The lipoprotein-associated phospholipase A2 inhibitor Darapladib sensitises cancer cells to ferroptosis by remodelling lipid metabolism.

Arachidonic and adrenic acids in the membrane play key roles in ferroptosis. Here, we reveal that lipoprotein-associated phospholipase A2 (Lp-PLA2) controls intracellular phospholipid metabolism and contributes to ferroptosis resistance. A metabolic drug screen reveals that darapladib, an inhibitor of Lp-PLA2, synergistically induces ferroptosis in the presence of GPX4 inhibitors. We show that darapladib is able to enhance ferroptosis under lipoprotein-deficient or serum-free conditions. Furthermore, we find that Lp-PLA2 is located in the membrane and cytoplasm and suppresses ferroptosis, suggesting a critical role for intracellular Lp-PLA2. Lipidomic analyses show that darapladib treatment or deletion of PLA2G7, which encodes Lp-PLA2, generally enriches phosphatidylethanolamine species and reduces lysophosphatidylethanolamine species. Moreover, combination treatment of darapladib with the GPX4 inhibitor PACMA31 efficiently inhibits tumour growth in a xenograft model. Our study suggests that inhibition of Lp-PLA2 is a potential therapeutic strategy to enhance ferroptosis in cancer treatment.

Lp-PLA2 inhibition prevents Ang II-induced cardiac inflammation and fibrosis by blocking macrophage NLRP3 inflammasome activation.

Macrophage-mediated inflammation plays an important role in hypertensive cardiac remodeling, whereas effective pharmacological treatments targeting cardiac inflammation remain unclear. Lipoprotein-associated phospholipase A2 (Lp-PLA2) contributes to vascular inflammation-related diseases by mediating macrophage migration and activation. Darapladib, the most advanced Lp-PLA2 inhibitor, has been evaluated in phase III trials in atherosclerosis patients. However, the role of darapladib in inhibiting hypertensive cardiac fibrosis remains unknown. Using a murine angiotensin II (Ang II) infusion-induced hypertension model, we found that Pla2g7 (the gene of Lp-PLA2) was the only upregulated PLA2 gene detected in hypertensive cardiac tissue, and it was primarily localized in heart-infiltrating macrophages. As expected, darapladib significantly prevented Ang II-induced cardiac fibrosis, ventricular hypertrophy, and cardiac dysfunction, with potent abatement of macrophage infiltration and inflammatory response. RNA sequencing revealed that darapladib strongly downregulated the expression of genes and signaling pathways related to inflammation, extracellular matrix, and proliferation. Moreover, darapladib substantially reduced the Ang II infusion-induced expression of nucleotide-binding oligomerization domain-like receptor with pyrin domain 3 (NLRP3) and interleukin (IL)-1ß and markedly attenuated caspase-1 activation in cardiac tissues. Furthermore, darapladib ameliorated Ang II-stimulated macrophage migration and IL-1ß secretion in macrophages by blocking NLRP3 inflammasome activation. Darapladib also effectively blocked macrophage-mediated transformation of fibroblasts into myofibroblasts by inhibiting the activation of the NLRP3 inflammasome in macrophages. Overall, our study identifies a novel anti-inflammatory and anti-cardiac fibrosis role of darapladib in Lp-PLA2 inhibition, elucidating the protective effects of suppressing NLRP3 inflammasome activation. Lp-PLA2 inhibition by darapladib represents a novel therapeutic strategy for hypertensive cardiac damage treatment.

Consumption of Enriched Yogurt with PAF Inhibitors from Olive Pomace Affects the Major Enzymes of PAF Metabolism: A Randomized, Double Blind, Three Arm Trial.

Platelet-activating factor (PAF), a proinflammatory lipid mediator, plays a crucial role in the formation of the atherosclerotic plaque. Therefore, the inhibition of endothelium inflammation by nutraceuticals, such as PAF inhibitors, is a promising alternative for preventing cardiovascular diseases. The aim of the present study was to evaluate the impact of a new functional yogurt enriched with PAF inhibitors of natural origin from olive oil by-products on PAF metabolism. Ninety-two apparently healthy, but mainly overweight volunteers (35-65 years) were randomly allocated into three groups by block-randomization. The activities of PAF's biosynthetic and catabolic enzymes were measured, specifically two isoforms of acetyl-CoA:lyso-PAF acetyltransferase (LPCATs), cytidine 5'-diphospho-choline:1-alkyl-2-acetyl-sn-glycerol cholinephosphotransferase (PAF-CPT) and two isoforms of platelet activating factor acetylhydrolase in leucocytes (PAF-AH) and plasma (lipoprotein associated phospholipase-A2, LpPLA2). The intake of the enriched yogurt resulted in reduced PAF-CPT and LpPLA2 activities. No difference was observed in the activities of the two isoforms of lyso PAF-AT. In conclusion, intake of yogurt enriched in PAF inhibitors could favorably modulate PAF biosynthetic and catabolic pathways.

Platelet activating-factor acetylhydrolase II: A member of phospholipase A2 family that hydrolyzes oxidized phospholipids.

Intracellular platelet activating-factor acetylhydrolase type II (PAF-AH II) is a 40-kDa monomeric enzyme. It was originally identified as an enzyme that hydrolyzes the acetyl group of PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine). As a member of phospholipase A2 super family, PAF-AH II has broad substrate specificity. It can hydrolyze phospholipids with relatively short-length or oxidatively modified sn-2 chains which endows it with various functions such as protection against oxidative stress, transacetylase activity and producing lipid mediators. PAF-AH II has been proven to be involved in several diseases such as allergic diseases, oxidative stress-induced injury and ischemia injury, thus it has drawn more attention from researchers. In this paper, we outline an entire summary of PAF-AH II, including its structure, substrate specificity, activity assay, inhibitors and biological activities.

Lipid Receptor G2A-Mediated Signal Pathway Plays a Critical Role in Inflammatory Response by Promoting Classical Macrophage Activation.

Macrophage polarization is a dynamic and integral process in tissue inflammation and remodeling. In this study, we describe that lipoprotein-associated phospholipase A2 (Lp-PLA2) plays an important role in controlling inflammatory macrophage (M1) polarization in rodent experimental autoimmune encephalomyelitis (EAE) and in monocytes from multiple sclerosis (MS) patients. Specific inhibition of Lp-PLA2 led to an ameliorated EAE via markedly decreased inflammatory and demyelinating property of M1. The effects of Lp-PLA2 on M1 function were mediated by lysophosphatidylcholine, a bioactive product of oxidized lipids hydrolyzed by Lp-PLA2 through JAK2-independent activation of STAT5 and upregulation of IRF5. This process was directed by the G2A receptor, which was only found in differentiated M1 or monocytes from MS patients. M1 polarization could be inhibited by a G2A neutralizing Ab, which led to an inhibited disease in rat EAE. In addition, G2A-deficient rats showed an ameliorated EAE and an inhibited autoimmune response. This study has revealed a mechanism by which lipid metabolites control macrophage activation and function, modification of which could lead to a new therapeutic approach for MS and other inflammatory disorders.

MicroRNA-212-5p and its target PAFAH1B2 suppress vascular proliferation and contraction via the downregulation of RhoA.

Vascular remodeling and contraction contribute to the development of hypertension. We investigated the role of miR-212-5p and its downstream target in vascular smooth muscle cell (VSMC) proliferation, migration, and contraction. MicroRNA microarray and PCR analyses showed that miR-212-5p expression was increased with angiotensin II treatment in vivo and in vitro. Moreover, miR-212-5p mimic treatment attenuated and miR-212-5p inhibitor treatment increased VSMC proliferation and migration. Additionally, miR-212-5p mimic treatment suppressed VSMC contraction and related gene expression [Ras homolog gene family member A (RhoA) and Rho-associated protein kinase 2], while miR-212-5p inhibitor treatment exerted opposite effects. Bioinformatics analysis revealed that platelet-activating factor acetylhydrolase 1B2 (PAFAH1B2) is a target of miR-212-5p. miR-212-5p mimic treatment significantly reduced and miR-212-5p inhibitor treatment increased PAFAH1B2 expression. Furthermore, PAFAH1B2 expression was decreased in angiotensin II-treated aortic tissues and VSMCs. PAFAH1B2 was ubiquitously expressed in most adult rat tissues. In the vasculature, PAFAH1B2 was only distributed in the cytoplasm. PAFAH1B2 overexpression decreased A10 cell proliferation, while PAFAH1B2 knockdown increased A10 cell proliferation and cyclin D1 mRNA levels. PAFAH1B2 knockdown stimulated VSMC contraction and RhoA expression. These results suggest that miR-212-5p and PAFAH1B2 are novel negative regulators of VSMC proliferation, migration, and contraction in hypertension.

Identification of Highly Selective Lipoprotein-Associated Phospholipase A2 (Lp-PLA2) Inhibitors by a Covalent Fragment-Based Approach.

Covalent ligands are of great interest as therapeutic drugs or biochemical tools. Here, we reported the discovery of highly selective and irreversible inhibitors of lipoprotein-associated phospholipase A2 (Lp-PLA2) using a covalent fragment-based approach. The crystal structure of Lp-PLA2 in complex with a covalent fragment not only reveals the covalent reaction mechanism but also provides a good starting point to design compound 8, which has a more than 130,000-fold and 3900-fold increase in potency and selectivity, respectively, compared to those of the covalent fragment. Furthermore, fluorescent probes with high selectivity and sensitivity are developed to characterize Lp-PLA2 and its enzymatic activity in vitro or even in living cells in a way more convenient than immunoblotting tests or immunofluorescence imaging. Overall, we provide a paradigm for application of the covalent fragment-based strategy in covalent ligand discovery and the advantage of enol-cyclocarbamate as a new warhead in designing covalent inhibitors of serine hydrolases.

Sesame lignans reduce LDL oxidative susceptibility by downregulating the platelet-activating factor acetylhydrolase.

OBJECTIVE: Low-density lipoprotein (LDL) oxidative susceptibility is recognized as a risk factor for atherosclerosis. We previously reported that the ingestion of a supplement containing sesame lignans (sesamin/episesamin) for 4 weeks reduced LDL oxidative susceptibility in humans. MATERIALS AND METHODS: To elucidate the mechanisms underlying this observation, 12-week-old New Zealand White rabbits were fed a fat/cholesterol-enriched diet (100 g/day) for 6 weeks followed by oral administration of vehicle (control) or sesame lignans (50 mg/kg) for 4 weeks with the fat/cholesterol-enriched diet. RESULTS: The results showed that the ingestion of sesame lignans prolonged LDL oxidation lag time, regardless of the existence of the anti-oxidative catechol metabolite of sesamin/episesamin in LDL. Plasma platelet-activating factor acetylhydrolase (PAF-AH) activity was significantly reduced by sesame lignans. The prolongation of LDL oxidation lag time was abolished by the addition of a PAF-AH inhibitor. The expression level of pro-inflammatory cytokines and macrophage infiltration observed in the liver following the feeding of the fat/cholesterol-enriched diet were also significantly reduced by sesame lignans. CONCLUSIONS: These results indicate that sesame lignans reduce LDL oxidative susceptibility by downregulating plasma PAF-AH activity via the reduction of inflammation in the liver induced by fat/cholesterol-enriched diets.

Decreasing angiogenesis vasa vasorum through Lp-PLA2 and H2O2 inhibition by PSP from Ganoderma lucidum in atherosclerosis: in vivo diabetes mellitus type 2.

Background Type 2 diabetes mellitus (T2DM) is a major risk factor of atherosclerosis. Hyperglycemia in T2DM causes advanced formation of glycation end products (AGE) which leads to oxidative stress and chronic inflammation. Oxidative stress occurs due to increased levels of reactive oxygen species (ROS) such as H2O2. On the other hand, lipoprotein-associated phospholipase (Lp-PLA2) has pro-inflammatory effects, which cause instability of atherosclerosis plaques. This condition causes hypoxemic cells to stimulate HIFα induced vasa vasorum angiogenesis. This study aims to understand the potential of PSP as an anti-angiogenic agent through decreased levels of H2O2 and Lp-PLA2 leading to the decline of vasa vasorum angiogenesis in diabetic rat model. In addition, this study also measured the lipid profile of diabetic rat model in relation to vasa vasorum angiogenesis. Methods True laboratory experiment with randomized post-test control of group design using 25 wistar rats (Rattus norvegicus) were divided into five groups; one normal group and four group with High Fat Diet (HFD) and low dose streptozotocin (30 mg/kgBW) injection sc, treated with placebo and three various doses of PSP 50, 150, 300 mg/kgBW. Results ANOVA test (p < 0.05) shows that there is a significant influence of polysaccharide peptide (PSP) feeding on the decreased amount of vasa vasorum angiogenesis (p = 0.00), lipid profile (cholesterol total and triglyceride; p = 0.01, p = 0.001), and amount of H202 (p = 0.003). The amount of Lp-PLA2 declined to (p = 0.184). This result indicates that PSP prevents inflammation in atherosclerosis. Conclusions PSP of Ganoderma lucidum is an anti-angiogenic agent in T2DM.

NuMA1 promotes axon initial segment assembly through inhibition of endocytosis.

Axon initial segments (AISs) initiate action potentials and regulate the trafficking of vesicles between somatodendritic and axonal compartments. However, the mechanisms controlling AIS assembly remain poorly defined. We performed differential proteomics and found nuclear mitotic apparatus protein 1 (NuMA1) is downregulated in AIS-deficient neonatal mouse brains and neurons. NuMA1 is transiently located at the AIS during development where it interacts with the scaffolding protein 4.1B and the dynein regulator lissencephaly 1 (Lis1). Silencing NuMA1 or protein 4.1B by shRNA disrupts AIS assembly, but not maintenance. Silencing Lis1 or overexpressing NuMA1 during AIS assembly increased the density of AIS proteins, including ankyrinG and neurofascin-186 (NF186). NuMA1 inhibits the endocytosis of AIS NF186 by impeding Lis1's interaction with doublecortin, a potent facilitator of NF186 endocytosis. Our results indicate the transient expression and AIS localization of NuMA1 stabilizes the developing AIS by inhibiting endocytosis and removal of AIS proteins.

Reduction in Vasa Vasorum Angiogenesis by Lp-PLA2 Selective Inhibitor Through The HIF-1α and VEGF Expression Under Dyslipidemic Conditions in Atherosclerosis Pathogenesis.

BACKGROUND: Atherosclerosis is a chronic inflammatory disease which may lead to major cardiovascular events. The primary cause of atherosclerosis is Dyslipidemia. The increased level of lipid profile triggers endothelial dysfunction. This results in inflammation with the recruitment of monocyte, macrophage, T lymphocyte, and Mast cells secreted by an Lp-PLA2 enzyme which causes binding between macrophage and oxidized LDL. This binding results in the formation of foam cells and also the migration of smooth muscle cells. Following that, an Lp-PLA2 receptor hydrolizes OxPC which results in LysoPC and OxNEFA, bioactive compounds which stimulate the progression of atherosclerosis plaques. This process leads to cell hypoxia, which may result in the increase of HIF-1α and VEGF expressions and induction of vasa vasorum angiogenesis. Employing darapladib as an agent of Lp-PLA2 selective inhibitors, this study aimed to find out the effect of darapladib as an Lp- PLA2 selective inhibitor agent on the formation of vasa vasorum angiogenesis and the decrease of HIF-1α and VEGF expression in aortic tissue of rats with dyslipidemia. METHOD: A true laboratory experiment with a randomized post-test control group design used 30 male spraque dowley rats as animal models which were divided into 6 groups: Normal 8 weeks, Normal 16 weeks, Dyslipidemia (DL) 8 weeks, Dyslipidemia (DL) 16 weeks, Dyslipidemia with darapladib treatment (DLDP) 8 weeks and Dyslipidemia with darapladib treatment (DLDP) 16 weeks. The data measured in this study were the lipid profile (total cholesterol, HDL, and LDL). Using EnzyChrom TM kit, hematoxylin eosin, and double-labelling immunofluorescene, the levels of lipid profile, vasa vasorum, HIF-1α and VEGF were measured. RESULTS: The study results which were analyzed using NOVA test showed that with darapladib administration, there was a significant decrease in vasa vasorum angiogenesis (p=0.000), HIF-1α (p=0.005) and VEGF (p=0.009) expression in each time series. This result proves that Lp-PLA2 inhibitor reduces inflammatory process. CONCLUSION: Darapladib injection as an Lp-PLA2 selective inhibitor correlates with the decreasing vasa vasorum angiogenesis through alteration in HIF-1α and VEGF expressions in the aorta of high fat diet rats. We recommend further experiments to determine the effectiveness of darapladib with earlier time series in the atherosclerosis process.

Amelioration of atherosclerosis in apolipoprotein E-deficient mice by combined RNA interference of lipoprotein-associated phospholipase A2 and YKL-40.

To test the hypothesis that combined RNA interference (RNAi) of lipoprotein-associated phospholipase A2 (Lp-PLA2) and YKL-40 is superior to RNAi of Lp-PLA2 or YKL-40 alone in ameliorating atherosclerosis. A total of 120 apolipoprotein E-deficient mice (apoE-/- mice) were randomly divided into five groups, including the vehicle alone, scrambled RNAi, Lp-PLA2 RNAi, YKL-40 RNAi, and combined Lp-PLA2 and YKL-40 RNAi groups. Constrictive collars were used to induce plaque formation. Lp-PLA2 RNAi and YKL-40 RNAi viral suspensions were transduced into carotid plaques of the mice. Carotid plaques were harvested for histological analysis four weeks after viral vector transduction. Inflammatory gene expression in the plasma and atherosclerotic plaques was determined by ELISA and real-time PCR. Four weeks after RNAi, the serum concentration and plaque mRNA expression of Lp-PLA2 and YKL-40 were remarkably attenuated, leading to reduced inflammatory gene expression. Plaques from the Lp-PLA2 or YKL-40 RNAi group showed lower lipid content, higher collagen content, increased fibrous cap thickness, and lower mRNA expressions of MCP-1 and MMP-8 than than those in the vehicle and scramble groups. When compared with the isolated Lp-PLA2 or YKL-40 RNAi group, the combined Lp-PLA2 and YKL-40 RNAi group exhibited higher collagen content and fibrous cap thickness, and lower lipid content and local inflammation. The beneficial effects of RNAi were independent of the plasma lipoprotein profile. Combined RNAi of Lp-PLA2 and YKL-40 is superior to RNAi of Lp-PLA2 or YKL-40 alone in ameliorating atherosclerosis.

Platelet-activating factor acetylhydrolase 1B3 (PAFAH1B3) is required for the formation of the meiotic spindle during in vitro oocyte maturation.

Platelet-activating factor (PAF) is a well-described autocrine growth factor involved in several reproductive processes and is tightly regulated by its hydrolysing enzyme, PAF acetylhydrolase 1B (PAFAH1B). This intracellular enzyme consists of three subunits: one regulatory, 1B1, and two catalytic, 1B2 and 1B3. PAFAH1B3 has remained uncharacterised until now. Here, we report that PAFAH1B3 is present during the different stages of the first meiotic division in bovine, murine and human oocytes. In these species, the PAFAH1B3 subunit was clearly present in the germinal vesicle, while at metaphase I and II, it localised primarily at the meiotic spindle structure. In cattle, manipulation of the microtubules of the spindle by nocodazole, taxol or cryopreservation revealed a close association with PAFAH1B3. On the other hand, disruption of the enzyme activity either by P11, a selective inhibitor of PAFAH1B3, or by PAFAH1B3 antibody microinjection, caused arrest at the MI stage with defective spindle morphology and consequent failure of first polar body extrusion. In conclusion, our results show that one of the catalytic subunits of PAFAH1B, namely PAFAH1B3, is present in bovine, murine and human oocytes and that it plays a functional role in spindle formation and meiotic progression during bovine oocyte maturation.

Effect of lipoprotein-associated phospholipase A2 inhibitor on insulin resistance in streptozotocin-induced diabetic pregnant rats.

This paper aims to investigate the influence of lipoprotein-associated phospholipase A2 (Lp-PLA2) inhibitor, darapladib, on insulin resistance (IR) in streptozotocin (STZ)-induced diabetic pregnant rats. The rat models were divided into Control (normal pregnancy), STZ + saline (STZ-induced diabetic pregnant rats), STZ + Low-dose and STZ + High-dose darapladib (STZ-induced diabetic pregnant rats treated with low-/high-dose darapladib) groups. Pathological changes were observed by Hematoxylin-eosin (HE) and Immunohistochemistry staining. Lp-PLA2 levels were determined by enzyme-linked immunosorbent assay (ELISA). An automatic biochemical analyzer was used to measure the serum levels of biochemical indicators, and homeostatic model assessment for insulin resistance (HOMA-IR) and insulin sensitivity index (ISI) were calculated. Western blot was applied to determine levels of inflammatory cytokines. Compared with Control group, rats in the STZ + saline group were significantly decreased in body weight, the number of embryo implantation, the number of insulin positive cells and pancreatic islet size as well as the islet endocrine cells, and high-density lipoprotein (HDL-C) level, but substantially increased in Lp-PLA2, low-density lipoprotein (LDL-C), fatty acids (FFA), serum total cholesterol (TC), triglyceride (TG) levels. Moreover, the increased fasting plasma glucose (FPG) and HOMA-IR and inflammatory cytokines but decreased fasting insulin (FINS) and ISI were also found in diabetic pregnant rats. On the contrary, rats in the darapladib-treated groups were just opposite to the STZ + saline group, and STZ + High-dose group improved better than STZ + Low-dose group. Thus, darapladib can improve lipid metabolism, and enhance insulin sensitivity of diabetic pregnant rats by regulating inflammatory cytokines.

Chronic inhibition of lipoprotein-associated phospholipase A2 does not improve coronary endothelial function: A prospective, randomized-controlled trial.

AIMS: Lipoprotein-associated phospholipase A2 (Lp-PLA2), a novel biomarker for vascular inflammation, is associated with coronary endothelial dysfunction (CED) and independently predicts cardiovascular events. The current study aimed to determine whether darapladib, an orally administered Lp-PLA2 inhibitor, improved CED. METHODS AND RESULTS: Fifty-four patients with CED were enrolled in a double-blinded randomized placebo-controlled trial, and were randomized to receive oral darapladib, 160mg daily, or placebo. Coronary angiography and invasive coronary endothelial function assessment were performed at baseline and post-6months of treatment. Primary endpoints were change in coronary artery diameter and coronary blood flow in response to acetylcholine. Additionally, Lp-PLA2 activity was measured at baseline and on follow-up to evaluate for adherence and drug effect. Fifty-four patients were randomized to placebo (n=29) and darapladib (n=25). Mean age in darapladib group was 55.2.±11.7years vs. 54.0±10.5years (p=0.11). On follow-up, there was no significant difference in the percent response to acetylcholine of coronary artery diameter in treatment vs. placebo group (+3 (IQR -9, 15) vs. +3 (-12, 19); p=0.87) or coronary blood flow (-5 (IQR -24, 54) vs. 39 (IQR -26, 67); p=0.41). There was significant reduction in Lp-PLA2 activity in the treatment arm vs. placebo (-76 (IQR -113, -52) vs. -7(-21, -7); p<0.001). DISCUSSION: Lp-PLA2 inhibition with darapladib did not improve coronary endothelial function, despite significantly reduced Lp-PLA2 activity with darapladib. This study suggests endogenous Lp-PLA2 may not play a primary role in coronary endothelial function in humans. CLINICALTRIALS. GOV IDENTIFIER: NCT01067339.

Lipoprotein-associated Phospholipase A2 and Coronary Heart Disease.

In the last decades, the role of inflammation in the pathogenesis of atherosclerosis has been the topic of intense research. Several markers of inflammation have shown predictive value for first and recurrent coronary events in patients without and with established Coronary Heart Disease (CHD). Among these markers, lipoprotein- associated phospholipase A2 (Lp-PLA2) has recently received considerable attention. In the present review, the potential role of Lp-PLA2 as a marker of CHD risk and as a therapeutic target is discussed. Elevated Lp- PLA2 mass and activity appears to be associated with increased risk for CHD, both in the general population and in patients with established CHD. However, it is unclear whether the measurement of Lp-PLA2 improves risk discrimination when incorporated in models that include traditional cardiovascular risk factors. Moreover, the lack of effect on CHD events of darapladib, a potent, selective Lp-PLA2 inhibitor, in two large, randomized, placebo-controlled trials and the mostly negative findings of genetic association studies suggest that Lp-PLA2 is unlikely to represent a causal factor in atherogenesis. Therefore, it is doubtful whether Lp-PLA2 will constitute a therapeutic target for the prevention of CHD.

Regioselectivity of thiouracil alkylation: Application to optimization of Darapladib synthesis.

Darapladib is one of the most potent Lp-PLA2 (Lipoprotein-associated phospholipase A2) inhibitor with an IC50 of 0.25 nM. We demonstrate that a crucial step of Darapladib synthesis was not correctly described in the literature, leading to the production of wrong regioisomers. Moreover we show that the inhibitory activity is directly linked to the position on N1 since compounds bearing alkylation on different sites have potentially less interaction within the active site of Lp-PLA2.

Structure-Guided Discovery of Novel, Potent, and Orally Bioavailable Inhibitors of Lipoprotein-Associated Phospholipase A2.

Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a promising therapeutic target for atherosclerosis, Alzheimer's disease, and diabetic macular edema. Here we report the identification of novel sulfonamide scaffold Lp-PLA2 inhibitors derived from a relatively weak fragment. Similarity searching on this fragment followed by molecular docking leads to the discovery of a micromolar inhibitor with a 300-fold potency improvement. Subsequently, by the application of a structure-guided design strategy, a successful hit-to-lead optimization was achieved and a number of Lp-PLA2 inhibitors with single-digit nanomolar potency were obtained. After preliminary evaluation of the properties of drug-likeness in vitro and in vivo, compound 37 stands out from this congeneric series of inhibitors for good inhibitory activity and favorable oral bioavailability in male Sprague-Dawley rats, providing a quality candidate for further development. The present study thus clearly demonstrates the power and advantage of integrally employing fragment screening, crystal structures determination, virtual screening, and medicinal chemistry in an efficient lead discovery project, providing a good example for structure-based drug design.

Performance characteristics of lipoprotein-associated phospholipase A2 activity assay on the Dimension Vista analyser and preliminary study of a healthy Italian population.

INTRODUCTION: Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a marker of vascular inflammation associated with coronary heart disease and stroke. We evaluated analytical performance of the PLAC® Activity Test on Siemens Dimension Vista® 1500 analyzer and measured Lp-PLA2 activity in Italian adults to establish reference intervals (RIs) and evaluate correlation with circulating lipids and age. MATERIALS AND METHODS: The evaluation protocol consisted of precision, linearity, sensitivity, method comparison, substrate depletion ("hook") effect and interference assessment. Inhibitor (Darapladib) effect was also evaluated. Lp-PLA2 activity was measured in 250 healthy donors (123 males, 127 females, aged 18-70 years). Central 95% RIs were established using nonparametric statistics. RESULTS: Intra-assay and inter-assay precision showed CVs of 0.6% - 1.4% and 0.9% - 2.0%, respectively. Linearity replicates showed R2 > 0.98. Limit of quantitation was 5.8 U/L (CV = 9.4%). Bland Altman plot showed bias - 0.9, 95% limits of agreement -6.5 - 4.72. Passing-Bablok regression showed excellent correlation (Slope = 1.02, 95% CI: 1.01 to 1.03; Intercept = - 1.86, 95% CI: - 3.08 to - 1.26; R2 = 0.999). No "hook effect" was observed at Lp-PLA2 activities ≤ 1000 U/L. Average Lp-PLA2 activity in 250 healthy donors was 182 ± 44 U/L (mean ± SD). Males showed statistically significant higher activities than females (P < 0.001). RIs were 107 - 265 U/L for males and 84 - 225 U/L for females. Moderate significant correlation (r = 0.29, P < 0.001) was found between Lp-PLA2 activity and total cholesterol. CONCLUSIONS: The PLAC® Activity Test shows very good performance characteristics on Dimension Vista® 1500.

Pharmacogenetic meta-analysis of baseline risk factors, pharmacodynamic, efficacy and tolerability endpoints from two large global cardiovascular outcomes trials for darapladib.

Darapladib, a lipoprotein-associated phospholipase A2 (Lp-PLA2) inhibitor, failed to demonstrate efficacy for the primary endpoints in two large phase III cardiovascular outcomes trials, one in stable coronary heart disease patients (STABILITY) and one in acute coronary syndrome (SOLID-TIMI 52). No major safety signals were observed but tolerability issues of diarrhea and odor were common (up to 13%). We hypothesized that genetic variants associated with Lp-PLA2 activity may influence efficacy and tolerability and therefore performed a comprehensive pharmacogenetic analysis of both trials. We genotyped patients within the STABILITY and SOLID-TIMI 52 trials who provided a DNA sample and consent (n = 13,577 and 10,404 respectively, representing 86% and 82% of the trial participants) using genome-wide arrays with exome content and performed imputation using a 1000 Genomes reference panel. We investigated baseline and change from baseline in Lp-PLA2 activity, two efficacy endpoints (major coronary events and myocardial infarction) as well as tolerability parameters at genome-wide and candidate gene level using a meta-analytic approach. We replicated associations of published loci on baseline Lp-PLA2 activity (APOE, CELSR2, LPA, PLA2G7, LDLR and SCARB1) and identified three novel loci (TOMM5, FRMD5 and LPL) using the GWAS-significance threshold P≤5E-08. Review of the PLA2G7 gene (encoding Lp-PLA2) within these datasets identified V279F null allele carriers as well as three other rare exonic null alleles within various ethnic groups, however none of these variants nor any other loci associated with Lp-PLA2 activity at baseline were associated with any of the drug response endpoints. The analysis of darapladib efficacy endpoints, despite low power, identified six low frequency loci with main genotype effect (though with borderline imputation scores) and one common locus (minor allele frequency 0.24) with genotype by treatment interaction effect passing the GWAS-significance threshold. This locus conferred risk in placebo subjects, hazard ratio (HR) 1.22 with 95% confidence interval (CI) 1.11-1.33, but was protective in darapladib subjects, HR 0.79 (95% CI 0.71-0.88). No major loci for tolerability were found. Thus, genetic analysis confirmed and extended the influence of lipoprotein loci on Lp-PLA2 levels, identified some novel null alleles in the PLA2G7 gene, and only identified one potentially efficacious subgroup within these two large clinical trials.