Identification and analyses of inhibitors targeting apolipoprotein(a) kringle domains KIV-7, KIV-10, and KV provide insight into kringle domain function.

Increased plasma concentrations of lipoprotein(a) (Lp(a)) are associated with an increased risk for cardiovascular disease. Lp(a) is composed of apolipoprotein(a) (apo(a)) covalently bound to apolipoprotein B of low-density lipoprotein (LDL). Many of apo(a)'s potential pathological properties, such as inhibition of plasmin generation, have been attributed to its main structural domains, the kringles, and have been proposed to be mediated by their lysine-binding sites. However, available small-molecule inhibitors, such as lysine analogs, bind unselectively to kringle domains and are therefore unsuitable for functional characterization of specific kringle domains. Here, we discovered small molecules that specifically bind to the apo(a) kringle domains KIV-7, KIV-10, and KV. Chemical synthesis yielded compound AZ-05, which bound to KIV-10 with a Kd of 0.8 µm and exhibited more than 100-fold selectivity for KIV-10, compared with the other kringle domains tested, including plasminogen kringle 1. To better understand and further improve ligand selectivity, we determined the crystal structures of KIV-7, KIV-10, and KV in complex with small-molecule ligands at 1.6-2.1 Å resolutions. Furthermore, we used these small molecules as chemical probes to characterize the roles of the different apo(a) kringle domains in in vitro assays. These assays revealed the assembly of Lp(a) from apo(a) and LDL, as well as potential pathophysiological mechanisms of Lp(a), including (i) binding to fibrin, (ii) stimulation of smooth-muscle cell proliferation, and (iii) stimulation of LDL uptake into differentiated monocytes. Our results indicate that a small-molecule inhibitor targeting the lysine-binding site of KIV-10 can combat the pathophysiological effects of Lp(a).

Discovery of a Series of Indole-2 Carboxamides as Selective Secreted Phospholipase A2 Type X (sPLA2-X) Inhibitors.

In order to assess the potential of sPLA2-X as a therapeutic target for atherosclerosis, novel sPLA2 inhibitors with improved type X selectivity are required. To achieve the objective of identifying such compounds, we embarked on a lead generation effort that resulted in the identification of a novel series of indole-2-carboxamides as selective sPLA2-X inhibitors with excellent potential for further optimization.

Effect of broad-spectrum matrix metalloproteinase inhibition on atherosclerotic plaque stability.

OBJECTIVE: Matrix metalloproteinases (MMPs) form a large family of enzymes that collectively can degrade all components of the extracellular matrix, and there is widespread interest in developing MMP inhibitors for the prevention of atherosclerotic plaque rupture. We have therefore investigated the effects of a broad-spectrum MMP inhibitor, RS-130830, on plaque development and stability. This compound inhibits a wide range of MMPs at concentrations below 20 nmol/L. METHODS: Apolipoprotein E knockout mice were fed a Western diet. Dietary administration of RS-130830 commenced at the same time as fat-feeding and continued for 8, 12, 26 or 36 weeks. To investigate the effect of RS-130830 on established plaques, mice were fed high-fat diet for 16 weeks before initiation of drug treatment and were terminated 20 weeks after this. RESULTS: Broad-spectrum MMP inhibition was associated with a significant increase in plaque area, but there was no change in the incidence of plaque rupture. There were unfavourable changes in phenotypic characteristics associated with plaque instability, such as an increased lipid content and decreased collagen content. CONCLUSIONS: These data suggest that broad-spectrum MMP inhibition RS-130830 does not have a beneficial effect on atherosclerosis in the apolipoprotein E knockout mouse model, and indicate that more selective compounds would be preferable.

Discovery of AZD6642, an inhibitor of 5-lipoxygenase activating protein (FLAP) for the treatment of inflammatory diseases.

A drug discovery program in search of novel 5-lipoxygenase activating protein (FLAP) inhibitors focused on driving a reduction in lipophilicity with maintained or increased ligand lipophilic efficiency (LLE) compared to previously reported compounds led to the discovery of AZD6642 (15b). Introduction of a hydrophilic tetrahydrofuran (THF) ring at the stereogenic central carbon atom led to a significant shift in physicochemical property space. The structure-activity relationship exploration and optimization of DMPK properties leading to this compound are described in addition to pharmacokinetic analysis and an investigation of the pharmacokinetic (PK)-pharmacodynamic (PD) relationship based on ex vivo leukotriene B4 (LTB4) levels in dog. AZD6642 shows high specific potency and low lipophilicity, resulting in a selective and metabolically stable profile. On the basis of initial PK/PD relation measured, a low dose to human was predicted.

The potential link between atherosclerosis and the 5-lipoxygenase pathway: investigational agents with new implications for the cardiovascular field.

The 5-lipoxygenase pathway is responsible for the production of leukotrienes--inflammatory lipid mediators that have a role in innate immunity, but that can also have pathological effects in inflammatory diseases. Recently, a potential link between leukotriene production and atherosclerosis has been proposed. The expression of leukotriene biosynthetic enzymes and leukotriene receptors has been identified in coronary and carotid atherosclerotic plaques, and the levels of biosynthetic enzymes have been correlated with the clinical symptoms of unstable plaques. Genetic variants in 5-lipoxygenase pathway genes have also been associated with a relative risk of developing myocardial infarction and stroke. On the basis of these discoveries, antileukotriene compounds are now being evaluated for the treatment of cardiovascular disease. Several tool compounds have been shown to limit the progression of lesion development in preclinical models of atherosclerosis, and three compounds, including two drugs previously developed for asthma, are undergoing clinical trials in patients with acute coronary syndromes.