Targeting vascular adhesion protein-1 and myeloperoxidase with a dual inhibitor SNT-8370 in preclinical models of inflammatory disease

Abstract

Inflammatory diseases are a major source of morbidity and mortality world-wide, the pathogenesis of which are characterised by the interplay of key pro-inflammatory and oxidative enzymes. Here, we report the development of a small molecule dual inhibitor targeting vascular adhesion protein-1 (VAP-1) and myeloperoxidase (MPO), two clinically relevant pro-inflammatory/oxidative enzymes that play complementary pathogenic roles in various inflammatory diseases. This agent, SNT-8370 [(E)-3-(3-((2-(aminomethyl)-3-fluoroallyl)oxy)benzyl)-2-thioxo-1,2,3,7-tetrahydro-6H-purin-6-one)], irreversibly inhibits VAP-1 and MPO activity with equivalent and enhanced nanomolar potency, respectively, when compared to benchmark clinical VAP-1 and MPO inhibitors. SNT-8370 is selective, exhibiting >100-1000-fold more potency for VAP-1 and MPO versus other mammalian (per)oxidases and shows no significant off-target activity in established preclinical screening panels. In vivo, SNT-8370 is metabolically stable, exhibits a favourable pharmacokinetic/pharmacodynamic profile without CNS penetration, and effectively inhibits VAP-1 and MPO activities. Moreover, compared to monotherapy, SNT-8370 more effectively inhibits leukocyte infiltration in mouse peritonitis, carrageenan air pouch, and lipopolysaccharide-induced lung injury models of acute inflammation. SNT-8370 is also protective in preclinical models of myocardial ischemia-reperfusion injury and unilateral-ureteral-obstruction-induced nephropathy. Collectively, our results support SNT-8370 as a first-in-class, mechanism-based dual inhibitor of VAP-1 and MPO, and as a promising therapeutic for the clinical treatment of inflammatory disorders.

Fig. 1. Biological roles and interaction of VAP-1 and MPO.

During inflammation, VAP-1 expressed on the luminal membrane of the activated endothelium facilitates the recruitment, adhesion and transmigration of leucocytes (e.g. neutrophils) into inflamed tissues, via its enzyme activity, which includes the local production of the redox signalling oxidant, hydrogen peroxide (H₂O₂). Recruited neutrophils release MPO in response to inflammatory stimuli, which upon activation by its co-substrate H₂O₂ (potentially derived from VAP-1 activity) catalyses a variety of oxidative reactions that result in the oxidation, chlorination and nitration of an array of biomolecules within the inflamed tissue (e.g. atherosclerotic lesion), leading to an inflammatory feedback loop, whereby additional leucocytes are recruited from the circulation. The inhibition of VAP-1 reduces leucocyte influx and the local generation of H₂O₂, leading to the reduced release of MPO. Inhibition of MPO reduces local oxidative tissue damage and inhibits the inflammatory feedback loop.

Fig. 2. Building a hybrid VAP-1/MPO dual inhibitor from known structures for each target.

Representative examples of clinically investigated VAP-1 and MPO inhibitors showing the starting templates for drug-drug conjugate design. Selected examples of explored hybrid molecules (Compounds A–E), including the structure of SNT-8370 (Compound E). Colour-coding showing the active warheads for VAP-1 (Blue) and MPO (Green, Pink).

Fig. 3. Impact of inhibitors on MPO-catalysed oxidative reactions.

A–D Inhibitors were added at the indicated concentrations to PBS (pH-7.4) supplemented with 5 mM taurine, followed by incubation at room temperature with mixing. H₂O₂ (50 μM) was added and the change in H₂O₂ concentration was continuously monitored using an H₂O₂-specific electrode. After a maximal steady-state H₂O₂ concentration was achieved, MPO (10 nM) was added and the rate of MPO-catalysed H₂O₂ consumption was measured over the ensuing 200–800 s. Representative H₂O₂ consumption curves for A SNT-8370, B verdiperstat and C PF06282999. D Dose-dependent inhibition curves of MPO-catalysed H₂O₂ consumption by SNT-8370, verdiperstat and PF06282999. Data is expressed as a % of the rate of MPO-catalysed H₂O₂ consumption in the absence of inhibitors (control) and represents the mean ± SEM of n = 3 (SNT-8370, verdiperstat) or n = 4 (PF06282999) independent experiments. E MPO (10 nM) was incubated in the absence or presence of the indicated concentrations of inhibitors in PBS (pH-7.4) supplemented with 5 mM taurine. MPO was activated by H₂O₂ (50 μM) addition, the reaction terminated after 6 min and the level of MPO-catalysed HOCl production assessed by the iodide-catalysed TMB assay. Results show the dose-dependent inhibition curves of MPO chlorination activity. Data is expressed as a % of MPO-catalysed HOCl production in the absence of inhibitors (control) and represents the mean ± SEM of n = 3 (PF06282999, verdiperstat) or n = 7 (SNT-8370) independent experiments. F LDL (0.2 mg/mL) was incubated with 30 nM MPO, 500 μg/mL glucose, 100 ng/mL glucose oxidase and 0.5 mM NaNO₂ in the absence (control) or presence of the indicated concentrations of SNT-8370 (n = 4) or verdiperstat (n = 4). Reactions were incubated at 37 °C for 2 h and LDL cholesteryl ester hydro(per)oxides (CE-O(O)H) levels measured. Data is expressed as a % of MPO-dependent CE-O(O)H formation in the absence of inhibitors (control) and represents the mean ± SEM of n = 4 (SNT-8370, verdiperstat) independent experiments. Data was analysed as area under the curve (AUC) by (D, E) one-way ANOVA (Tukey’s post hoc) or (F) Student’s t-test (two-tailed). The p values shown are versus the corresponding SNT-8370 concentration curve.

Fig. 4. Molecular modelling of the interaction of SNT-8370 within the MPO active site.

In silico modelling of SNT-8370 (yellow) bound to the heme group (purple) in the active site of activated MPO, showing (A) the active site pocket with SNT-8370 orientated in two lipophilic clefts (green, white and yellow colouring), B the molecular interactions as calculated by the Molecular Operating Environment (MOE) platform, showing the interaction between the allylamine portion of the molecule and the Glu116 residue at the entrance to the active site, and C a 3-D representation of this interaction in PYMOL, where a distance of 1.71 Å was measured between the SNT-8370 protonated amine and the negatively charged Glu116 residue acid group (shown in green stick form).

Fig. 5. SNT-8370 is an efficient mechanism-based irreversible dual-inhibitor of VAP-1 and MPO.

A–C For partition ratio, MPO (60 nM) was incubated with 2 μM H₂O₂ in the presence of DMSO (vehicle control) or relevant inhibitor (0.13–5 μM). After 15 min, the mixture was diluted 300-fold into assay buffer containing 2 μM H₂O₂ and 30 μM Amplex Red, and MPO activity immediately measured as the rate of H₂O₂ consumption. Data is the mean ± SEM of n = 3 independent experiments and represents % control MPO activity graphed as a function of [inhibitor]/[MPO]. Partition ratios of A SNT-8370 (n = 3), B verdiperstat (n = 3) and C PF06282999 (n = 3) were calculated as the X-axis-intercept of the line of best fit (green). D MPO (500 pM) was incubated with H₂O₂ (100 μM) in the presence of varying concentrations of BMS-250 and MPO activity measured as the rate of H₂O₂ consumption by the Amplex Red assay. Data is the mean value of n = 2 independent experiments. E For MPO jump dilution experiments, MPO (30 nM) was incubated with H₂O₂ (2 μM) in the absence (DMSO) and presence of SNT-8370 or BMS-250 at 300-fold their recorded IC₅₀ to achieve maximal enzyme inhibition. The mixture was diluted 300-fold and the recovery of MPO activity measured upon enzyme reactivation. Data is expressed as % control activity and represents the mean ± SEM of n = 3 independent experiments. F VAP-1 (0.25 μg/mL) was incubated in the presence of varying concentrations of ‘des-fluoro’ SNT-8370 and VAP-1 activity measured as the rate of H₂O₂ generation by the Amplex Red assay. Data is the mean value of n = 2 independent experiments. G For VAP-1 Jump dilution experiment, VAP-1 (700 nM) was incubated in the absence (DMSO) or presence of SNT-8370 or ‘des-fluoro’ SNT-8370 at 30-fold their recorded IC₅₀ to achieve maximal enzyme inhibition. The mixture was diluted 50-fold, and the recovery of VAP-1 activity measured upon enzyme reactivation. Data is expressed as % control and represents the mean ± SEM of n = 3 independent experiments. Data was analysed by one-way ANOVA (Tukey’s post hoc).

Fig. 6. Measurement of pharmacodynamics of MPO inhibition by inhibitors in a mouse peritonitis model.

A Peritoneal inflammation and leucocyte recruitment was induced in male C57Bl/6J mice by i.p. injection of thioglycolate broth for 20 h. Control mice (Con) received i.p. injection of PBS (n = 7). After 20 h, thioglycolate broth-treated mice received SNT-8370 by oral gavage at 30 (n = 4) or 60 mg/kg (n = 6), verdiperstat at 30 (n = 3) or 60 mg/kg (n = 6), or vehicle control (n = 7). One hour later, mice were administered zymosan A i.p. to promote leucocyte activation and MPO release. After 4 h, the inflammatory exudates were collected via peritoneal lavage and analysed for MPO activity and protein levels. B Effect of oral drug treatment on peritoneal MPO activity in thioglycolate broth/zymosan A-treated mice with peritonitis. Results are expressed as a % of MPO activity apparent in thioglycolate broth/zymosan A, vehicle-treated mice with peritonitis and represent the mean ± SEM for non-treated, control (Con) mice (n = 7) or peritonitis mice treated with vehicle control (n = 7), SNT-8370 at 30 mg/kg (n = 4) or 60 mg/kg (n = 6), and verdiperstat at 30 mg/kg (n = 3) or 60 mg/kg (n = 6). Data was analysed by one-way ANOVA (Dunnett’s post hoc). p values shown are versus vehicle control mice with peritonitis. C MPO protein levels in the peritoneum were determined by ELISA. Data represents the mean ± SEM (n = 3 for all treatments). The concentrations of D SNT-8370 and E verdiperstat present in the peritoneal lavage fluid at 5 h after oral gavage were measured by mass spectrometry. Data represent the mean ± SEM for D SNT-8370 at 10 (n = 3), 30 (n = 3), or 60 (n = 3) mg/kg and E verdiperstat at 10 (n = 3), 30 (n = 3) or 60 (n = 6) mg/kg.

Fig. 7. SNT-8370 attenuates leucocyte infiltration and oedema in a murine air pouch model of acute inflammation.

A Male BALB/c mice were injected with a sterile air pouch on the nape of their necks at day 0 and 3. On day 6, at 30 min prior to administration of the λ-carrageenan solution into the sterile air pouch, the animals were given a single oral dose of either control vehicle (n = 8), SNT-8370 (3 [n = 8], 10 [n = 8] or 30 [n = 8] mg/kg), verdiperstat (15 mg/kg [n = 8]) or PXS-4707 (6 mg/kg [n = 8]). Dexamethasone (10 mg/kg [n = 8]) was used as a positive anti-inflammatory control. Mice were re-dosed with vehicle or test drugs at 8 h post-λ-carrageenan challenge. Untreated control (Con) mice received de-ionised water i.p (instead of λ-carrageenan). The impact of the test drugs on λ-carrageenan-induced leucocyte infiltration into the inflamed sterile air pouch was assessed at (B) 4 h or (C) 12 h post-λ-carrageenan challenge and (D) sterile air pouch exudate volume at 12 h post-λ-carrageenan challenge. B, C Data is expressed as mean ± SEM of the total number of white blood cells (WBC) in the sterile air pouch at (B) 4 h for Con (n = 8), λ-carrageenan alone (n = 8), dexamethasone (n = 6), PXS-4707 (n = 8), verdiperstat (n = 8), SNT-8370 at 30 mg/kg (n = 7) and (C) 12 h for Con (n = 7), λ-carrageenan alone (n = 8), dexamethasone (n = 8), PXS-4707 (n = 7), verdiperstat (n = 8) and SNT-8370 at 3 (n = 7), 10 (n = 8), or 30 (n = 7) mg/kg. D Data is expressed as mean ± SEM of sterile air pouch exudate volume at 12 h post-λ-carrageenan challenge (n = 8 mice for all treatments). Data was analysed by a one-way ANOVA (Dunnett’s post hoc).

Fig. 8. SNT-8370 protects in preclinical models of acute lung injury and myocardial infarction.

A–C Acute Lung Injury. A Female BALB/cJUnib mice were non-treated (control, con) or treated with vehicle, SNT-8370 (30 mg/kg), verdiperstat (30 mg/kg) or PXS-4707 (10 mg/kg) 1 h prior to intranasal administration of LPS. Mice were then euthanised at 6 h after LPS instillation and the impact of the drugs on lung infiltration (BALF) of (B) total leucocytes (con n = 6, vehicle n = 5, SNT-8370 n = 6, verdiperstat n = 6, PXS-4707 n = 6) and (C) neutrophils (con n = 6, vehicle n = 5, SNT-8370 n = 5, verdiperstat n = 6, PXS-4707 n = 6) measured. Data is expressed as the number of cells/mL of BALF and represents the mean ± SEM. Data was analysed by one-way ANOVA (Tukey’s post hoc). p values shown are versus LPS + vehicle group. D–I Myocardial Infarction. D Male Sprague Dawley rats were administered control vehicle or SNT-8370 (60 mg/kg) by oral gavage just prior to LAD occlusion or sham surgery. The LAD occlusion was released after 30 min and rats were treated twice daily with vehicle or SNT-8370 (60 mg/kg) by oral gavage for a further 28 days before E ejection fraction and F fractional shortening were measured via cardiac ultrasound. E, F Data represents the mean ± SEM (Sham n = 5, LAD/control vehicle n = 6, LAD/SNT-8370 n = 5). Data was analysed by one-way ANOVA (Dunnett’s post-hoc). Cardiac fibrosis was measured in left ventricle tissue sections by G picrosirius red (LAD/control vehicle n = 7, LAD/SNT-8370 n = 6), H Masson’s trichrome (LAD/control vehicle n = 8, LAD/SNT-8370 n = 5) and I periostin (LAD/control vehicle n = 5, LAD/SNT-8370 n = 5) staining. Data is expressed as a % of positive staining per left ventricle tissue area and represents the mean ± SEM. Data was analysed by Student’s t test (two-tailed). p values shown are versus the LAD/control group.