Experimental details are described under “Experimental Procedures.” AU, arbitrary units.

FIGURE 5.

Determination of binding kinetics of MPO inhibitors using SPR. MPO was immobilized to a CM5 sensor chip, and compound binding was evaluated for HX1, HX2, and HX3. The panels show specific binding traces representative of three to six separate experiments. Each panel shows the overlay of multiple serial sensorgram traces for nine different compound concentrations in a series of 3-fold dilutions ranging from 0.3 μm to 30 pm for HX1 and HX2 and from 30 μm to 3 nm for HX3. From t = 0, compound was continuously perfused over the sensor chip leading to a clear net association of compound to the immobilized MPO. Subsequently, compound perfusate was replaced with buffer at t = 60–210 s, leading to a loss of response due to net compound dissociation. RU, response units.

FIGURE 6.

X-ray crystal structure of the MPO-HX1 complex and electron density maps. A, the enzyme-inhibitor complex has HX1 (orange stick representation) bound in the active site pocket above the MPO heme group (green). MPO side chains are shown for residues within 5 Å from the ligand in a stick representation (yellow). Hydrogen bonds between HX1 and MPO and solvent are indicated with dashed lines. The beige surface outlines the solvent-accessible area of the active site. Red atoms indicate the oxygen of water molecules. B, electron density maps for HX1. The F_o − F_c omit map was contoured at 2.5 σ and calculated in the absence of HX1, and the 2F_o − F_c map was contoured at 1 σ and calculated for the final model where occupancy of HX1 was set to 0.5.

FIGURE 7.

Inhibition of the rate of NADH bromohydrin formation. MPO (20 nm) was incubated at room temperature in 20 mm phosphate buffer, pH 7.4 containing 100 μm NADH with either 50 μm H₂O₂ ± 0.1 μm HX1 with varying concentrations of NaBr (A) or 5 mm NaBr ± 0.1 μm HX1 with varying concentrations of H₂O₂ (B). The formation of NADH bromohydrin was detected by absorbance at 275 nm, and initial rates were determined within the 1st min. Data shown are means ± S.E. (error bars) of triplicates in the absence (●) or presence (○) of HX1 and are representative of three separate experiments.

FIGURE 8.

Effect of HX1 and HX2 on the absorption spectrum of MPO. A, MPO (1.8 μm) was incubated with 10 μm HX2 in 50 mm phosphate buffer, pH 7.4 (gray). H₂O₂ (40 μm) was then added, and spectra were recorded at 10 s (black) and 5 min (dashed). B, the difference spectrum between the first observable spectrum after H₂O₂ (A, black) and ferric MPO (A, gray). C, MPO (2.75 μm) was incubated with 62.5 μm HX2 in 50 mm phosphate buffer, pH 7.4 (gray). H₂O₂ (50 μm) was added, and the spectral changes were recorded (black). The new spectrum with peaks at 468 and 637 nm formed within 30 s and was stable for approximately 3 min. D, spectral changes in the UV region after adding H₂O₂ to MPO and HX2. The arrows indicate the direction of the spectral changes observed at 30-s intervals. All results are typical of three experiments.

FIGURE 9.

Proposed mechanism for the inhibition of MPO by hydroxamates, and their concurrent oxidation. Ferric MPO is bound by hydroxamate (RC(O)NHOH) forming an inactive complex (top left), thereby abrogating the cycling of ferric MPO via Compounds I and II. This is the inhibition pathway. Hydroxamates can to varying degrees also serve as substrates of MPO Compound I to form transient nitroxide radical RC(O)NHO^•. This in turn can reduce ferric MPO to ferrous MPO (Fe(II)) to yield nitrosyl ferrous MPO (NO-Fe(II)) upon binding of released NO. The oxidized product of RC(O)NHO^• is subject to hydrolysis (dotted line), yielding the carboxylic acid and HNO, a source of NO.