2-(Alkylthio)pyrimidin-4-ones as novel, reversible inhibitors of lipoprotein-associated phospholipase A2

Starting from two weakly active hits from high throughput screening, a novel series of 2-(alkylthio)-pyrimidin-4-ones with high potency and selectivity for lipoprotein-associated phospholipase A2 has been designed. In contrast to previously known inhibitors, these have been shown to act by a non-covalent and substrate competitive mechanism.     

Potent inhibitors of lipoprotein-associated phospholipase A(2): benzaldehyde O-heterocycle-4-carbonyloxime

A series of multi-substituted oximes were prepared and their potencies for inhibiting lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) activity were evaluated in vitro. Among them, compounds 3a, 3b, and 3m were identified to display a micromolar potency for inhibiting Lp-PLA(2) in whole human plasma and isolated human LDL. Based on these results, structure-activity relationship was studied on modification of three parts of R(1), R(2), and R(3) to identify a potent pharmacophore for Lp-PLA(2). In an attempt to introduce various functional groups at R(2) and R(3), we discovered that replacement of less lipophilic groups led to an increase of inhibitory activity. Among the tested oxime derivatives, cyano- and morpholino-substituted analogue 4f at R(2) and R(3) had the highest potency with an IC(50) value of 0.05 microM in whole human plasma.     

Selective inhibitors and tailored activity probes for lipoprotein-associated phospholipase A2

Lipoprotein-associated phospholipase A₂ (Lp-PLA₂ or PLA₂G7) binds to low-density lipoprotein (LDL) particles, where it is thought to hydrolyze oxidatively truncated phospholipids. Lp-PLA₂ has also been implicated as a pro-tumorigenic enzyme in human prostate cancer. Several inhibitors of Lp-PLA₂ have been described, including darapladib, which is currently in phase 3 clinical development for the treatment of atherosclerosis. The selectivity that darapladib and other Lp-PLA₂ inhibitors display across the larger serine hydrolase family has not, however, been reported. Here, we describe the use of both general and tailored activity-based probes for profiling Lp-PLA₂ and inhibitors of this enzyme in native biological systems. We show that both darapladib and a novel class of structurally distinct carbamate inhibitors inactivate Lp-PLA₂ in mouse tissues and human cell lines with high selectivity. Our findings thus identify both inhibitors and chemoproteomic probes that are suitable for investigating Lp-PLA₂ function in biological systems.     

1-(Arylpiperazinylamidoalkyl)-pyrimidones: orally active inhibitors of lipoprotein-associated phospholipase A(2).

The lipophilic 1-substituent in a series of 1-((amidolinked)-alkyl)-pyrimidones, inhibitors of recombinant lipoprotein-associated phospholipase A(2), has been modified to give inhibitors of high potency in human plasma and enhanced physicochemical properties. Phenylpiperazineacetamide derivative 23 shows very promising oral activity.     

Benzene-di-N-octylcarbamates as conformationally constrained phospholipase A(2) inhibitors

Conformationally constrained 1,2-, 1,3-, and 1,4-benzene-di-N-octylcarbamates are potent reversible competitive inhibitors of Naja mocambique mocambique phospholipase A(2) with the K(i) values of 11, 4, and 15 microM, respectively. With the angle of 120(o) between two C(benzene)-O bonds, 1,3-benzene-di-N-octylcarbamate mimics the preferable eclipsed C(sn-2)-O/C(sn-3)-O conformer of phospholipid in the enzyme-phospholipid complex. Further, a three-step phospholipase A(2) inhibition mechanism by the inhibitor is proposed.     

Potent, orally active inhibitors of lipoprotein-associated phospholipase A(2): 1-(biphenylmethylamidoalkyl)-pyrimidones.

A series of 1-(biphenylmethylamidoalkyl)-pyrimidones has been designed as nanomolar inhibitors of recombinant lipoprotein-associated phospholipase A(2) with high potency in whole human plasma. 5-(Pyrazolylmethyl) derivative 16 and 5-(methoxypyrimidinylmethyl) derivative 27 demonstrated excellent pharmacodynamic profiles which correlated well with their pharmacokinetic effects.     

Snake inhibitors of phospholipase A(2) enzymes

Fragment 53--103 of bovine alpha-lactalbumin, prepared by limited peptic digestion of the protein at low pH, is a 51-residue polypeptide chain crosslinked by two disulfide bonds encompassing helix C (residues 86--98) of the native protein. Refolding of the fully reduced fragment (four--SH groups) is expected to lead to three fully oxidized isomers, the native (61--77, 73--91) and the two misfolded species named ribbon (61--91, 73--77) and beads (61--73, 77--91) isomers. The fragment with correct disulfide bonds was formed in approx. 30% yield when refolding was conducted in aqueous solution at neutral pH in the presence of the redox system constituted by reduced and oxidized glutathione. On the other hand, when the reaction was conducted in 30% (v/v) trifluoroethanol (TFE), the oxidative refolding to the native isomer was almost quantitative. To provide an explanation of the beneficial effect of TFE in promoting the correct oxidative folding, the conformational features of the various fragment species were analyzed by far-UV circular dichroism measurements. The fully reduced fragment is largely unfolded in water, but it becomes helical in aqueous TFE. Correctly refolded fragment is produced most when the helical contents of the reduced and oxidized fragment in aqueous TFE are roughly equal. It is proposed that 30% TFE promotes a native-like format of the fragment and thus an efficient and correct pairing of disulfides. Higher concentrations of TFE, instead, promote some non-native helical secondary structure in the fragment species, thus hampering correct folding.     

Suicide-inhibitory bifunctionally linked substrates (SIBLINKS) as phospholipase A2 inhibitors. Mechanistic implications

Novel phospholipids that function as mechanism-based inhibitors for phospholipase A2 (PLA2) are described. PLA2-catalyzed hydrolysis of the sn-2 ester of these suicide-inhibitory bifunctionally linked substrates (SIBLINKS) followed by a cyclization reaction generates a cyclic anhydride at the active site of the enzyme which leads to inhibition. Structure/activity relationships for the SIBLINKS substituents in the sn-1 and sn-2 position are delineated. Time courses and efficiency of SIBLINKS inhibition are reported and compared for extracellular PLA2s obtained from Naja naja naja, porcine pancreas, bee venom, Crotalus atrox and Crotalus adamanteus. SIBLINKS-inhibited PLA2s cannot process either monomeric or micellar substrates consistent with inhibition at the catalytic site. Some SIBLINKS efficiently inactivate 1 mol of N. naja naja and C. adamanteus PLA2/6-10 mol of SIBLINKS hydrolyzed. Inhibition of N. naja naja PLA2 can be reversed by hydroxylamine, suggesting that a tyrosine residue is acylated.     

N-1 substituted pyrimidin-4-ones: novel, orally active inhibitors of lipoprotein-associated phospholipase A2

From two related series of 2-(alkylthio)-pyrimidones, a novel series of 1-((amidolinked)-alkyl)-pyrimidones has been designed as nanomolar inhibitors of human lipoprotein-associated phospholipase A2. These compounds show greatly enhanced activity in isolated plasma. Selected derivatives such as compounds 51 and 52 are orally active with a good duration of action.     

抗人脂蛋白相关磷脂酶A2单抗制备及应用

本研究旨在制备抗人脂蛋白相关磷脂酶A2 (Lp-PLA2)单克隆抗体,对单抗进行初步评价,采用免疫层析方法学,建立一种可在社区医疗机构应用的Lp-PLA2快速检测方法。首先从NCBI获得人Lp-PLA2全长基因序列构建表达载体,在CHO-K1细胞中表达Lp-PLA2重组蛋白。以获得的重组蛋白免疫BALB/c小鼠,利用细胞融合技术筛选杂交瘤细胞,小鼠腹水诱生单克隆抗体。通过SDS-PAGE、ELISA等方法评价抗体性能。利用双抗夹心法制备Lp-PLA2量子点荧光免疫层析检测试剂,并使用便携式检测仪器评估试剂性能。制备并筛选得到亲和力达到1×10~(–8)的配对单克隆抗体PLA1与PLA5,抗体亚类为IgG1,能特异性识别血液中Lp-PLA2蛋白。自制Lp-PLA2量子点荧光免疫检测试剂可检测线性范围为20–2000ng/mL,与进口试剂的检测相关系数R达到0.99。综上,本研究制备得到一对高亲和力、高特异性的抗人Lp-PLA2单克隆抗体,并建立了Lp-PLA2免疫层析检测方法,该方法检测准确、操作简便,适合Lp-PLA2检测应用于社区医疗机构,为居民心血管健康管理提供了新途径。     

Novel delta2-isoxazolines as group II phospholipase A2 inhibitors

The synthesized imidazolyl substituted delta2-isoxazolines were subjected to Phospholipase A(2) (PLA(2)) enzyme inhibitory activity against snake venom source and their structure-activity relationship with respect to different groups attached to this moiety is reported for the first time. The crystal structure of the compound 2-butyl-5-chloro-3H-imidazolyl-4-carbaldehyde oxime 2, an intermediate for the construction of isoxazolines is reported. These compounds exerted a significant PLA(2) enzyme inhibitory activity against group II PLA(2). The in vivo activity on mice of selected compounds 3bI and 3bIV shows the comparable anti-inflammatory activity with the known standard ursolic acid.     

Heterologous expression of lipoprotein-associated phospholipase A2 in different expression systems

Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) is a key enzyme involved in atherosclerosis, and has been considered as a new target for drug discovery. The major difficulty for high-throughput screening of Lp-PLA(2) inhibitors and for functional studies was their fast and efficient production. Purification of native Lp-PLA(2) from human plasma was complicated and produced a very low yield. We herein examined the feasibility of expressing and purifying recombinant Lp-PLA(2) in different heterologous expression systems. The fusion Lp-PLA(2) was expressed at high levels and exhibited strong enzyme activity in insect cell-baculovirus expression system. The functional enzyme could also be produced in Pichia pastoris. The inclusion of a Kozak sequence increased greatly the expression level of recombinant Lp-PLA(2) in insect cells, but had little effect on the expression of recombinant Lp-PLA(2) in P. pastoris and Escherichia coli. P. pastoris-produced Lp-PLA(2) could be purified rapidly and conveniently through a one-step procedure, while baculovirus-produced Lp-PLA(2) could be efficiently purified through a two-step procedure. This ability to readily produce recombinant Lp-PLA(2) could provide a screening model for Lp-PLA(2) inhibitors and will facilitate further studies on this enzyme.     

4-Phenyl-7-azaindoles as potent and selective IKK2 inhibitors

The synthesis and SAR of a novel series of IKK2 inhibitors are described. Modification around the hinge binding region of the 7-azaindole led to a series of potent and selective inhibitors with good cellular activity.     

Novel 3-dodecanoylindole-2-carboxylic acid inhibitors of cytosolic phospholipase A(2)

Derivatives of 1-[2-(4-carboxyphenoxy)ethyl]-3-dodecanoylindole-2-carboxylic acid (4) with modified substituents at the indole-1-position were synthesized and evaluated for their ability to inhibit the arachidonic acid release in human platelets mediated by the cytosolic phospholipase A(2). One of the most active compounds obtained was 26 with an IC(50) of 0.44 microM.     

Inhibition of lipoprotein-associated phospholipase A2 reduces complex coronary atherosclerotic plaque development

Increased lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) activity is associated with increased risk of cardiac events, but it is not known whether Lp-PLA(2) is a causative agent. Here we show that selective inhibition of Lp-PLA(2) with darapladib reduced development of advanced coronary atherosclerosis in diabetic and hypercholesterolemic swine. Darapladib markedly inhibited plasma and lesion Lp-PLA(2) activity and reduced lesion lysophosphatidylcholine content. Analysis of coronary gene expression showed that darapladib exerted a general anti-inflammatory action, substantially reducing the expression of 24 genes associated with macrophage and T lymphocyte functioning. Darapladib treatment resulted in a considerable decrease in plaque area and, notably, a markedly reduced necrotic core area and reduced medial destruction, resulting in fewer lesions with an unstable phenotype. These data show that selective inhibition of Lp-PLA(2) inhibits progression to advanced coronary atherosclerotic lesions and confirms a crucial role of vascular inflammation independent from hypercholesterolemia in the development of lesions implicated in the pathogenesis of myocardial infarction and stroke.     

Therapeutic application of natural inhibitors against snake venom phospholipase A(2)

Natural inhibitors occupy an important place in the potential to neutralize the toxic effects caused by snake venom proteins and enzymes. It has been well recognized for several years that animal sera, some of the plant and marine extracts are the most potent in neutralizing snake venom phospholipase A(2) (svPLA(2)). The implication of this review to update the latest research work which has been accomplished with svPLA(2) inhibitors from various natural sources like animal, marine organisms presents a compilation of research in this field over the past decade and revisiting the previous research report including those found in plants. In addition to that the bioactive compounds/inhibitor molecules from diverse sources like aristolochic alkaloid, flavonoids and neoflavonoids from plants, hydrocarbones -2, 4 dimethyl hexane, 2 methylnonane, and 2, 6 dimethyl heptane obtained from traditional medicinal plants Tragia involucrata (Euphorbiaceae) member of natural products involved for the inhibitory potential of phospholipase A(2) (PLA(2)) enzymes in vitro and also decrease both oedema induced by snake venom as well as human synovial fluid PLA(2). Besides marine natural products that inhibit PLA(2) are manoalide and its derivatives such as scalaradial and related compounds, pseudopterosins and vidalols, tetracylne from synthetic chemicals etc. There is an overview of the role of PLA(2) in inflammation that provides a rationale for seeking inhibitors of PLA(2) as anti-inflammatory agents. However, more studies should be considered to evaluate antivenom efficiency of sera and other agents against a variety of snake venoms found in various parts of the world. The implications of these new groups of svPLA(2) toxin inhibitors in the context of our current understanding of snake biology as well as in the development of new novel antivenoms therapeutics agents in the efficient treatment of snake envenomations are discussed.     

Design and synthesis of conformationally restricted phospholipids as phospholipase A2 inhibitors

The use of conformationally restricted phospholipids 1 and 2 has been employed to understand the conformational preference of phospholipase A2 (PLA2) for substrate phospholipids. Inhibition of porcine pancreatic PLA2 with 1 and 2 indicated a two- to fivefold preference for the distal isomer 2 over the proximal isomer 1. Based upon these studies, both side-chains of the substrate phospholipid appear to occupy the lipid binding domains near the active site with the side-chains further apart most preferred by PLA2.     

Identification of group X secretory phospholipase A(2) as a natural ligand for mouse phospholipase A(2) receptor

Phospholipase A(2) receptor (PLA(2)R) mediates various biological responses elicited by group IB secretory phospholipase A(2) (sPLA(2)-IB). The recently cloned group X sPLA(2) (sPLA(2)-X) possesses several structural features characteristic of sPLA(2)-IB. Here, we detected a specific binding site of sPLA(2)-X in mouse osteoblastic MC3T3-E(1) cells. Cross-linking experiments demonstrated its molecular weight (180 kDa) to be similar to that of PLA(2)R. In fact, sPLA(2)-X was found to bind the recombinant PLA(2)R expressed in COS-7 cells, and its specific binding detected in mouse lung membranes was abolished by the deficiency of PLA(2)R. These findings demonstrate sPLA(2)-X to be one of the high-affinity ligands for mouse PLA(2)R.     

A new class of acyclic 2-alkyl-1,2-diaryl (E)-olefins as selective cyclooxygenase-2 (COX-2) inhibitors

A new class of (E)-2-alkyl-2-(4-methanesulfonylphenyl)-1-phenylethenes were designed for evaluation as selective cyclooxygense-2 (COX-2) inhibitors. The target olefins were synthesized, via a Takeda olefination reaction, followed by oxidation of the respective thiomethyl olefinic intermediate. In vitro COX-1/COX-2 inhibition studies identified (E)-2-(4-methanesulfonylphenyl)-1-phenyloct-1-ene (8d) as a potent (IC(50)=0.77 microM) and selective (Selectivity Index>130) COX-2 inhibitor.     

Modulation of oxidative stress, inflammation, and atherosclerosis by lipoprotein-associated phospholipase A2

Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)), also known as platelet-activating factor acetylhydrolase (PAF-AH), is a unique member of the phospholipase A(2) superfamily. This enzyme is characterized by its ability to specifically hydrolyze PAF as well as glycerophospholipids containing short, truncated, and/or oxidized fatty acyl groups at the sn-2 position of the glycerol backbone. In humans, Lp-PLA(2) circulates in active form as a complex with low- and high-density lipoproteins. Clinical studies have reported that plasma Lp-PLA(2) activity and mass are strongly associated with atherogenic lipids and vascular risk. These observations led to the hypothesis that Lp-PLA(2) activity and/or mass levels could be used as biomarkers of cardiovascular disease and that inhibition of the activity could offer an attractive therapeutic strategy. Darapladib, a compound that inhibits Lp-PLA(2) activity, is anti-atherogenic in mice and other animals, and it decreases atherosclerotic plaque expansion in humans. However, disagreement continues to exist regarding the validity of Lp-PLA(2) as an independent marker of atherosclerosis and a scientifically justified target for intervention. Circulating Lp-PLA(2) mass and activity are associated with vascular risk, but the strength of the association is reduced after adjustment for basal concentrations of the lipoprotein carriers with which the enzyme associates. Genetic studies in humans harboring an inactivating mutation at this locus indicate that loss of Lp-PLA(2) function is a risk factor for inflammatory and vascular conditions in Japanese cohorts. Consistently, overexpression of Lp-PLA(2) has anti-inflammatory and anti-atherogenic properties in animal models. This thematic review critically discusses results from laboratory and animal studies, analyzes genetic evidence, reviews clinical work demonstrating associations between Lp-PLA(2) and vascular disease, and summarizes results from animal and human clinical trials in which administration of darapladib was tested as a strategy for the management of atherosclerosis.