Self-Assembling Cyclic d,l‑α-Peptides as Modulators of Plasma HDL Function. A Supramolecular Approach toward Antiatherosclerotic Agents

There is great interest in developing new modes of therapy for atherosclerosis to treat coronary heart disease and stroke, particularly ones that involve modulation of high-density lipoproteins (HDLs). Here, we describe a new supramolecular chemotype for altering HDL morphology and function. Guided by rational design and SAR-driven peptide sequence enumerations, we have synthesized and determined the HDL remodeling activities of over 80 cyclic d,l-α-peptides. We have identified a few distinct sequence motifs that are effective <i>in vitro</i> in remodeling human and mouse plasma HDLs to increase the concentration of lipid-poor pre-beta HDLs, which are key initial acceptors of cholesterol in the reverse cholesterol transport (RCT) process, and concomitantly promote cholesterol efflux from macrophage cells. Functional assays with various control peptides, such as scrambled sequences, linear and enantiomeric cyclic peptide variants, and backbone-modified structures that limit peptide self-assembly, provide strong support for the supramolecular mode of action. Importantly, when the lead cyclic peptide c­[wLwReQeR] was administered to mice (ip), it also promoted the formation of small, lipid-poor HDLs in vivo, displayed good plasma half-life (∼6 h), did not appear to have adverse side effects, and exerted potent anti-inflammatory effects in an acute in vivo inflammation assay. Given that previously reported HDL remodeling peptides have been based on α-helical apoA-I mimetic architectures, the present study, involving a new structural class, represents a promising step toward new potential therapeutics to combat atherosclerosis

Abdominal Aortic Aneurysms.

<p><b>A</b>) Incidence of aneurysm development in the abdominal aorta. <b>B</b>) Representative images of abdominal aortic aneurysms for P2Y₆ KO mice and P2Y₆ wild-type mice. Aneurysm-positive and -negative aortas are given for P2Y₆ KO wild type mice. Aneurysms are demarcated by the white boxes. <b>C</b>) Trichrome-stained histological sections of aneurysms taken from the supra-renal section of the abdominal aorta. Sections were cut from the center of the aneurysm mass. The single asterisk denotes the highly inflamed and remodeled adventitial mass. The double asterisk denotes a region of apparent hemorrhage. Neo-intimal plaque is shown by the arrows. The image at the far right shows an abdominal aorta cross-section taken from P2Y₆ wild type mice. The magnification scale for each image is shown.</p

Discovery of Potent and Selective Quinoxaline-Based Protease-Activated Receptor 4 (PAR4) Antagonists for the Prevention of Arterial Thrombosis

PAR4 is a promising antithrombotic target with potential for separation of efficacy from bleeding risk relative to current antiplatelet therapies. In an effort to discover a novel PAR4 antagonist chemotype, a quinoxaline-based HTS hit 3 with low μM potency was identified. Optimization of the HTS hit through the use of positional SAR scanning and the design of conformationally constrained cores led to the discovery of a quinoxaline-benzothiazole series as potent and selective PAR4 antagonists. The lead compound 48, possessing a 2 nM IC50 against PAR4 activation by γ-thrombin in platelet-rich plasma (PRP) and greater than 2500-fold selectivity versus PAR1, demonstrated robust antithrombotic efficacy and minimal bleeding in the cynomolgus monkey models

Discovery of Potent and Selective Quinoxaline-Based Protease-Activated Receptor 4 (PAR4) Antagonists for the Prevention of Arterial Thrombosis

PAR4 is a promising antithrombotic target with potential for separation of efficacy from bleeding risk relative to current antiplatelet therapies. In an effort to discover a novel PAR4 antagonist chemotype, a quinoxaline-based HTS hit 3 with low μM potency was identified. Optimization of the HTS hit through the use of positional SAR scanning and the design of conformationally constrained cores led to the discovery of a quinoxaline-benzothiazole series as potent and selective PAR4 antagonists. The lead compound 48, possessing a 2 nM IC50 against PAR4 activation by γ-thrombin in platelet-rich plasma (PRP) and greater than 2500-fold selectivity versus PAR1, demonstrated robust antithrombotic efficacy and minimal bleeding in the cynomolgus monkey models

Atherosclerosis development with P2Y₆-deficient bone marrow reconstitution.

<p><b>A</b>) Quantification of en face lesion area with oil red O staining in the whole aorta from the ascending aorta to the iliac bifurcation and <b>B</b>) aortic arch. <b>C</b>) Representative images of aortic arch atherosclerosis. Group sizes: n = 9 P2Y₆ WT transplanted mice; n = 14 P2Y₆ KO transplanted mice. Significance vs. control: *p<0.05. <i>Not significant, n.s.</i></p

Accelerated Atherosclerosis development with Angiotensin II infusion.

<p>Atherosclerosis development measured in the aorta with oil red O. <b>A</b>) Quantification of en face lesion area in the thoracic aorta (ascending aorta to the diaphragm). Regional atherosclerosis in <b>B</b>) aortic arch and <b>C</b>) descending thoracic aorta segments. Group sizes: n = 9 P2Y₆ WT mice; n = 14 P2Y₆ KO mice. The fold change in P2Y₆ lesion area is given relative to P2Y₆ wild type levels. <i>Not significant, n.s.</i></p

P2Y₆ knockout mice exhibit reduced responsiveness to inflammatory challenge.

<p>Generation and evaluation of P2Y₆ KO mice. <b>A</b>) Targeting strategy used to replace the 3^rd exon of the P2Y₆ gene containing the entire open reading frame with a pKOS-17 vector containing β-galactosidase (LacZ) reporter and neomycin resistance (Neo) cassette. The second and third exon of the P2Y₆ gene are depicted as grey boxes with indicated translational start and stop codons. Location of Southern Blot probes is shown in green and blue, and PCR genotyping primers are shown in red. <b>B</b>) MPMs from P2Y₆ knockout mice (KO) and wild type littermates (WT) were incubated with increasing concentrations of 3P-UDP to generate calcium concentration response curves. Calculated EC₅₀ value for WT MPMs is shown. <b>C</b>) P2Y₆ KO males and WT male littermates (n =  4–5 per group) were subjected to intra-peritoneal challenge with vehicle, MSU or combination of MSU and 3P-UDP (1mg/mouse each) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111385#s2" target="_blank">Materials and Methods</a>. Blood was collected 1 hr after injection and cytokines in plasma measured by multiplex ELISA. Cytokines that displayed differential responses in KO mice are shown. Baseline (unchallenged mice) and vehicle treated mice are also depicted for MIP-1α. Significance KO vs WT: **p<0.01 *p<0.05. <i>Not significant, n.s.</i></p

P2Y₆ expression mediates release of calcium and pro-inflammatory cytokines in 1321N1 cells.

<p>Evaluation of calcium release and cytokine secretion to cell culture media in 1321N1 astrocytoma cells stably expressing human P2Y₆ receptor. <b>A</b>) Isolated 1321N1 clones expressing varying levels of P2Y₆ mRNA and mock transfected cells (Parental) were incubated with increasing concentrations of UDP and 3P-UDP. Calcium mobilization was measured to generate concentration response curves with examples shown for 3G10 clone and parental cell line. For each clone, achieved maximal calcium response is expressed as percentage relative to 3G10 clone (set as 100%) together with calculated EC₅₀ values (ND  =  not determined) and relative P2Y₆ mRNA levels (relative to parental cells, which had very low but detectable P2Y₆ mRNA). <b>B</b>) 1321N1 clones were incubated for 20 hr with increasing concentrations of 3P-UDP and cytokines in cell culture medium quantified using multiplex ELISA as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111385#s2" target="_blank">Materials and Methods</a>. Each cytokine was normalized to cellular protein content. The data are presented as mean +/− SD of replicate wells.</p

Atherosclerosis development with Western Diet Feeding.

<p>En face plaque quantification of excised mouse aortas using oil red O. <b>A</b>) Lesion area measured in the whole aorta from the ascending aorta to the iliac bifurcation. <b>B</b>) Regional atherosclerosis in the aortic arch. The percentage of positive stained area with respect to total analyzed aortic area. Group sizes: n = 12 P2Y₆ WT mice; n = 13 P2Y₆ KO mice.</p

PK/PD Disconnect Observed with a Reversible Endothelial Lipase Inhibitor

Screening of a small set of nonselective lipase inhibitors against endothelial lipase (EL) identified a potent and reversible inhibitor, <i>N</i>-(3-(3,4-dichlorophenyl)­propyl)-3-hydroxy-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxamide (<b>5</b>; EL IC₅₀ = 61 nM, EL_HDL IC₅₀ = 454 nM). Deck mining identified a related hit, <i>N</i>-(3-(3,4-dichlorophenyl)­propyl)-4-hydroxy-1-methyl-5-oxo-2,5-dihydro-1<i>H</i>-pyrrole-3-carboxamide (<b>6a</b>; EL IC₅₀ = 41 nM, EL_HDL IC₅₀ = 1760 nM). Both compounds were selective against lipoprotein lipase (LPL) but nonselective versus hepatic lipase (HL). Optimization of compound <b>6a</b> for EL inhibition using HDL as substrate led to <i>N</i>-(4-(3,4<b>-</b>dichlorophenyl)­butan-2-yl)-1-ethyl-4-hydroxy-5-oxo-2,5-dihydro-1<i>H</i>-pyrrole-3-carboxamide (<b>7c</b>; EL IC₅₀ = 148 nM, EL_HDL IC₅₀ = 218 nM) having improved PK over compound <b>6a</b>, providing a tool molecule to test for the ability to increase HDL-cholesterol (HDL-C) levels in vivo using a reversible EL inhibitor. Compound <b>7c</b> did not increase HDL-C in vivo despite achieving plasma exposures targeted on the basis of enzyme activity and protein binding demonstrating the need to develop more physiologically relevant in vitro assays to guide compound progression for in vivo evaluation