Myeloperoxidase Inhibitor Research Articles

Environment-sensitive turn-on fluorescent probe enables live cell imaging of myeloperoxidase activity during NETosis.

Myeloperoxidase (MPO) plays an important role in the immune response of human neutrophils and has been implicated in autoimmune conditions, cardiovascular disorders, and neurodegeneration. Current methods to detect MPO activity rely on the detection of HOCl using activatable probes or require challenging experimental procedures. Therefore, these tools provide limited information about the dynamics and localization of MPO in complex molecular processes such as NETosis in real time. In this study, we report a ''turn-on" activity-based probe that fluoresces exclusively upon binding to MPO, exhibits minimal background fluorescence in buffered aqueous media, and is blocked by MPO inhibitors. Our probe facilitates real-time imaging of direct MPO activity in human neutrophils and HL-60-derived granulocytes during NETosis under wash-free conditions. Furthermore, it allows for the discrimination between different triggers of NETosis in human neutrophils. These findings hold promise for advancing our understanding of the role of MPO in immune responses and inflammatory conditions.

Targeting Myeloperoxidase to Reduce Neuroinflammation in X-Linked Dystonia Parkinsonism.

Although the genetic locus of X-linked dystonia parkinsonism (XDP), a neurodegenerative disease endemic in the Philippines, is well-characterized, the exact mechanisms leading to neuronal loss are not yet fully understood. Recently, we demonstrated an increase in myeloperoxidase (MPO) levels in XDP postmortem prefrontal cortex (PFC), suggesting a role for inflammation in XDP pathogenesis. Therefore, we hypothesized that inhibiting MPO could provide a therapeutic strategy for XDP. MPO activity was measured by using an MPO-activatable fluorescent agent (MAFA) in human postmortem PFC. Reactive oxygen species (ROS) and MPO activity were measured in XDP-derived fibroblasts and SH-SY5Y cells following MPO inhibition. MPO activity was significantly increased in XDP PFC. Additionally, treatment of cell lines with postmortem XDP PFC resulted in a significant increase in ROS levels. To determine whether increases in MPO activity caused increases in ROS, MPO content was immunodepleted from XDP PFC, which resulted in a significant decrease in ROS in SH-SY5Y cells. Consistently, the treatment with verdiperstat, a potent and selective MPO inhibitor, significantly decreased ROS in both XDP-derived fibroblasts and XDP PFC-treated SH-SY5Y cells. Collectively, our results suggest that MPO inhibition mitigates oxidative stress and may provide a novel therapeutic strategy for XDP treatment.

MPO interacts with hRSV particles, contributing to the virucidal effects of NETs against clinical and laboratory hRSV isolates

Human Respiratory syncytial virus (hRSV) mainly affects immunosuppressed patients requiring hospitalization. No specific treatment is financially accessible, and available vaccines do not cover all risk groups. During hRSV infection, there is a robust neutrophilic influx into the airways. hRSV-activated neutrophils release substantial neutrophil extracellular traps (NETs) in lung tissue, comprising DNA, histones, cytosolic, and granular proteins. NETs form mucus buildup in the lungs, compromising respiratory capacity and neutralizing viral particles. Understanding responsible NETs molecules requires improvement. We evaluated NETs interacting with hRSV particles and their contribution to anti-hRSV NET effects. Immunoblotting, immunoprecipitation, and peptide sequencing assays confirmed hRSV binding to a 50–75 kDa NET protein, Myeloperoxidase (MPO). MPO, a microbicide enzyme in NETs, interacts with hRSV, likely at F0 protein (site IV) on the viral surface. Additionally, MPO (32 μM) and NETs (0.4 μg/mL) reduced in vitro replication of clinical (hRSV A and B) and laboratory (Long) hRSV isolates by approximately 30 %, reversible by selective MPO inhibitor (PF-06281355; 48 μM). Thus, MPO contributes to virucidal NET effects on diverse hRSV strains, enhancing comprehension of NETs' role in infection and aiding treatment strategies for respiratory diseases.

Protective effect of (E)-(2,4-dihydroxy)-α-aminocinnamic acid, a hydroxy cinnamic acid derivative, in an ulcerative colitis model induced by TNBS.

Ulcerative colitis (UC) is a multifactorial disease that causes long-lasting inflammation and ulcers in the digestive tract. UC is the most common form of inflammatory bowel disease (IBD). The current treatment for mild-to-moderate UC involves the use of 5-aminosalicylates (5-ASA), but much of this compound is unabsorbed and metabolized by N-acetylation. Several efforts have since been made to evaluate new molecules from synthetic or natural sources. Recently, it was reported that (E)-(5-chloro-2-hydroxy)-α-aminocinnamic acid (2c) and (E)-(2,4-dihydroxy)-α-aminocinnamic acid (2f) are as good or better myeloperoxidase (MPO) inhibitors and antioxidants than 5-ASA. Then, the present study aimed to evaluate the protective effects of 2c and 2f on a rat model of UC induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS). The results showed that TNBS caused the induction of colonic ulcers, as well as a significant increase in MPO activity and malondialdehyde (MDA) and a decrease in glutathione (GSH) content. The administration of 2f, 2c and 5-ASA, decreased the ulcers presence, inhibited MPO peroxidation activity and MPO presence (as determined by immunofluorescence), and increased GSH and reduced MDA content. However, 2f was better than 2c and 5-ASA, then, the principal mechanism by which 2f presented a protective effect in a UC model induced by TNBS in rats is by inhibiting MPO activity and due to its antioxidant activity.

Endoplasmic Reticulum Stress in Bronchopulmonary Dysplasia: Contributor or Consequence?

Bronchopulmonary dysplasia (BPD) is the most common complication of prematurity. Oxidative stress (OS) and inflammation are the major contributors to BPD. Despite aggressive treatments, BPD prevalence remains unchanged, which underscores the urgent need to explore more potential therapies. The endoplasmic reticulum (ER) plays crucial roles in surfactant and protein synthesis, assisting mitochondrial function, and maintaining metabolic homeostasis. Under OS, disturbed metabolism and protein folding transform the ER structure to refold proteins and help degrade non-essential proteins to resume cell homeostasis. When OS becomes excessive, the endogenous chaperone will leave the three ER stress sensors to allow subsequent changes, including cell death and senescence, impairing the growth potential of organs. The contributing role of ER stress in BPD is confirmed by reproducing the BPD phenotype in rat pups by ER stress inducers. Although chemical chaperones attenuate BPD, ER stress is still associated with cellular senescence. N-acetyl-lysyltyrosylcysteine amide (KYC) is a myeloperoxidase inhibitor that attenuates ER stress and senescence as a systems pharmacology agent. In this review, we describe the role of ER stress in BPD and discuss the therapeutic potentials of chemical chaperones and KYC, highlighting their promising role in future therapeutic interventions.

The Interaction of Myeloperoxidase with the Industrial Contaminant 6-PPD: A Potential Pathway for Reactive Metabolites.

6-PPD (N-[1,3-dimethylbutyl]-N'-phenyl-p-phenylenediamine) is an industrial antioxidant reported to be an environmental contaminant. It was found to be highly toxic to coho salmon and potentially other aquatic organisms. The toxicity of 6-PPD in humans, however, remains unknown. The neutrophil enzyme myeloperoxidase (MPO) is known to catalyze xenobiotic metabolism; therefore, its role in 6-PPD cytotoxicity was investigated using the MPO-rich HL-60 cell line. UV-visible spectroscopy and liquid chromatography-mass spectrometry (LC/MS) were performed to investigate the MPO-mediated oxidation of 6-PPD and identify possible metabolites in the absence and presence of glutathione (GSH). 6-PPD's cytotoxicity, effect on mitochondrial membrane potential (MMP), and GSH-depleting ability in HL-60 cells were assessed. Electron paramagnetic resonance (EPR) was used to determine GSH radical formation using DMPO, and mitochondrial-derived superoxide was assessed with the mito-TEMPO-H probe. Evaluation of the 6-PPD-induced cellular injury pathways was performed by preincubating an antioxidant and an MPO inhibitor with HL-60 cells. UV-vis analysis of MPO-catalyzed oxidation of 6-PPD demonstrated changes in the 6-PPD spectrum, whereas the addition of GSH altered the spectrum, indicating possible GSH conjugate formation. LC/MS showed the formation of multiple products, including GSH-6-PPD conjugates and a GSH conjugate to a 4-hydroxydiphenylamine (a known 6-PPD degradant), which could potentially induce cytotoxicity. 6-PPD demonstrated concentration-dependent cytotoxicity, and cellular GSH levels were decreased by 6-PPD. Similarly, the level of MMP decreased, suggesting mitochondrial depolarization. Furthermore, the EPR spin probe for mitochondrial superoxide showed a positive relationship with 6-PPD concentration, and EPR spin-trapping demonstrated 6-PPD concentration-dependent GSH radical signal intensity using MPO/H2O2. The GSH precursor, NAC, demonstrated partial cytoprotection against 6-PPD; however, the MPO inhibitor PF-1355 surprisingly showed no significant cytoprotective effect. Our results suggest that MPO could be a potential catalyst for 6-PPD toxicity in humans. However, MPO inhibition did not significantly affect cellular viability, suggesting an MPO-independent toxicity pathway. These findings warrant a deeper investigation to determine 6-PPD mammalian toxicity pathways.

An integrated approach combining experimental, informatics and energetic methods for solid form derisking of PF-06282999

The landscapes of observed and predicted three-dimensional crystal packing arrangements of small-molecule drug candidates can be complex. The possible appearance of a more thermodynamically stable solid form during drug development has led to the digital workflow of informatics-based risk assessments, named a Solid Form Health Check. Herein, we describe the use of a combined approach consisting of experiments, informatics together with energetic calculations in analysis of four competing polymorphs of PF-06282999, a myeloperoxidase (MPO) inhibitor with conformational flexibility and multiple plausible hydrogen bond networks. This combined approach offered a comprehensive understanding of the solid form structure, properties, and performance, ensuring robust solid form derisking and selection.

Temporal Dynamics of Oxidative Stress and Inflammation in Bronchopulmonary Dysplasia.

Bronchopulmonary dysplasia (BPD) is the most common lung complication of prematurity. Despite extensive research, our understanding of its pathophysiology remains limited, as reflected by the stable prevalence of BPD. Prematurity is the primary risk factor for BPD, with oxidative stress (OS) and inflammation playing significant roles and being closely linked to premature birth. Understanding the interplay and temporal relationship between OS and inflammation is crucial for developing new treatments for BPD. Animal studies suggest that OS and inflammation can exacerbate each other. Clinical trials focusing solely on antioxidants or anti-inflammatory therapies have been unsuccessful. In contrast, vitamin A and caffeine, with antioxidant and anti-inflammatory properties, have shown some efficacy, reducing BPD by about 10%. However, more than one-third of very preterm infants still suffer from BPD. New therapeutic agents are needed. A novel tripeptide, N-acetyl-lysyltyrosylcysteine amide (KYC), is a reversible myeloperoxidase inhibitor and a systems pharmacology agent. It reduces BPD severity by inhibiting MPO, enhancing antioxidative proteins, and alleviating endoplasmic reticulum stress and cellular senescence in a hyperoxia rat model. KYC represents a promising new approach to BPD treatment.

Inhibition of myeloperoxidase to treat left ventricular dysfunction in non-ischaemic cardiomyopathy.

Non-ischaemic cardiomyopathy (NICMP), an incurable disease terminating in systolic heart failure (heart failure with reduced ejection fraction [HFrEF]), causes immune activation, however anti-inflammatory treatment strategies so far have failed to alter the course of this disease. Myeloperoxidase (MPO), the principal enzyme in neutrophils, has cytotoxic, pro-fibrotic and nitric oxide oxidizing effects. Whether MPO inhibition ameliorates the phenotype in NICMP remains elusive. Prognostic information from MPO was derived from proteomic data of a large human cardiovascular health cohort (n = 3289). In a murine model of NICMP, we studied the mechanisms of MPO in this disease. In a case series, the MPO inhibitor was also evaluated in NICMP patients. Individuals with increased MPO revealed higher long-term mortality and worsening of heart failure, with impaired prognosis when MPO increased during follow-up. MPO infusion attenuated left ventricular ejection fraction (LVEF) in mice with NICMP, whereas genetic ablation or inhibition of MPO decreased systemic vascular resistance (SVR, 9.4 ± 0.7 mmHg*min/ml in NICMP vs. 6.7 ± 0.8 mmHg*min/ml in NICMP/Mpo-/-mice, n = 8, p = 0.006, data expressed as mean ± standard error of the mean) and improved left ventricular function (LVEF 30.3 ± 2.2% in NICMP vs. 40.7 ± 1.1% in NICMP/Mpo-/- mice, n = 16, p < 0.0001). Four patients diagnosed with NICMP and treated with an MPO inhibitor over 12 weeks showed increase in LVEF, decline in natriuretic peptides and improved 6-min walking distance. MPO inhibitor-related changes in the proteome of NICMP patients predicted reduced mortality when related to the changes in the proteome of the above referenced cardiovascular health cohort. Myeloperoxidase predicts long-term outcome in HFrEF and its inhibition elicits systemic anti-inflammatory and vasodilating effects which translate into improved left ventricular function. MPO inhibition deserves further evaluation as a novel, complementary treatment strategy for HFrEF.

Aging Intensifies Myeloperoxidase Activity after Ischemic Stroke.

Abnormally elevated oxidative stress underlies many diseases and contributes to aging. The myeloid enzyme myeloperoxidase (MPO) generates oxidative stress and contributes to damage after stroke. How aging changes MPO in stroke has not been studied. We aimed to determine the effects aging has on MPO and how these changes contribute to age-related differences in outcomes after ischemic stroke. To investigate tissue MPO activity we developed MPO Activatable Fluorescent Agent (MAFA). We found that aged mice exhibited worse neurological outcomes and higher mortality within the first few days after stroke. Accordingly, neuronal loss was higher in aged mice on day 3. MAFA imaging revealed that aged brains have markedly higher MPO activity that increased after stroke on day 3 compared to young adult brains. Correspondingly, we found more Iba1+ cells in aged brains compared to young adult brains before and after stroke. Interestingly, we found decreased percentage of MPO+ cells and lower MPO protein levels in aged on day 3, suggesting that most Iba1+ cells in aged mice have degranulated and secreted MPO in response to stroke. By day 10 MPO activity and Iba1+ cells decreased in both age groups, although MPO activity remained higher in aged mice. MPO inhibition in aged mice decreased MAFA signal and Iba1+ cells and improved neurobehavioral outcomes to near young adult stroke mice levels and improved mortality rate. While aging is an unmodifiable risk, by uncovering the connection between aging and MPO-related changes after stroke, new therapies can be developed to mitigate these adverse changes brought upon by aging.

Polymorphisms in myeloperoxidase and tissue inhibitor of metalloproteinase-1 genes and their association with preeclampsia in the Chinese Han population

Hypertensive disorders of pregnancy (HDP) are multifaceted syndromes unique to pregnancy, characterized by increased blood pressure, edema, and proteinuria. Patients with HDP exhibit signs of endothelial dysfunction, possibly linked to increased myeloperoxidase (MPO) level and aberrant oxidative stress. Additionally, altered level of tissue inhibitor of metalloproteinase-1 (TIMP1) protein is associated with placental ischemia, hypoxia, and maternal vascular endothelial damage. Preeclampsia (PE) represents a critical stage of HDP that poses severe threats to maternal and fetal safety. This study aimed to determine the relationship between MPO and TIMP1 polymorphisms and the risk of PE in the Chinese Han population. Single nucleotide polymorphisms (SNPs), including MPO rs7208693, MPO rs2243828, and TIMP1 rs6609533, were genotyped in 170 patients with PE and 303 control participants. No significant association was observed between MPO polymorphisms (rs7208693 and rs2243828) and the risk of PE, whereas significant association between the TIMP1 rs6609533 A > G SNP and PE susceptibility was found. Specifically, individuals with the GG or AG genotypes had elevated risk of PE compared to those harboring the AA genotype. Furthermore, in the PE group, patients carrying the G allele were more likely to experience fetal growth restriction (FGR). In the non-PE group, the association between the G allele and the risk of FGR was not evident. In conclusion, the TIMP1 rs6609533 G allele in Chinese Han women was identified as a risk factor for PE. Our results indicated that the TIMP1 rs6609533 SNP can serve as a biomarker for the clinical diagnosis and treatment of PE.

Elucidation of γ-glutamyl-β-cyanoalanylglycine biosynthesis in mammalian cells by LC-QTOF-MS.

γ-Glutamyl-β-cyanoalanylglycine (gEcnAG) is a glutathione analog in which the cysteine moiety in glutathione is replaced with β-cyanoalanine, a known plant cyanide metabolite. Previously, gEcnAG was detected in the liver of rats and chicks exposed to β-cyanoalanine. We reported the detection of gEcnAG in naïve mammalian cells using liquid chromatography coupled with tandem quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). LC-QTOF-MS analysis enabled high-resolution confirmation (exact mass determination and MS/MS fragmentation) of the gEcnAG structure. The detection of gEcnAG in rat pheochromocytoma (PC12) cells that were not exposed to β-cyanoalanine suggests its endogenous production. Furthermore, the inhibition of myeloperoxidase, an enzyme potentially required for endogenous cyanide generation, decreased gEcnAG levels in PC12 cells. This supports the notion that PC12 cells intrinsically produce cyanide, unlike HepG2 cells, which exhibited lower intracellular gEcnAG levels. Notably, β-cyanoalanine was undetectable in PC12 cells. Moreover, depleting glutathione with buthionine sulfoximine reduced intracellular gEcnAG levels, whereas supplementation with glutathione reduced ethyl ester increased them. These observations suggest that endogenous gEcnAG may be generated from glutathione, potentially through its reaction with endogenous cyanide. Our findings implicate gEcnAG as a possible metabolite of endogenous cyanide.

The effect of severe renal impairment on the pharmacokinetics, safety and tolerability of mitiperstat.

Mitiperstat is a novel, highly potent myeloperoxidase inhibitor being evaluated in patients with cardio-metabolic disease (phase 2). These patients often have impaired renal function, which may affect mitiperstat pharmacokinetics. This study assessed mitiperstat pharmacokinetics, safety and tolerability in participants with severe renal impairment and normal renal function, to inform inclusion of participants with estimated glomerular filtration rate (eGFR) < 30 mL/min/1.73 m2 in phase 3. Participants with severe renal impairment (eGFR ≥15 and <30 mL/min/1.73 m2) who were not on dialysis (n = 10) and group-matched controls (eGFR ≥90 mL/min/1.73 m2; n = 10) received a single mitiperstat 2.5mg oral tablet. Blood samples were collected at intervals for 2weeks and urine samples for 24 h post-dose. Total apparent mitiperstat clearance was 10.83 L/h in the severe renal impairment cohort and 25.62 L/h in the control cohort. The area under the plasma concentration-time curve was 2.37-fold higher (90% confidence interval [CI]: 1.79, 3.12) in the severe renal impairment cohort than in the control cohort, with longer elimination half-life and similar maximum concentration. Non-renal clearance was similar between the cohorts. Mitiperstat apparent clearance was approximately twofold lower in individuals with severe renal impairment than in those with normal renal function. Lower clearance was driven by reduced renal clearance; non-renal clearance was similar. Mitiperstat was generally well tolerated by participants with severe renal impairment and normal renal function. These findings, together with efficacy and safety/tolerability data from phase 2b, will guide the dosing regimen for phase 3.

Mixed culture fermentation using Lactobacillus delbrueckii and novel Lactiplantibacillus plantarum strains isolated from traditional chhurpi from Sikkim Himalayan region for potential probiotic and improved functional properties

The aim of the study was to isolate potential probiotic lactic acid bacteria (LAB) strains from the traditional fermented milk products, Chhurpi of the Sikkim Himalayan region and to prepare curd using mixed culture fermentation. A preliminary probiotic assessment of 110 LAB isolates including antimicrobial activity, bile and acid tolerance, surface hydrophobicity, auto-aggregation and co-aggregation, and adhesion to Caco-2 cells, was performed. Lactiplantibacillus plantarum strains N4, CHDE2, CHDE4, and SS22 demonstrated better viability in bile (0.3%) and acid (pH 2.5), adherence with hydrophobicity (13.93–92.85%), auto-aggregation (72.44–89.74%), adhesion to Caco-2 cells (45–73.61%) and antimicrobial activity against food borne pathogens. These strains were used for mixed culture fermentation with a previously screened curd forming strain Lactobacillus delbrueckii WS4. Curd produced using mixed culture fermentation of Lb. delbrueckii WS4 and the selected Lp. plantarum strains had a relatively higher acid production (pH 3.97–4.01), protein hydrolysis, and antioxidant activity. Further, mixed culture fermentation also resulted in the increase of HOCl scavenging activity (12.22–32.96%), myeloperoxidase (MPO) inhibition activity (22.38–42.94%) and angiotensin converting enzyme (ACE) inhibition (15.61–33.03%) as compared to that of WS4 fermentation (P < 0.05). This is the first report on identification of potential probiotic strains from the traditional fermented milk products of Sikkim and application in mixed culture fermentation with enhanced multifunctional potential.

The role of NETosis in heart failure.

The hallmark of heart failure (HF) is structural myocardial remodeling including cardiomyocyte hypertrophy, fibrosis, cardiomyocyte cell death, and a low-grade aseptic inflammation. The initiation and maintenance of persistent chronic low-grade inflammation in HF are not fully understood. Oxidative stress-mediated neutrophil extracellular traps (NETs) are the main immune defense mechanism against external bacterial infections. Furthermore, NETs play important roles in noninfectious diseases. In the settings of myocardial infarction, myocarditis, or cardiomyopathies, neutrophils infiltrate the cardiac tissue and undergo NETosis that further aggravate the inflammation. A number of stimuli may cause NETosis that is a form of programmed cell death of neutrophils that is different from apoptosis of these cells. Whether NETosis is directly involved in the pathogenesis and development of HF is still unclear. In this review, we analyzed the mechanisms and markers of NETosis, especially placing the accent on the activation of the neutrophil-specific myeloperoxidase (MPO), elastase (NE), and peptidylarginine deiminase 4 (PAD4). These conclusions are supported by the recent genetic and pharmacological studies which demonstrated that MPO, NE, and PAD4 inhibitors are effective at least in the settings of post-myocardial infarction adverse remodeling, cardiac valve diseases, cardiomyopathies, and decompensated left ventricular hypertrophy whose deterioration can lead to HF. This is essential for understanding NETosis as a contributor to pathophysiology of HF and developments of new therapies of HF.

Role of Myeloperoxidase, Oxidative Stress, and Inflammation in Bronchopulmonary Dysplasia.

Bronchopulmonary dysplasia (BPD) is a lung complication of premature births. The leading causes of BPD are oxidative stress (OS) from oxygen treatment, infection or inflammation, and mechanical ventilation. OS activates alveolar myeloid cells with subsequent myeloperoxidase (MPO)-mediated OS. Premature human neonates lack sufficient antioxidative capacity and are susceptible to OS. Unopposed OS elicits inflammation, endoplasmic reticulum (ER) stress, and cellular senescence, culminating in a BPD phenotype. Poor nutrition, patent ductus arteriosus, and infection further aggravate OS. BPD survivors frequently suffer from reactive airway disease, neurodevelopmental deficits, and inadequate exercise performance and are prone to developing early-onset chronic obstructive pulmonary disease. Rats and mice are commonly used to study BPD, as they are born at the saccular stage, comparable to human neonates at 22-36 weeks of gestation. The alveolar stage in rats and mice starts at the postnatal age of 5 days. Because of their well-established antioxidative capacities, a higher oxygen concentration (hyperoxia, HOX) is required to elicit OS lung damage in rats and mice. Neutrophil infiltration and ER stress occur shortly after HOX, while cellular senescence is seen later. Studies have shown that MPO plays a critical role in the process. A novel tripeptide, N-acetyl-lysyltyrosylcysteine amide (KYC), a reversible MPO inhibitor, attenuates BPD effectively. In contrast, the irreversible MPO inhibitor-AZD4831-failed to provide similar efficacy. Interestingly, KYC cannot offer its effectiveness without the existence of MPO. We review the mechanisms by which this anti-MPO agent attenuates BPD.

Targeting myeloperoxidase to reduce neuroinflammation in X-linked dystonia parkinsonism.

MPO activity is increased in XDP post-mortem prefrontal cortex.MPO activity is increased in cellular models of XDP.MPO increases reactive oxygen species (ROS) in vitro.Inhibiting MPO mitigates ROS in XDP.The MPO inhibitor, verdiperstat, dampens ROS suggesting a potential therapeutic strategy for XDP.

Novel Mouse Model of Myocardial Infarction, Plaque Rupture, and Stroke Shows Improved Survival With Myeloperoxidase Inhibition.

Thromboembolic events, including myocardial infarction (MI) or stroke, caused by the rupture or erosion of unstable atherosclerotic plaques are the leading cause of death worldwide. Although most mouse models of atherosclerosis develop lesions in the aorta and carotid arteries, they do not develop advanced coronary artery lesions. Moreover, they do not undergo spontaneous plaque rupture with MI and stroke or do so at such a low frequency that they are not viable experimental models to study late-stage thrombotic events or to identify novel therapeutic approaches for treating atherosclerotic disease. This has stymied the development of more effective therapeutic approaches for reducing these events beyond what has been achieved with aggressive lipid lowering. Here, we describe a diet-inducible mouse model that develops widespread advanced atherosclerosis in coronary, brachiocephalic, and carotid arteries with plaque rupture, MI, and stroke. We characterized a novel mouse model with a C-terminal mutation in the scavenger receptor class B, type 1 (SR-BI), combined with Ldlr knockout (designated SR-BI∆CT/∆CT/Ldlr-/-). Mice were fed Western diet (WD) for 26 weeks and analyzed for MI and stroke. Coronary, brachiocephalic, and carotid arteries were analyzed for atherosclerotic lesions and indices of plaque stability. To validate the utility of this model, SR-BI∆CT/∆CT/Ldlr-/- mice were treated with the drug candidate AZM198, which inhibits myeloperoxidase, an enzyme produced by activated neutrophils that predicts rupture of human atherosclerotic lesions. SR-BI∆CT/∆CT/Ldlr-/- mice show high (>80%) mortality rates after 26 weeks of WD feeding because of major adverse cardiovascular events, including spontaneous plaque rupture with MI and stroke. Moreover, WD-fed SR-BI∆CT/∆CT/Ldlr-/- mice displayed elevated circulating high-sensitivity cardiac troponin I and increased neutrophil extracellular trap formation within lesions compared with control mice. Treatment of WD-fed SR-BI∆CT/∆CT/Ldlr-/- mice with AZM198 showed remarkable benefits, including >90% improvement in survival and >60% decrease in the incidence of plaque rupture, MI, and stroke, in conjunction with decreased circulating high-sensitivity cardiac troponin I and reduced neutrophil extracellular trap formation within lesions. WD-fed SR-BI∆CT/∆CT/Ldlr-/- mice more closely replicate late-stage clinical events of advanced human atherosclerotic disease than previous models and can be used to identify and test potential new therapeutic agents to prevent major adverse cardiac events.

Prediction of the liver safety profile of a first-in-class myeloperoxidase inhibitor using quantitative systems toxicology modeling

The novel myeloperoxidase inhibitor verdiperstat was developed as a treatment for neuroinflammatory and neurodegenerative diseases. During development, a computational prediction of verdiperstat liver safety was performed using DILIsym v8A, a quantitative systems toxicology (QST) model of liver safety. A physiologically-based pharmacokinetic (PBPK) model of verdiperstat was constructed in GastroPlus 9.8, and outputs for liver and plasma time courses of verdiperstat were input into DILIsym. In vitro experiments measured the likelihood that verdiperstat would inhibit mitochondrial function, inhibit bile acid transporters, and generate reactive oxygen species (ROS); these results were used as inputs into DILIsym, with two alternate sets of parameters used in order to fully explore the sensitivity of model predictions. Verdiperstat dosing protocols up to 600 mg BID were simulated for up to 48 weeks using a simulated population (SimPops) in DILIsym. Verdiperstat was predicted to be safe, with only very rare, mild liver enzyme increases as a potential possibility in highly sensitive individuals. Subsequent Phase 3 clinical trials found that ALT elevations in the verdiperstat treatment group were generally similar to those in the placebo group. This validates the DILIsym simulation results and demonstrates the power of QST modelling to predict the liver safety profile of novel therapeutics.

Staphylococcal peroxidase inhibitor (SPIN): Investigation of the inhibitory N-terminal domain via a stabilizing disulfide insertion

Staphylococcus aureus secretes an array of small proteins that inhibit key enzyme-catalyzed reactions necessary for proper function of the human innate immune system. Among these, the Staphylococcal Peroxidase Inhibitor, SPIN, blocks the activity of myeloperoxidase (MPO) and thereby disrupts the HOCl-generating system of neutrophils. Previous studies on S. aureus SPIN have shown that it relies on a C-terminal α-helical bundle domain to mediate initial binding to MPO, but requires a disordered N-terminal region to fold into a β-hairpin conformation to inhibit MPO activity. To further investigate the structure/function relationship of SPIN, we introduced two cysteine residues into its N-terminal region to trap SPIN in its MPO-bound conformation and characterized the modified protein, which we refer to here as SPIN-CYS. Although control experiments confirmed the presence of the disulfide bond in SPIN-CYS, solution structure determination revealed that the N-terminal region of SPIN-CYS adopted a physically constrained series of lariat-like structures rather than a well-defined β-hairpin. Nevertheless, SPIN-CYS exhibited a gain in inhibitory potency against human MPO when compared to wild-type SPIN. This gain of function persisted even in the presence of deleterious mutations within the C-terminal α-helical bundle domain. Surface plasmon resonance studies showed that the gain in potency arose through an increase in apparent affinity of SPIN-CYS for MPO, which was driven primarily by an increased association rate with MPO when compared to wild-type SPIN. Together, this work provides new information on the coupled binding and folding events required to manifest biological activity of this unusual MPO inhibitor.