Structural and Thermodynamic Characterization of ProteinâLigand

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Structural and Thermodynamic Characterization of Protein-Ligand Interactions formed between Lipoprotein-Associated Phospholipase A2 and Inhibitors Qiufeng Liu, Xinde Chen, Wuyan Chen, Xiao-Jing Yuan, Haixia Su, Jianhua Shen, and Ye-Chun Xu J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00282 • Publication Date (Web): 14 Apr 2016 Downloaded from http://pubs.acs.org on April 15, 2016

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Journal of Medicinal Chemistry

Structural and Thermodynamic Characterization of Protein−Ligand Interactions formed between Lipoprotein-Associated Phospholipase A2 and Inhibitors Qiufeng Liu||#‡, Xinde Chen⊥‡, Wuyan Chen||, Xiaojing Yuan||, Haixia Su||, Jianhua Shen⊥, and Yechun Xu||* ||

CAS Key Laboratory of Receptor Research, ⊥State Key Laboratory of Drug Research, Drug Discovery and Design Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (CAS), Shanghai 201203, China.

#

School of Life Science and Technology, ShanghaiTech University, Shanghai 200031, China

KEYWORDS: Lp-PLA2-inhibitor complex, crystal structure, thermodynamic characterization, binding pocket, and interaction mode.

ABSTRACT: Lipoprotein-associated phospholipase A2 (Lp-PLA2) represents a promising therapeutic target for atherosclerosis and Alzheimer’s disease. Here we reported the first crystal structure of Lp-PLA2 bound with two reversible inhibitors and the thermodynamic characterization of complexes. High rigidity of Lp-PLA2 structure and similar binding modes of inhibitors with completely different scaffolds are revealed. It not only provides the molecular basis for inhibitory activity but also sheds light on the essential features of Lp-PLA2 recognition with reversible inhibitors.

INTRODUCTION Lipoprotein-associated phospholipase A2 (Lp-PLA2, also named the group-VIIA PLA2), was first characterized as the platelet-activating factor (PAF) acetylhydrolase.1 Lp-PLA2 belongs to the serine phospholipases superfamily.2 The crystal structure revealed that Lp-PLA2 has a classic lipase α/β-hydrolase fold and contains a catalytic triad of S273, H351, and D296.3 It preferentially cleaves the ester bond at the sn-2 position of phospholipid substrates with a short sn-2 chain, such as PAF and the oxidation products of phosphatidylcholine. In human plasma, the vast majority of Lp-PLA2 is bound to low-density lipoprotein (LDL) particles where the enzyme hydrolyzes the oxidatively modified phosphatidylcholine. The resulted oxidized non-esterified fatty acids and lysophosphatidylcholine activate the inflammatory and pro-atherogenic pathways in the vascular wall.4, 5 Acting as a pro-inflammatory lipid-modifying enzyme and due to its high expression within the necrotic core and apoptotic macrophages in human coronary plaques, Lp-PLA2 serves as one of the lead anti-inflammatory atherosclerosis drug targets.5 Several lines of evidence suggest Lp-PLA2 as a cardiovascular risk marker and a predictor of ischemic stroke.6 In addition, elevated Lp-PLA2 is associated with risk of developing dementia7 and aggressive prostate cancer.8 It was also reported that inhibition of Lp-PLA2 has beneficial effects

against Alzheimer’s disease (AD) and diabetic macular edema (DME).9, 10 Owing to the interest in Lp-PLA2 as a promising therapeutic target, several classes of Lp-PLA2 inhibitors have been reported.11-18 GlaxoSmithKline (GSK) is the pioneer and leader in this field. The Lp-PLA2 inhibitors in clinical trials belong to GSK. Among them, darapladib,12 was once regarded as a potential first in class drug against the inflammatory component of atherosclerosis, however it missed its primary end points in two mega phase-III trials focusing on coronary heart disease.19,20 The other Lp-PLA2 inhibitor rilapladib is in Phase II trials for the treatment of AD. We recently reported a series of imidazo[1,2a]pyrimidine derivatives as novel Lp-PLA2 inhibitors with excellent pharmacokinetic profiles and potent inhibitory efficacy in vivo.17 And our continued chemical efforts based on the structure−activity relationship (SAR) of imidazo[1,2-a]pyrimidine derivatives resulted in novel LpPLA2 inhibitors containing a pyrimidone scaffold.18 The best compound from these pyrimidone derivatives significantly inhibited retinal thickening in STZ-induced diabetic Sprague−Dawley rats as a model of DME after oral dosing for 4 weeks.18 In contrast to the fact that a couple of Lp-PLA2 inhibitors have run into clinical trials, the inhibitory mechanism and in particular the binding mode of these inhibi-

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tors to this enzyme at the molecular level remains unknown. The crystal structure of human Lp-PLA2 in complex with any reversible inhibitor is not available as yet, albeit the structures of Lp-PLA2 in a ligand-free form and covalently bound with organophosphorus (OP) agents including paraoxon, diisopropylfluorophosphate (DFP), sarin, soman, and tabun were solved a few years ago.3, 21 SAR interpretation and structure-based design of LpPLA2 inhibitors are thus hampered. In the present study, the first crystal structure of LpPLA2 in complex with reversible inhibitor was reported. We determined crystal structures of human Lp-PLA2 bound with two potent inhibitors, darapladib and 4-((4(4-chloro-3-(trifluoromethyl)phenoxy)-3,5difluorobenzyl)-oxy)-6-(1,1-dioxidothiomorpholino)-1methylpyrimidin-2(1H)-one (compound 1). Compound 1 is a novel inhibitor of Lp-PLA2 with a pyrimidone scaffold. The design and synthesis of compound 1 have been reported in our recent publication in which it is named with 13p.18 The reported IC50 of darapladib and compound 1 against recombinant human Lp-PLA2 are 0.7 and 1.7 nM, respectively (Table 1)18. Notably, the scaffold as well as the size of two inhibitors is significantly different (Fig. 1). Moreover, isothermal titration calorimetry (ITC) experiments were carried out to determine thermodynamic properties of Lp-PLA2 binding with two inhibitors. On the basis of the structural information of the complexes combined with the thermodynamic data, the binding mechanism and detailed interactions of reversible inhibitors with Lp-PLA2 were revealed for the first time, providing valuable information for further design and development of novel Lp-PLA2 inhibitors.

Figure 1. 2D structures of two Lp-PLA2 inhibitors. RESULTS To determine the binding mode of reversible inhibitor to Lp-PLA2, we soaked darapladib and compound 1 into the apo crystals of Lp-PLA2 with space group of C121. Crystals of Lp-PLA2 in complex with darapladib and 1 were diffracted to 2.7 and 2.37 Å, respectively. There are two crystallographically independent Lp-PLA2/inhibitor complexes in the solved structures. The details of data correction and refinement of two crystal structures are summarized in Table S1 in Supporting Information. The Binding Pocket of Inhibitors. As expected, the overall structure of Lp-PLA2 seen in two complex structures is similar to that shown in the previously determined structures.3, 21 Two inhibitors fitted very well into the ordered electron-density difference maps (Figure S1), implying that stable complexes of Lp-PLA2 with two inhibitors were formed. The binding pocket of the inhibitor mainly relies on a gorge that initiates from the catalytic

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triad and extends to two solvent-accessible α-helices (residues 114-126 and 362-369) which are proved to participate in binding to the lipoprotein particles LDL or/and HDL (Figures S1 and 2).3, 22, 23 According to the ligand sites predicted by the graphic program PyMOL,24 residues build up the molecular surface of the binding pocket include L107, F110, L111, L121, F125, G152, L153, G154, A155, L159, Y160, H272, S273, F274, G275, W298, F322, H351, Q352, A355, F357, L369, and L371 (Figure 3A). Two catalytic residues S273 and H351 are directly involved into construction of the pocket. A subtle difference exists between the binding pockets of two complexes. In the case of darapladib, one more residues, K370, is included, while S185 and D356 are added in the pocket of compound 1. Both inhibitors fully occupied the pocket and their binding location is overlapped well (Figures 2 and 3).

Figure 2. The crystal structures of Lp-PLA2 in complex with darapladib (A, B) and compound 1 (C, D). The electrostatic surface of Lp-PLA2 is shown from the same view in A and C. Two inhibitors, darapladib (green) and 1(yellow), and the catalytic triad (S273, H351 and D296) were shown in stick. B and D is the enlarged view of the inhibitor at the binding pocket shown in A and C, respectively. Interactions between Inhibitors and Lp-PLA2. As shown in Figure 2, two inhibitors nestled into the binding gorge except that the diethylamine group of darapladib projects out of the active site toward the protein surface. The distance between the nitrogen of this group and the backbone carbonyl oxygen of F110 is 4.3 Å (Figures 2B and 3B), indicating that electrostatic interactions occur especially after the amine group is protonated. Beside this, the carbonyl oxygen of the cyclopenta-pyrimidone ring of darapladib simultaneously makes two hydrogen bonds (H-bonds) with the backbone amide of F274 and L153, with a distance of 2.8 and 2.6 Å, respectively (Figure 3B). These two residues act as the oxyanion hole of the enzyme to stabilize the negative charge of a tetrahedral intermediate of the esterolysis reaction or H-bond to the double bonded phosphor-oxygen of OPs.3, 21 In contrast to


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Figure 3. Interactions between Lp-PLA2 and two inhibitors. (A) Residues contributed to the molecular surface of the ligand binding pocket in the complex of Lp-PLA2/darapladib (green) and Lp-PLA2/1 (yellow). (B, C) The H-bonding and hydrophobic interactions formed between Lp-PLA2 and darapladib (B) or 1 (C). Residues are shown in stick and inhibitors are rendered in ball and stick. Distances of two polar atoms which form a H-bond are labeled in Å.

Table 1. Thermodynamic Data of Two Inhibitors Binding to Lp-PLA2 Inhibitors darapladib a

1

Kd (M) -8 (4.97±0.44)×10 -7

(1.85±0.18)×10

∆G (KJ/mol) -42.29±0.22

∆H (KJ/mol) -53.67±1.12

-T∆S (KJ/mol) 11.38±1.32

IC50 (nM) 18 0.7

-38.99±0.24

-41.43±0.04

2.45±0.27

1.7

18

and F322) are added while L111 and L369 are missed (Table S2). Notably, F357 contributes 9 hydrophobic-interacting atom pairs within 4 Å to the terminal benzene ring of compound 1, resulting in an edge to face π-π interactions formed between these two aromatic rings (Table S2 and Figure 3C).

Figure 4. Representative ITC results and fitting curve of darapladib(A) and 1(B)binding to Lp-PLA2 in solution.

few H-bonds, multiple residues like L371, L369, F357, A355, Q352, H351, W298, S273, L159, G154, L153, L121, L111, and F110, have hydrophobic interactions with darapladib (Figure 3B and Table S2). Among them, L371, F357, W298, L153, L111, and F110 contribute the major interactions with darapladib (Table S2). The Lp-PLA2/1 complex shares many features of protein-ligand interactions of Lp-PLA2 with darapladib, including two H-bonds with the oxyanion hole and hydrophobic interactions with multiple residues (Figure 3C). In addition to two conserved H-bonds, a third and unique H-bond is formed between the oxygen linked to the pyrimidone ring and the side chain of Q352. Residues participating in hydrophobic interactions with compound 1 are almost identical to those presented in the complex of LpPLA2/darapladib, except that two more residues (G152

Rigid Structure of Lp-PLA2. There are two copies of complex in the asymmetric unit of both structures. An overall RMSD of two copies is 0.37 and 0.36 Å for Cα atoms, and 0.70 and 0.61 Å for all atoms of the enzyme in Lp-PLA2/darapladib and Lp-PLA2/1 complexes, respectively. This shows that protein conformations of two copies are virtually identical. The details about RMSD calculations are described in Supporting Information. Subsequently, structure alignment of chain A of the apo enzyme, five OP-bound complexes and the present two complexes were performed. The calculated Cα RMSDs of every two of these eight structures were listed in Table S3. All values are smaller than 0.5 Å, suggesting that the framework of Lp-PLA2 in eight structures is virtually unchanged. In another word, the present two inhibitorbound complexes are similar to the previously reported structures including the apo form.21 It is not surprising that OP-bound complexes are very similar to the ligandfree form because the size of OPs is very small and they only covalently linked to S273. The size of darapladib and 1 is pretty large as compared to OPs and their scaffold is quite different, however, the Cα RMSDs of two complex structures to the apo form of Lp-PLA2 are 0.23 and 0.17 Å, respectively (Table S3). Moreover, the RMSDs of all heavy atoms of Lp-PLA2 between every two structures are less than 1 Å (Table S4), meaning that conformational changes of the side-chains among eight structures are small too.



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Accordingly, the structure of Lp-PLA2 is very rigid and the binding of darapladib and 1 into Lp-PLA2 does not change the overall conformation of the protein and even the side-chain of residues at the binding pocket (Figure 3A). Thermodynamic and Binding Properties. To gain insight into the binding properties of inhibitors with LpPLA2, the thermodynamic behavior of darapladib and 1 binding to the enzyme in solution were investigated by an ITC study. The averaged dissociation constant (Kd), binding free energy (ΔG), enthalpy (ΔH), and entropy term (−TΔS) resulted from three independent ITC measurements on each inhibitor are listed in Table 1. Figure S3 displays the representative ITC results and fitting curve of each inhibitor binding to Lp-PLA2. In support of the multiple protein-inhibitor interactions depicted in Figures 3B and 3C, the ΔH for darapladib and 1 reaches -53.67 and 41.43 KJ/mol, respectively. However, the ΔG of two compounds is close to each other. The reason for this is that the entropy loss is severer in the complex of LpPLA2/darapladib. The higher flexibility of darapladib compared to compound 1 is responsible for its entropy loss. Therefore, the binding of two inhibitors to Lp-PLA2 is predominantly driven by the enthalpic term, while the influence of entropy on the binding is negative. The resulting similar binding free energies of two inhibitors to the enzyme agree well with the proximal values of two IC50. DISCUSSION Even though the definitive roles of Lp-PLA2 in various diseases are not completely addressed as yet, there is a need for the discovery of potent and selective Lp-PLA2 inhibitors serving as chemical probes to explore pathological mechanism of Lp-PLA2 or novel medicinal agents for effective treatments of diseases. Given the important contribution of protein-ligand complex structure to drug design, we herein presented the first complex structure of Lp-PLA2 bound with a potent reversible inhibitor. The structures of Lp-PLA2 in complex with darapladib and 1 not only allow us to clearly visualize how a reversible inhibitor blocks the activity of the enzyme at the atomic level but also identify the binding pocket as well as key interactions of inhibitors with the enzyme. The previously determined structures of Lp-PLA2 reveal that an open channel or gorge connects the catalytic triad and the lipoprotein particles binding interface. It has been speculated that substrate like PAF accesses the active site through this channel.3 However, it is not clearly yet whether small molecules inhibit the hydrolysis activity of the enzyme by interacting with this channel. Our complex structures show that both darapladib and compound 1 are embedded into the channel, not only occupied the active site but also the entrance of the substrate from the lipoprotein (Figure 2). The inhibitor binding pocket is fairly open and large so that Lp-PLA2 can accommodate a large inhibitor like darapladib and a compound with completely different scaffold such as 1. Another feature is that most residues involved in the binding

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pocket are amino acid with a hydrophobic side-chain (Figure 3A). As a result, the hydrophobic interactions play a major role in the recognition of Lp-PLA2 with these two inhibitors. Notably, the completely hydrophobic subpocket constructed by residues L371, F357, L369, F125, L121, and L111 perfectly matches with the trifluoromethyl substituted benzene ring of both darapladib and 1 (Figure 3A). Thirdly, to our surprise, the binding of both reversible and irreversible inhibitors has almost no effect on the conformation of the binding pocket. If it is too small for the OPs, but the binding of larger inhibitors such as darapladib still could not induce Lp-PLA2 to adopt a new conformation. To our experience, such a high rigidity of the binding pocket as well as the entire protein is not often observed with enzymes. Further development of Lp-PLA2 inhibitor would benefit by utilization of such a structural rigidity.

Figure 5. The binding modes of darapladib (green) and 1 (yellow) to Lp-PLA2 resulted from the superimposition of two complex structures. The black dash lines represent Hbonds. Important pharmacophores are indicated by shadow regions colored grey, salmon and purple, respectively.

The superimposition of two complex structures revealed that the bounded darapladib and 1 are greatly overlapped although their scaffolds are significantly different (Figure 5). The pyrimidone ring of two inhibitors, the fluorobenzyl sulfane group of darapladib and the 3,5difluorobenzyl group of 1, and the trifluoromethyl substituted benzene ring of darapladib and the 4-chloro-3(trifluoromethyl)phenoxy group of 1, were superimposed well, respectively. As a consequence, two inhibitors share common features in binding to Lp-PLA2 and have conserved interactions with the enzyme, which include two H-bonds with the amide groups of F274 and L153 and hydrophobic interactions with multiple residues. The major difference between two inhibitors is the diethylamine group of darapladib, a corresponding group of which is lacking in 1. The protonated nitrogen of this diethylamine group forms electrostatic interactions with the mainchain of F110 (Figures 2B and 3B), might increasing the binding affinity of darapladib to Lp-PLA2. The existence of such an electrostatic interaction was once speculated on the basis of SAR exploration of a series of darapladib analogues by replacing the amide group of darapladib with a triazole ring,16 which is proved in this study.


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The similar binding mode of two inhibitors and the rigid binding pocket as discussed above make it rational to construct a general pharmacophore model of Lp-PLA2 inhibitors. H-bond(s) with two oxyanion-hole residues and hydrophobic interactions with multiple residues in particular with L371, L357, W298, L153, and F110, are central to this model (Figure 5). While complex structures provide the structure basis for the inhibitory activities of darapladib and 1, thermodynamic properties of two inhibitors binding to Lp-PLA2 via ITC measurements was pursued as a further step to explore the protein-ligand binding properties. The data shown in Table 1 reveal that the binding of two inhibitors to Lp-PLA2 is driven by the enthalpic effects, which is consistent with the multiple protein-ligand interactions revealed by two complex structures. In light of the rigid conformation of the binding pocket, the higher flexibility of daralpladib leads to a severer entropy loss as compared to 1. Therefore, in conjunction with the structural information, the thermodynamic study gets deep insight into the binding properties of protein-inhibitor complex and serves as an excellent approach to rationalize structure and energy relationship of protein-inhibitor interactions. In summary, structural investigation into the LpPLA2/darapladib and Lp-PLA2/1 complexes identified a fairly open, large, relatively hydrophobic, and rigid binding pocket. Key interactions between inhibitors and LpPLA2 were revealed. In addition, structural analysis shows that the conformation of the protein and the ligand binding pocket as well are very rigid. The conserved interaction mode of two inhibitors with the enzyme together with the highly rigid binding pocket leads us to propose a general pharmacophore model of Lp-PLA2 inhibitors. Moreover, ITC experiments revealed that the binding of these two inhibitors to Lp-PLA2 is driven by the enthalpic effects while the influence of entropy on the binding is negative. Therefore, the structural and thermodynamic characterization of two potent inhibitors binding to LpPLA2 not only reveal the molecular basis of inhibitor activities but also provide valuable information for SAR exploration and further optimization of inhibitors or de novo design of novel inhibitors. We believe the results presented here are instructive for the further development of Lp-PLA2 inhibitors.

EXPERIMENTAL Compounds. Darapladib was purchased commercially (MedChem Express). Compound 1 was synthesized. The procedure to obtain this compound has been reported in the previous study.18 The purity of two compounds was >95% as confirmed by HPLC. Crystal Structure Determination. Production of the recombinant human Lp-PLA2 (47-429 aa) followed the protocols of Uttamkumar Samanta et al21 with certain modifications. The details of protein expression and purification are presented in Supporting Information. The purified proteins were concentrated to 4 mg/ml for crystallization. Crystallization of Lp-PLA2 was carried out by mixing a solution of the protein with an equal volume of

precipitant solution (0.1 M MOPS pH 6.6, 0.4 M Li2SO4, 27% (w/v) (NH4)2SO4, 1 M Na-Ac, 1.4% (v/v) 1,4butanediol). Crystals were obtained by the vapourdiffusion method in hanging drops at 20°C. The proteininhibitor complex crystal was prepared by soaking crystals of apo Lp-PLA2 into the reservoir solution containing 0.2 mM inhibitor overnight. Subsequently, crystals were directly flash frozen in liquid nitrogen. Data were collected at 100 K on beamline at the Shanghai Synchrotron Radiation Facility (SSRF), and were processed with the XDS software packages.25 The structures were solved by molecular replacement, using the program PHASER26 with the search model of PDB code 3D59.3 The structures were refined with the simulated-annealing protocol implemented in the program PHENIX.27 With the aid of the program Coot,28 compounds and water molecules were fitted into to the initial Fo-Fc map. The complete statistics, as well as the quality of the solved structures, are shown in Table S1. Thermodynamic Data Determination. The ITC measurements were conducted in the gel filtration buffer at 30℃ with an iTC200 instrument (Microcal, Malvern). The details of each titration are described in Supporting Information. Titrations were run in triplicate to ensure reproducibility. In all the cases a single binding site mode was employed and a nonlinear least-squares algorithm was used to obtain best-fit values of the stoichiometry (n), change in enthalpy (∆H), and binding constant (K). Thermodynamic parameters were subsequently calculated with the formula ∆G = ∆H–T∆S = -RTlnK, where T, R, ∆G, and ∆S are the experimental temperature, the gas constant, the changes in free energy, and entropy of binding, respectively.

ASSOCIATED CONTENT PDB ID Codes 5I9I (Lp-PLA2/darapladib) and 5I8P (Lp-PLA2/compound 1). Authors will release the atomic coordinates and experimental data upon article publication. Supporting Information. Figures S1-S2 , Table S1-S4, RMSD calculations, Protein expression and purification, and ITC measurements. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * Y.X.: email, [email protected], phone, +86-21-50801267.

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally.

Funding Sources Funding was provided by the CAS-Novo Nordisk Great Wall Professorship to Y.X., the National Natural Science Foundation of China (Grant No. 81422047, 81402850 and 81502987).



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Notes The authors declare no competing financial interest.

ABBREVIATIONS Lp-PLA2, lipoprotein-associated phospholipase A2; PAF, the platelet-activating factor; LDL, low-density lipoprotein; HDL, high-density lipoprotein; AD, Alzheimer’s disease; SAR, structure-activity relationship; OP, organophosphorus; DFP, diisopropylfluorophosphate; H-bond, hydrogen bond; ITC, isothermal titration calorimetric; RMSD, root mean square deviation.

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14. Hu, Y.; Lin, E. C.; Pham, L. M.; Cajica, J.; Amantea, C. M.; Okerberg, E.; Brown, H. E.; Fraser, A.; Du, L.; Kohno, Y.; Ishiyama, J.; Kozarich, J. W.; Shreder, K. R. Amides of 4-hydroxy-8methanesulfonylamino-quinoline-2-carboxylic acid as zincdependent inhibitors of Lp-PLA(2). Bioorg. Med. Chem. Lett. 2013, 23, 1553-1556. 15. Nagano, J. M.; Hsu, K. L.; Whitby, L. R.; Niphakis, M. J.; Speers, A. E.; Brown, S. J.; Spicer, T.; Fernandez-Vega, V.; Ferguson, J.; Hodder, P.; Srinivasan, P.; Gonzalez, T. D.; Rosen, H.; Bahnson, B. J.; Cravatt, B. F. Selective inhibitors and tailored activity probes for lipoproteinassociated phospholipase A(2). Bioorg. Med. Chem. Lett. 2013, 23, 839-843. 16. Wang, K.; Xu, W.; Zhang, W.; Mo, M.; Wang, Y.; Shen, J. Triazole derivatives: a series of Darapladib analogues as orally active Lp-PLA2 inhibitors. Bioorg. Med. Chem. Lett. 2013, 23, 2897-2901. 17. Chen, X.; Xu, W.; Wang, K.; Mo, M.; Zhang, W.; Du, L.; Yuan, X.; Xu, Y.; Wang, Y.; Shen, J. Discovery of a novel series of imidazo[1,2a]pyrimidine derivatives as potent and orally bioavailable lipoprotein-associated phospholipase A2 inhibitors. J. Med. Chem. 2015, 58, 8529-8541. 18. Chen, X.; Wang, K.; Xu, W.; Ma, Q.; Chen, M.; Du, L.; Mo, M.; Wang, Y.; Shen, J. Discovery of potent and orally active lipoproteinassociated pospholipase A2 (Lp-PLA2) inhibitors as a potential therapy for diabetic macular edema. J. Med. Chem. 2016, 59, 26742687. 19. Investigators, S.; White, H. D.; Held, C.; Stewart, R.; Tarka, E.; Brown, R.; Davies, R. Y.; Budaj, A.; Harrington, R. A.; Steg, P. G.; Ardissino, D.; Armstrong, P. W.; Avezum, A.; Aylward, P. E.; Bryce, A.; Chen, H.; Chen, M. F.; Corbalan, R.; Dalby, A. J.; Danchin, N.; De Winter, R. J.; Denchev, S.; Diaz, R.; Elisaf, M.; Flather, M. D.; Goudev, A. R.; Granger, C. B.; Grinfeld, L.; Hochman, J. S.; Husted, S.; Kim, H. S.; Koenig, W.; Linhart, A.; Lonn, E.; Lopez-Sendon, J.; Manolis, A. J.; Mohler, E. R., 3rd; Nicolau, J. C.; Pais, P.; Parkhomenko, A.; Pedersen, T. R.; Pella, D.; Ramos-Corrales, M. A.; Ruda, M.; Sereg, M.; Siddique, S.; Sinnaeve, P.; Smith, P.; Sritara, P.; Swart, H. P.; Sy, R. G.; Teramoto, T.; Tse, H. F.; Watson, D.; Weaver, W. D.; Weiss, R.; Viigimaa, M.; Vinereanu, D.; Zhu, J.; Cannon, C. P.; Wallentin, L. Darapladib for preventing ischemic events in stable coronary heart disease. N. Engl. J. Med. 2014, 370, 1702-1711. 20. Mullard, A. GSK's darapladib failures dim hopes for antiinflammatory heart drugs. Nat. Rev. Drug Discovery 2014, 13, 481-482. 21. Samanta, U.; Kirby, S. D.; Srinivasan, P.; Cerasoli, D. M.; Bahnson, B. J. Crystal structures of human group-VIIA phospholipase A2 inhibited by organophosphorus nerve agents exhibit non-aged complexes. Biochem. Pharmacol. 2009, 78, 420-429. 22. Cao, J.; Hsu, Y. H.; Li, S.; Woods, V. L.; Dennis, E. A. Lipoproteinassociated phospholipase A2 interacts with phospholipid vesicles via a surface-disposed hydrophobic α-helix. Biochemistry 2011, 50, 53145321. 23. Cao, J.; Hsu, Y. H.; Li, S.; Woods, V. L.; Dennis, E. A. Structural basis of specific interactions of Lp-PLA2 with HDL revealed by hydrogen deuterium exchange mass spectrometry. J. Lipid Res. 2013, 54, 127-133. 24. The PyMOL Molecular Graphics System, Version 1.2 Schrödinger, LLC. 25. Kabsch, W. Xds. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 125-132. 26. McCoy, A. J.; Grosse-Kunstleve, R. W.; Adams, P. D.; Winn, M. D.; Storoni, L. C.; Read, R. J. Phaser crystallographic software. J. Appl. Crystallogr. 2007, 40, 658-674. 27. Adams, P. D.; Afonine, P. V.; Bunkoczi, G.; Chen, V. B.; Davis, I. W.; Echols, N.; Headd, J. J.; Hung, L. W.; Kapral, G. J.; GrosseKunstleve, R. W.; McCoy, A. J.; Moriarty, N. W.; Oeffner, R.; Read, R. J.; Richardson, D. C.; Richardson, J. S.; Terwilliger, T. C.; Zwart, P. H. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 213-221. 28. Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K. Features and development of Coot. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 486-501.


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Table of Contents



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Figure 1. 2D structures of two Lp-PLA2 inhibitors. 66x26mm (300 x 300 DPI)


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Figure 2. The crystal structures of Lp-PLA2 in complex with darapladib (A, B) and compound 1 (C, D). The electrostatic surface of Lp-PLA2 is shown from the same view in A and C. Two inhibitors, darapladib (green) and 1(yellow), and the catalytic triad (S273, H351 and D296) were shown in stick. B and D is the enlarged view of the inhibitor at the binding pocket shown in A and C, respectively. 141x132mm (300 x 300 DPI)



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Figure 3. Interactions between Lp-PLA2 and two inhibitors. (A) Residues contributed to the molecular surface of the ligand binding pocket in the complex of Lp-PLA2/darapladib (green) and Lp-PLA2/1 (yellow). (B, C) The H-bonding and hydrophobic interactions formed between Lp-PLA2 and darapladib (B) or 1 (C). Residues are shown in stick and inhibitors are rendered in ball and stick. Distances of two polar atoms which form a H-bond are labeled in Å. 211x86mm (300 x 300 DPI)


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Figure 4. Representative ITC results and fitting curve of darapladib(A) and 1(B)binding to Lp-PLA2 in solution. 169x116mm (300 x 300 DPI)



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Figure 5. The binding modes of darapladib (green) and 1 (yellow) to Lp-PLA2 resulted from the superimposition of two complex structures. The black dash lines represent H-bonds. Important pharmacophores are indicated by shadow regions colored grey, salmon and purple, respectively. 127x90mm (300 x 300 DPI)


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TOC graphic 96x55mm (300 x 300 DPI)


Structural and Thermodynamic Characterization of ProteinâLigand

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