Computational Investigation of Darapladib and Rilapladib Binding to Platelet Activating Factor Receptor . A Possible Mechanism of Their Involvement in Atherosclerosis

Platelet Activating Factor (PAF), the most potent inflammatory mediator, is involved in a wide range of pathophysiological actions. PAF signal transduction is mediated through PAF receptors (PAFR) that are coupled with several isoforms of G-proteins. PAF hydrolysis is mediated through specific enzymes clustered as PAF acetylhydrolases (PAF-AH). The plasma isoform is known as lipoprotein-associated PLA2 (Lp-PLA2), and is considered a marker, or a mediator in the mechanism of atherosclerosis. Darapladib and rilapladib are selective Lp-PLA2 inhibitors. They are, thus, proposed as a novel therapeutic approach for cardiovascular disease (CVD). The data derived from the computational methods used in this paper suggest that darapladib and rilapladib are potential PAFR antagonists, predicted to bind inside the PAF-binding site with a comparable binding affinity to the endogenous agonist (ΔG = –11.1 Kcal mol). Darapladib (ΔG = –10.6 Kcal mol) exhibited a higher affinity than rilapladib (ΔG = –8.2 Kcal mol). The fact that darapladib down-regulates PAFR expression, while PAFR inhibitors down-regulate the expression of CD36, could be the biochemical explanation in the observed necrotic core reduction, both in animals and humans. The reported results in conjunction with bibliographical data lead to the hypothesis that the involvement of darapladib and rilapladib in atherosclerosis could be through direct inhibition of PAF activity as well as modification of PAF metabolism.


Platelet Activating Factor 1.Platelet Activating Factor General Data
Platelet Activating Factor (PAF), a phosphoglycerylether lipid (Scheme 1), is the most potent inflammatory mediator involved in a wide range of pathophysiological actions.PAF is a considered a cell-to-cell messenger acting both intercellular and intracellular (Antonopoulou, Nomikos, Karantonis, Fragopoulou, & Demopoulos, 2008).Even though the majority of ether lipids have been replaced with their esterified analogues during evolution, PAF and some minor phosphoglycerylether lipids were conserved in various organisms due to their important biological roles (Kulikov & Muzya, 1997).While the term PAF was initially attributed to 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (Demopoulos, Pinckard, & Hanahan, 1979), today it is clear that PAF is a member of a large family or structurally related phospholipids with similar pathophysiological activities.These molecules are produced due to enzymatic and chemical oxidation and their main common structural feature is the short chain at sn-2 position (Montrucchio, Alloatti, & Camussi, 2000).

Platelet Activating Factor Metabolism
PAF is synthesized by two distinct pathways, namely the "remodeling" and "de novo" (Snyder, 1995), while it is hydrolyzed by PAF acetylhydrolase (PAF AH).PAF AH is a PLA2 belonging to groups VII and VIII.The plasma isoform, also known as lipoprotein-associated PLA2 (Lp-PLA2, EC 3.1.1.47),has been classified as group VIIA PLA2 (PLA2G7), is calcium independent and circulates bound with LDL and HDL.Two intracellular PAF AHs, namely PAF AH Ib and PAF AH II, have been also characterized (Snyder, 1995;Tselepis & John Chapman, 2002).Increased Lp-PLA2 activity is associated with increased risk of cardiac events, but it is not known whether Lp-PLA2 is a causative agent.
1.1.3Platelet Activating Factor Receptor PAF signal transduction is mediated through PAF receptors (PAFR).PAFR is coupled with several isoforms of G-proteins.The type of cell and ligand determines the G-protein isoform(s) activated by PAFR each time.The ability of PAF to stimulate distinct signaling pathways via multiple G-proteins may explain the diverse biological responses of human cells to it (Ishii & Shimizu, 2000;Honda, Ishii, & Shimizu, 2002).PAFR are "serpentine receptors", with a seven a-helical domains that wave in and out of the plasma membrane seven times.PAFR have been found in all the blood cells and several tissue and organ cells.Soon after the cloning of PAFR, the expression levels of PAFR mRNA in various tissues and organs were determined.
The internalization and desensitization of PAFR is regulated through a phosphorylation site consisted of serine and threonine residues in the C-terminal cytoplasmic tail.PAFR is modified posttranslational by disulfide bonding at C90-C173 and glycosylation at N169, and modifications are necessary for the surface exposure of PAFR (Prescott, Zimmerman, Stafforini, & McIntyre, 2000;Honda et al., 2002).PAF and PAF-like lipids bearing a short oxidized acyl chain at the sn-2 position readily bind to PAFR, but PAFR also interacts with components of the bacterial wall, such as lipopolysaccharides (LPS) (Nakamura et al., 1992) and phosphorylcholine (Cundell, Gerard, Gerard, Idanpaan-Heikkila, & Tuomanen, 1995).These interactions are thought to be an alternative recognition system for innate immunity inducing inflammatory responses to the immune cells.
In humans, the PAFR mRNA is most abundant in neutrophils, monocytes, placenta, lung, dendritic cells and endothelial cells and is the same isoform for all the cell types.The PAFR was initially cloned from the guinea-pig lung by functional expression in Xenopus laevis oocytes.Subsequently, the cloning of human, rat, mouse, porcine, bovine and caprine PAFR was reported (Ishii & Shimizu, 2000;Honda et al., 2002).

Platelet Activating Factor and Atherosclerosis
PAF interplays in critical stages of atherogenesis including thrombosis, inflammation and oxidation.PAF promotes oxidation by stimulating human monocytes/macrophages and neutrophils to produce superoxide anions and hydrogen peroxide that cause LDL oxidation, creating a positive feedback effect, as PAF itself is produced during LDL oxidation.Lp-PLA2 protects LDL against the production and activity of Ox-LDLs by facilitating hydrolysis of PAF-like lipids (Demopoulos, Karantonis, & Antonopoulou, 2003).
In vitro studies involving PAF-like lipids that are mostly fragmented and/or oxidized sn-2 fatty acyl groups and their hydrolysis products, have shown that these molecules can act both as pro-and anti-inflammatory mediators (Berliner, Leitinger, & Tsimikas, 2009;Feige, Mendel, George, Yacov, & Harats, 2010).These studies though do not take into consideration the fact that lyso-PC and free fatty acids are associated with lipoproteins and other plasma carriers making it difficult to calculate their exact concentration and bioavailability in plasma (Öörni & Kovanen, 2009;Rosenson & Stafforini, 2012).These results are further weakened by the fact that many of the observations for lyso-PC can be attributed to contaminating traces of PAF remaining in the lyso-PC preparations used in the studies (Marathe et al., 2001), and the fact that there is no evidence for a specific lyso-PC receptor.
Several PAF agonists/antagonists have been synthesized and isolated from natural sources (Antonopoulou et al., 2008).Some of them are isolated from Mediterranean foods and when co-administered with cholesterol in animals, were able to significantly reduce the amount of esterified cholesterol in aorta without affecting cholesterol plasma levels, and reduce PAF-induced early atherogenesis (Nomikos, Fragopoulou, & Antonopoulou, 2007), as well to cause regression of the existing plaques.
Darapladib and rilapladib (Scheme 1) are selective Lp-PLA2 inhibitors and produce sustained inhibition of plasma Lp-PLA2 activity.Darapladib is a potent, freely reversible, inhibitor of human Lp-PLA2, with an inhibition constant K i of 0.11 nM (Blackie et al., 2003).Rilapladib is also a potent and reversible inhibitor of human Lp-PLA2 with half maximal inhibitory concentration (IC 50 ) in the range of 0.1 to 10 nM (Patent publication numbers: WO2012080497 A2, WO 2012080497 A3 and US20130267544 A1).
Initially darapladib was tested in vivo in atherosclerosis animal models with promising results, as the medication reduced plaque and necrotic core area in swine (Wilensky et al., 2008), and plaque area and inflammatory burden in mice (Hu et al., 2011;Wang et al., 2011).These results suggest that the drug could move on to Phase II   ΔG = R T lnK d , where R = 1.987 cal K -1 mol -1 and T = 273 K. Visual inspection of the lowest energy and highest populated model of PAFR-rilapladib complex (Figure 2D) reveled that its lower affinity could be attributed to the lack of interaction between the oxoquinolinyl ring and His248/His249 residues.In such a configuration, the position of the phosphate group of PAF is occupied by the methoxyethyl piperidinyl moiety of rilapladib, which provides no hydrogen bonding interactions with either His248 or His249.The oxoquinolinyl ring is hydrogen bonded with Tyr177 and is buried inside the same site of PAF's choline moiety.The 2,3-difluorophenyl ring of rilapladib is stacked above the oxoquinolinyl moiety interacting with Phe98 and Phe152.Finally, the 4-trifluoromethyl biphenyl group is buried a bit deeper inside the hydrophobic channel with respect to the corresponding moiety of darapladib, with the -CF 3 group at the same position of C-10 of PAF's aliphatic chain.

Interaction of Darapladib and Rilapladib With Lp-PLA2 and PAFR
Taken together, the above results suggest that darapladib may be a potential PAFR antagonist, which is predicted to bind inside the PAF-binding site with a comparable binding affinity (ΔG = -10.6Kcal mol -1 ) with respect to the endogenous agonist (ΔG = -11.1 Kcal mol -1 ).Similarly, rilapladib could also serve as a PAFR antagonist, albeit with lower binding affinity (ΔG = -8.2Kcal mol -1 ).The predicted binding modes of both inhibitors with either Lp-PLA2 or PAFR indicate that their trifluromethyl biphenyl moiety is probably interacting within the same binding sites that accommodate the long alkyl chain of PAF, whereas the pyrimidinyl and oxoquinolinyl rings (of darapladib and rilapladib, respectively) interact with the catalytic residues of Lp-PLA2 and the hydrophilic PAF-binding site.

Lp-PLA2 and PAF Involvement in Atherosclerosis
There is a considerable amount of data reporting Lp-PLA2 as a marker of the risk of coronary heart disease (CHD), with controversial results.Even though Lp-PLA2 tends to be considered an independent marker for CVD, recent studies fail to establish its association with CVD event in apparently health subjects and in patients treated with statins that have their cholesterol levels managed (Rosenson & Stafforini, 2012).
According to our proposed theory where PAF is the initial cause of atherosclerosis and plaque formation (Demopoulos et al., 2003), and the literature data, darapladib and rilapladib are likely to inhibit/reduce atherosclerosis and its development process (therefore all its harmful consequences) also through the inhibition/reduction of PAF effects.
The deposition and binding of LDL-Cholesterol in the subintimal space is considered a key factor for the initiation and development of the atherosclerosis.After their binding to proteoglycans the LDL-Cholesterol are oxidativelly modified (ox-LDL) resulting in high concentrations of really potent inflammatory molecules, like PAF and PAF-like lipids.These molecules act through the specific PAF receptor present in almost all the cells involved in atherosclerosis like smooth muscle cells, cardiomyocytes, neutrophils, monocytes-macrophages, eosinophils, and Kupffer cells.It has also been shown that endothelial cells, express PAFR not only on the cell surface, but also in the large endosomal compartment (Montrucchio et al., 2000;Antonopoulou et al., 2008).
In addition, we and others have published (Nomikos, Fragopoulou, & Antonopoulou, 2007) that the PAF-inhibitors usually inhibit the key-PAF biosynthetic enzymes, and, additionally, either inhibit or activate PAF AH, the key-PAF catabolic enzyme.

Darapladib, Rilapladib and PAF Biological Activity as Well as PAF Levels
Therefore, darapladib and rilapladib are likely to inhibit/reduce atherosclerosis and its development process (therefore all its harmful consequences) through the inhibition/reduction of PAF effects, but also through inhibition of PAF biosynthesis that leads to reduced PAF levels, as do statins (Tsantila et al., 2011).
Unfortunately there are no in vitro experimental data concerning the regulation of the two major PAF biosynthetic enzymes by darapladib and rilapladib that could help us understand the mechanism of PAF levels regulation.
On the other hand darapladib and rilapladib are potent specific inhibitors of Lp-PLA 2 , which is considered the main PAF degrading enzyme.The effect of darapladib on PAF levels has only been studied in mice by two different research groups.Hu et al. showed that darapladib reduced plaque area in LDL receptor (LDLR) deficient mice, without affecting the lipid profile or PAF levels of the mice.The intervention reduced as expected Lp-PLA2 activity along with CRP and IL-6 levels and the expression of the inflammatory genes of MCP-1 and VCAM-1 (Hu et al., 2011).The same results were obtained by Wang et al. in Apo-E deficient mice that also had decreased macrophages' content and increased collagen content in the lesions of the darapladib group (Wang et al., 2011).
These controversial results can be explained by suggesting that PAF hydrolysis is not mediated only through Lp-PLA2.The PAF clearance was measured in mice and showed that the majority of the PAF molecules were hydrolyzed in liver and kidney by the intracellular PAF AH, after being transported as intact molecules.The fact that mice have an 8.6 times increased enzymatic activity relative to humans indicates that this is probably the PAF clearance pathway also in humans (Liu et al., 2011).
These observations give rise to the critical question of whether and to what extend Lp-PLA2 is responsible for the clearance of the other highly inflammatory PAF-like molecules present in oxLDL that have a smaller affinity to the enzyme compared to PAF, but are in much higher concentrations (Markakis et al., 2010).

Possible Actions of Darapladib and Rilapladib in Aorta, That Are Explained by PAFR Inhibition
One critical aspect of the in vivo darapladib administration to diabetic and hypercholesterolemic swine, which is not discussed by the authors, is the down-regulation of PAFR expression by 49% compared to control group not receiving the drug (Wilensky et al., 2008).
It is known that the expression of CD36 is directly associated with the uptake of oxLDL by macrophages (Febbraio & Silverstein, 2007;Rios, Gidlund, & Jancar, 2011) and this uptake is not regulated by intracellular levels of cholesterol, leading to continuous uptake of oxLDL and the differentiation of macrophages into foam cells.The oxLDL molecules increase CD36 expression, exerting a positive feedback effect on the expression of its receptor in human and mouse monocytes/macrophages (Feng et al., 2000;Rios, Jancar, Melo, Ketelhuth, & Gidlund, 2008), an effect that is reversed by PAFR antagonists.Moreover treatment of LDL receptor-deficient mice with PAFR antagonists reduced the formation of fatty streaks lesions (Subbanagounder, Leitinger, Shih, Faull, & Berliner, 1999).
PAFR down-regulation with a possible antagonistic effect from darapladib and rilapladib can give an explanation to the reduction of the nectrotic core in darapladib and rilapladib treated animals and humans.The mechanism can involve the reduction of oxLDL uptake from macrophages through CD36, thus inhibiting extensive foam cell formation and subsequently a smaller necrotic core.

PAF Levels and PAF AH, Lp-PLA2 Expression
Several studies have shown that PAF and PAF inhibitors levels can affect PAF AH expression.LPS and PAF stimulate expression of PAF AH via distinct signaling pathways (Howard, Abdel-Al, Ditmyer, & Patel, 2011).In another study involving human non-adherent monocyte-macrophage cells (Mono-Mac 6; MM6) it was found that both PAF and LPS were able to up-regulate the expression of PAF AH in a dose dependent manner.The specific PAFR inhibitor WEB2170 was able to completely block the PAF stimulated up-regulation of PAF AH and also inhibited the PAF AH production in the tested cell line but also in rats (Howard & Olson, 2000), after LPS stimulation.The p38 MAPK inhibitor, SB203580 inhibited by 60% the up-regulation of PAF AH after LPS stimulation while PAF stimulation was not affected.The co-administration of WEB2170 and SB203580 completely abolished PAF AH expression, indicating that the LPS-induced PAF AH mRNA levels present after SB203580 administration are the result of autocrine activation of the PAF receptor due to LPS-stimulated production of PAF, or the fact that LPS acts through the PAFR.

The PAFR Inhibition by Darapladib and Rilapladib Hypothesis, Concerning Hydrolysis Products of Lp-PLA 2
From the above it could be formulated in more detail the aforementioned hypothesis concerning the harmful consequences of the hydrolysis products of Lp-PLA2 as follows: It is well-known that the general body's response is to increase the levels of Lp-PLA2 when the levels of PAF are increased (i.e.PAF affects the gene expression of Lp-PLA2), as shown in a recent study involving 150 patients with CHD and 120 controls that found a strong positive relationship between elevated plasma PAF or Lp-PLA2 levels and the risk of CHD.This study also provided evidence that there was a strong correlation between plasma PAF and Lp-PLA2 levels, and between plasma PAF and Lp-PLA2 and inflammatory factors IL-6 and hs-CRP levels (Zheng et al., 2012).
So it could thus be suggested that: a) Darapladib and rilapladib through reduction of the PAF biological activity (as PAF inhibitors) and PAF levels (as possible inhibitors of PAF biosynthesis) could reduce Lp-PLA2 biosynthesis and prevent the possible adverse effects of Lp-PLA2 (the pro-inflammatory molecules, such as LPC and oxNEFA), and b) In addition, darapladib and rilapladib inhibit also the effects of the existing Lp-PLA2 and so the harmful consequences of the LPC and the oxNEFA.

Conclusion
In conclusion, the combination of computational modeling data presented in this paper with the bibliography on PAFR inhibitors and their effect on atherosclerosis, recommend that more experiments should be done in order to clarify our hypothesis that the implication of darapladib and rilapladib in atherosclerosis is through the inhibition of PAF actions, but moreover through the modification of PAF metabolism.These experiments must include measurement with basic commonly accepted methodology of the effect of darapladib and rilapladib first on PAFR in ex-vivo models of washed platelet aggregation and second on the in vitro PAF biosynthetic enzymes.
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