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Crystal Structures of mPGES-1 Inhibitor Complexes Form a Basis for the Rational Design of Potent Analgesic and Anti-inflammatory Therapeutics John Gately Luz, Stephen Antonysamy, Bradley Condon, Matthew Lee, Aiping Zhang, Marijane Russell, Shawn S. Chang, Dagart Allison, Matthew J. Fisher, Steven L. Kuklish, Xiao-Peng Yu, AShley Sloan, Ryan Backer, Anita Harvey, and Srinivasan Chandrasekhar J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 11 May 2015 Downloaded from http://pubs.acs.org on May 12, 2015

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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Medicinal Chemistry

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Crystal Structures of mPGES-1 Inhibitor Complexes Form a Basis for the Rational Design of Potent Analgesic and Anti-inflammatory Therapeutics

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John Gately Luz#, Stephen Antonysamy, Steven L. Kuklish, Bradley Condon, Matthew Lee, Dagart Allison, Xiao-Peng Yu, Srinivasan Chandrasekhar, Ryan Backer, Aiping Zhang, Marijane Russell, Shawn S. Chang, Anita Harvey, Ashley V. Sloan, Matthew J. Fisher#

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SD: Lilly Biotechnology Center San Diego, 10300 Campus Point Drive, Suite 200, San Diego, California 92121, United States

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# To whom correspondence should be addressed: JGL; Phone, 858-638-8801; Fax, 858-638-889; E-mail, [email protected]; MJF; Phone, 317-276-0632; Fax, 317-276-1417; E-mail, [email protected]

INDI: Lilly Research Laboratories, Lilly Corporate Center, 355 East Merrill Street, Indianapolis, Indiana, 46285, United States

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Abstract

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Microsomal prostaglandin-E synthase 1 (mPGES-1) is an α-helical homotrimeric integral

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membrane inducible enzyme that catalyzes the formation of prostaglandin-E2 (PGE2) from

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prostaglandin-H2 (PGH2). Inhibition of mPGES-1 has been proposed as a therapeutic strategy for

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the treatment of pain, inflammation, and some cancers. Interest in mPGES-1 inhibition can, in

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part, be attributed to the potential circumvention of cardiovascular risks associated with anti-

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inflammatory cyclooxygenase 2 inhibitors (coxibs) by targeting the prostaglandin pathway

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downstream of PGH2 synthesis and avoiding suppression of anti-thrombotic prostacyclin

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production. We determined the crystal structure of mPGES-1 bound to four potent inhibitors in

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order to understand their structure-activity relationships and provide a framework for the rational

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design of improved molecules. In addition, we developed a light scattering-based thermal

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stability assay to identify molecules for crystallographic studies.

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Introduction

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The prostanoid class of fatty acid derivatives, a subclass of the eicosanoinds, is

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comprised of the prostaglandins, prostacyclins, and thromboxanes. The prostanoid synthesis

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pathway is targeted by several nonsteroidal anti-inflammatory drugs (NSAIDs) including

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acetylsalicylic acid, ibuprofen, naproxen, indometacin, and COX-2 inhibitors (coxibs) such as

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rofecoxib and celecoxib. While acetaminophen (aka paracetamol), an analgesic and antipyretic,

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also targets the prostanoid pathway by selectively inhibiting COX-2,1 it is not classified as an

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NSAID due to its limited anti-inflammatory properties. In contrast, acetylsalicylic acid

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irreversibly inhibits COX-1 via covalent modification2 and redirects COX-2 to produce anti-

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inflammatory lipoxins.3-5 The effectiveness of NSAIDs has led to their broad clinical use; 2 ACS Paragon Plus Environment

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however, their side effects have inspired the continued search for equally, or more, effective

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molecules with improved safety profiles.

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The primary adverse side effects of acetylsalicylic acid are gastric bleeding and

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ulceration. Though rare, the potentially fatal Rye’s syndrome is associated with acetylsalicylic

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acid usage especially in children.6 Thus, use of acetylsalicylic acid by children under the age of

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twelve is generally discouraged. In addition, acetylsalicylic acid is contraindicated in gout,

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hemophilia, and glucose-6-phosphate dehydrogenase deficiency.

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developed to avoid the gastric side effects of acetylsalicylic acid by bypassing COX-1 inhibition.

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Though initially promising, coxibs themselves proved to have serious liabilities. Rofecoxib was

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removed from the market after it was found to be associated with an increased risk of vascular

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events including heart attack and stroke.7-10

Selective coxibs were

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Inhibition of mPGES-1, which catalyzes the synthesis of PGE2 from PGH2, has been

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proposed as a therapeutic mechanism for the control of inflammation and pain11 without

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disrupting COX-2 catalyzed synthesis of anti-thrombotic prostacyclin. Additional recent studies

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implicate mPGES-1 as having a significant role in tumor growth.12-18 Validation of mPGES-1

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inhibition as being antipyretic, analgesic and anti-inflammatory is derived from both genetic and

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pharmacological models. Deletion of the mPGES-1 gene in the brain epithelia of mice attenuated

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the febrile response.19 Attenuation of the febrile response was also observed in mPGES-1 null

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mice challenged with intraperitoneal injection of interleukin-1β20 and peripheral injection of

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bacterial cell-wall lipopolysaccharide.21 In other studies, mPGES-1 null mice demonstrated

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ameliorated neuropathic pain22 as well as diminished pain nociception, inflammatory

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responsiveness, and collagen antibody-induced arthritis.18,

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Various pharmacological studies



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have demonstrated that inhibition of mPGES-1 can reduce the inflammatory response in vivo, as

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previously observed via genetic ablation.24- 27

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mPGES-1 is a 152 amino-acid member of the MAPEG (Membrane-Associated Proteins

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involved in Eicosanoid and Glutathione metabolism) superfamily of proteins which includes 5-

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lipoxygenase activating protein (FLAP), leukotriene C-4 synthase (LTC4), and microsomal

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glutathione S-transferase 1, 2, and 3 (MGST). Crystal structures of FLAP,28 LTC4,29 and

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mPGES-130, 31 and the cryo-electron microscopy structure of MGST-132, 33 have been determined.

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The quarternary structure of MAPEG proteins consists of a homotrimeric protein complex with

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twelve membrane-spanning alpha helices, four transmembrane helices per monomer. The

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mPGES-1 homotrimer binds three glutathione (GSH) molecules with the GSHs bound at the

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interfaces between the monomers. While the mPGES-1 crystal structure has been determined,

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there is, as of yet, no published structure-based detailed analysis of the structure-activity

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relationships (SAR) associated with drug-like mPGES-1 inhibitors. Here we describe the crystal

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structures (Data, Table 1) of mPGES-1 bound to four distinct specific potent small molecule

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inhibitors (Figure 1), providing a rationale for understanding the associated structure-activity

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relationships and a structural context for species-associated selectivities (Figure 2). The four

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scaffolds presented in complex with mPGES-1 are a biarylimidazole (5),34 a phenanthrene

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imidazole (MF63),25 and two biarylindoles (3,30).35,

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biophysical screen using differential static light scattering for identifying small molecules which

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thermally stabilize mPGES-1.

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Results

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Differential Static Light Scattering of mPGES-1 ligand complexes

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We also describe a high-throughput


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To better support ongoing medicinal chemistry efforts, we sought to increase the

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efficiency of our crystallization process by developing, as a secondary screen for biochemical

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assays, a biophysical screening method to prioritize ligands for structure determination.

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Significant scaffold-dependent discrepancies were observed between the determined IC50s of

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mPGES-1 assays depending on whether membrane extracts or purified mPGES-1 were used as

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catalysts. In some cases, as much as a ~10,000 fold loss in inhibitory activity was observed

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between membrane extracts and purified protein. The IC50 of 63 (MF63) was 6 nM when

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measured using mPGES-1 containing membranes; however, using purified mPGES-1, the

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measured IC50 was 1.9 µM (Table 2). Similarly, for 5, the IC50 was determined to be 7.3 nM

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using membranes and >62 µM using purified enzyme. As a result, the potency of biochemical

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assays did not sufficiently correlate with the likelihood of obtaining crystalline enzyme-inhibitor

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complexes. In previous studies, differential static light scattering (DSLS) was demonstrated to be

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an effective thermal stability assay for integral membrane proteins because of its general lack of

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sensitivity to the presence of detergent.37 Therefore, DSLS (Stargazer, Harbinger Biotech) was

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used to detect ligand-induced shifts in the temperature-dependent aggregation of the mPGES-1

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protein in the presence of inhibitors. Incubation of all four compounds (100 µM) with mPGES-1

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resulted in increases in the aggregation temperature. Compounds 5, 63,25 30 and 3 imparted

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increases of the mPGES-1 aggregation temperature of 7.8, 10.4, 5.4, and 7.3oC, respectively.

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The Crystal Structure of mPGES-1 bound to a biarylimidazole

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A series of nanomolar biarylimidazole mPGES-1 inhibitors was previously reported.34

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We used a variant of this scaffold, compound 5, that was brominated on the core imidazole for

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our original crystallization and structure determination by SAD phasing as, at the time, the 5 ACS Paragon Plus Environment


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crystal structure of mPGES-1 had not been described. In the crystal structure of mPGES-1 bound

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to 5, the inhibitor is bound with the bis-ortho-chlorofluorophenyl inserted into a groove above

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GSH at the top of the pocket (Figure 3a,b) and forms hydrophobic and van der Waals

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interactions with A123 and S127 side chains (monomer 1) and R38, L39, F44, D49, H53, and

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R53 side chains (monomer 2). The orientation is similar to the dichlorophenyl observed

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previously (PDB code: BPM).31 The core imidazole, in a near orthogonal orientation relative to

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the chlorofluorophenyl, forms hydrogen bonds with the H53 and S127 side chains and a

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structured water molecule (W75), and interacts hydrophobically with the P124 side chain. A

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similar hydrogen bond is formed between H53 and the dichlorophenyl linker nitrogen in the

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previous complex.31 The bromine extends towards the solvent interacting with the side chain of

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R52. This orientation of the core imidazole dictates a trajectory for the pyridine, triple bond C-C

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linker, and trifluoromethylbenzene tail that emphasizes interactions with α-4 of monomer 1. The

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nitrogen of the pyridine is within hydrogen bonding distance of the side chain oxygen of T131

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(monomer 1). Hydrophobic and van Waals contacts with the aforementioned “tail” functional

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groups are made exclusively with α-4 of monomer 1 including interactions with the P124, S127,

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V128, T131, L132, and L135 side chains (monomer 1).

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In the previous study,34 modifications of the central azole ring significantly affected

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potency. Loss of all detectable activity was observed in the oxadiazole, which loses the ability to

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display an H-bond donor (carbon to oxygen substitution at position 5). Analysis of the crystal

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structure after preparation of structured waters explains this seemingly ambiguous SAR (Figure

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4).


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For the two imidazole tautomers, hydrogen atoms were added to either the position 3 or

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position 1 nitrogen: 5A (Figure 4a), directed towards S127 and the structured water, and 5B

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(Figure 4b), directed towards H53. The solvated complex adjacent to position 1 appears equally

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satisfied in both 5A and 5B. Two structured waters bridge the R52 guanidinium onto the H53

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imidazole, allowing for H53 to project either a lone pair or polar hydrogen to the imidazole 1-N,

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with optimal hydrogen bonding to the two bridging waters in both scenarios.

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In contrast, at imidazole position 3, W75 is better satisfied in 5A than in 5B. Left of W75,

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another ordered water molecule (W98) H-bonds to the backbone carbonyl of S127 as well as the

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T131 hydroxyl group. Compound 5’s pyridine lone pair attracts T131’s hydroxyl polar proton

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(pyridine-N to T131-O distance = 3.0 Å), leaving only hydroxyl lone pairs exposed to W98,

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along with a S127 backbone carbonyl lone pair. Thus, the preferred orientation of W98 directs its

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two protons towards the protein, leaving only oxygen lone pairs to H-bond with one of S127’s

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hydrogen atoms. Right of W75, the glutathione-SH H-bonds with a W75 lone pair. W98, S127

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and the glutathione combine to keep W75 highly structured. With an NH at imidazole position

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3, 5A, satisfies the second lone pair of the well-ordered W75 and the S127 hydroxyl lone pair,

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with all polar hydrogens and lone pairs in this region engaged in an H-bond. In order to avoid

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electrostatic repulsion with the lone pair from the 3-N of 5B, both the Ser127 hydroxyl and W75

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direct hydrogen atoms up, but at the cost of i) leaving the W75 hydrogen atom much more

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desolvated from bulk solvent in the bound state than in the model for 5A and ii) positioning two

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polar protons 2.0 Å apart from one another.

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In agreement with these observations as to how 5A and 5B would be differentially

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satisfied, a Molecular Mechanics Generalized Born38 (MMGB) binding energy calculation

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predicts 5A to be 1.8 kcal/mol (~20 fold) more favorable than 5B. Decomposition of these 7 ACS Paragon Plus Environment


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predicted binding energies shows, as expected, that the van der Waals components do not differ

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significantly (0.2 kcal/mol higher for 5A). 5B, with an additional H-bond to the solvated protein,

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exhibits Coulombic attraction 8.3 kcal/mol lower and more favorable than 5A, but also pays a

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commensurate desolvation penalty 10.3 kcal/mol higher than 5A, with the net electrostatics for

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5B being 2.0 kcal/mol less favorable (higher) than for 5A. In addition, the MMGB binding

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energy for W75 is more unfavorable in the 5B model than in the 5A model, also with a

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Coulombic term that is more favorable for 5B but with a desolvation penalty that is of opposite

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and greater in magnitude, leading to the net electrostatics for W75 being less favorable with 5B

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than 5A.

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The previous SAR studies also demonstrated that, when the central azole was an

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imidazole, the two regioisomers appear to have shown a ~5 fold separation in activity, with a

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preference for nitrogen atoms at the 1- and 3-positions (as with 5) over nitrogen atoms at the 5-

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and 3-positions.

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towards the structured W75, but at the 1-position, the less active of the two would then project an

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aromatic CH to H-bond with the H53 N-lone pair, while the more active would then project an

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N-lone-pair with the H53 NH. The less potent of the two forms an H-bond with H53 that

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involves more polarity imbalance among the two H-bonding heavy atoms; the ligand’s aromatic

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CH likely exhibits less polarity than the H53 imidazole N-lone pair. For the more potent of the

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two regioisomers, the two heavy H-bonding atoms are comparably polar, the ligand’s N-lone-

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pair and the H53 imidazole NH. Thus, careful modeling of hydrogen atoms of

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crystallographically resolved waters conveys a preference for the central azole ring to project an

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H-bond donor down towards W75, which is consistent with the precipitous loss of activity by the

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oxadiazole that lacks an H-bond donor.

Both regioisomers maintain the ability to project an NH at the 3-position


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Another interesting subtle aspect of the previous SAR34 was improvement in potency by

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replacement of the imidazole core with a triazole (~6 fold in enzyme assay). Addition of the

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position 5 nitrogen to generate a triazole would not be expected from the crystal structure to

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create any new intermolecular contacts between the inhibitor and the enzyme as this position

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faces towards the solvent. Therefore, its effects are expected to be largely indirect and act

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through altering the hydrogen bond forming tendencies of other positions in the ring or through

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altering the behavior of the bound ligand with respect to its exposure to solvent.

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Wu et al34 also discuss SAR at the imidazole 4-position. A 4-pyridyl group reduced

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activity by ~3 fold over the corresponding 4-phenyl group (23 nM vs. 8 nM in enzyme assay),

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data which appears to conflict with the crystallographically observed H-bond between the

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pyridine N of compound 5 and T131 hydroxyl group discussed above and shown in Figure 4. As

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seen in Figure 3, the crystal structure of 5 also reveals that further functionalization of the 4-

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phenyl/4-pyridyl group with alkyne linkage of hydrophobic groups para to the imidazole allows

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the ligand to traverse a non-polar groove between V128 and T131 and place the terminal

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hydrophobic functionality in a non-polar dimple between V128, T131, L132 and L135 on α-4 of

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monomer 1.

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linkage, do not allow for placement of the terminal functional group in the non-polar dimple,

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consistent with the reported SAR studies. Filling of this nonpolar groove was seen in the

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previous complex31 where the trifluoromethyl and amide linker traverse the α-4 helix.

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Interestingly, in that case, the oxygen of the amide linker forms a hydrogen bond with the T131

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side chain as seen with pyridine nitrogen in the mPGES-1/5 complex. These two structures

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provide contrasting chemistries for complementing the α-4 helix binding groove.

SP3 alkyl and SP2 alkene linkages, with different exit vectors than the alkyne



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The Crystal Structure of mPGES-1 bound to 63

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In roughly the same binding mode as 5, the substituted phenanthrene imidazole 6325

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occupies the extreme upper region of the binding pocket above GSH (Figure 5). The planar

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chloro-phenanthrene extends over a flat surface of α-4 of monomer 1 (P124, S127 & V128 of

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monomer 1), with one face of the aromatic tetracycle facing solvent. Similar to the bis-ortho-

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chlorofluorophenyl of 5, a slightly larger 2,6-dicyano-phenyl points inward, clamped between

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the two protein chains, with one nitrile on the backside directed straight towards and forming a

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3.6 Å van der Waals contact with the Cβ of A123 (monomer 1) and a 3.2 Å interaction with the

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side chain hydroxyl of S127 (monomer 1). The second nitrile packs against the L39 side chain

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(monomer 2) while engaging a network of structured waters in the front of the binding site.

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Carbons of the 2,6-dicyano-phenyl form hydrophobic contacts with the sidechains of R38, L39,

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F44 and D49 (monomer 2), while the imidazole forms H-bonds with H53 (monomer 2) and a

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structured water as for 5. Côté et al. also reported SAR around imidazole replacements25 and, in

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agreement with our analysis of the SAR for the azoles of 5, found that eliminating H-bond

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donors from the azole led to significant loss of activity: ~20-fold with an oxazole and >100-fold

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with a thiazole.

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Analogous to the SAR pattern seen with the regioisomers for des-bromo-imidazole

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analogues of 5, the pyrrole (CH at 1-position of azole) showed an 8 fold loss of activity

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compared to the imidazole (N-lone-pair at 1-position of azole), comparable to the 5 fold loss

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discussed above. In addition, Côté et al showed data for an N-methyl imidazole,25 with the Me at

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the 3-position and the N-lone-pair at the 1-position, which lost all measurable activity (> 10 µM),

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illustrating the importance of H-bonds from the azole to both H53 and to the structured water.


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Subsequent elaboration of the 63 scaffold yielded more potent molecules that were active

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in a guinea pig model of analgesia.39 However, in that study, the substituents added to the

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aromatics flanking the central benzimidazole would appear from the crystal structure to derive

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their additional potency largely through non-specific effects. Loss of 63 activity in mice and

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rats,25 where H53 is replaced by arginine (Figure 2, Figure 5c), may result from the much longer

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arginine side chain sterically clashing with 63 and possibly less favorable hydrogen bonding

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geometry. Another critical species difference is the P124R substitution in mouse and rat. In

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human mPGES-1, P124, as discussed earlier, provides a flat hydrophobic surface for the planar

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phenanthrene tetracycle to lay against (Figure 5c). In addition, V128, which is within van der

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Waals distance of the phenanthrene chloro is replaced by glycine in rat and mouse mPGES-1

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(Figure 5c). Thus, it is evident from the crystal structure that the binding mode of 63 is highly

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selective with respect to species.

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The Crystal Structure of mPGES-1 Bound to 30, an MK-886 Derivative

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Compound 3035 is bound in an elongated orientation with the long axis of the molecule

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parallel to the transmembrane helices which form the pocket (Figure 6). Since its binding mode

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is more centrally located with respect to the two helices which form the binding groove, it

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interacts with mPGES-1 more extensively than do the other three inhibitors. The carboxylate is

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at the top of the binding site and forms a pair of salt bridges with the side chain of R52

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(monomer 2). The gem-di-methyl, while forming no contacts with mPGES-1, helps 30 bind in a

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low energy conformation: in the absence of the gem-di-methyl, the carboxylate would prefer to

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sit roughly orthogonal to the orientation in the bound-state conformation. A bridging water is

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found between the 30 carboxylate and the H53 imidazole. In addition, an aromatic CH from the

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indole and another from the fluorophenyl H-bond with the T131 hydroxyl lone pairs (monomer 11 ACS Paragon Plus Environment


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2). These are the only hydrogen bonds observed to the inhibitor. The chlorophenyl attached to

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the nitrogen of the indole ring extends inward between two helical turns at the top of α-4 from

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monomer 1 and α-1 from monomer 2 and places the halogen on top of the glutathione sulfur, in

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contrast to the 2,6-dicyano-phenyl of 63 where the aromatic ring is located over the glutathione

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sulfur. Hydrophobic van der Waals contacts are formed between the chlorophenyl and R38,

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L39, and F44 side chains (monomer 2) and GSH. The indole ring sits flatly across the binding

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site facing, but not contacting, GSH. The fluorinated biaryl extends downward from the indole

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with the fluorophenyl clearly represented by two conformations in the electron density maps. In

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one conformation, the fluorine points inwards into a small groove between the Y130 and T131

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side chains (monomer 1; Figure 6), and, in the second conformation, the fluorine points towards

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solvent. The fluorophenyl ring itself is slotted into the groove formed between the Y130 and

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T131 side chains (monomer 1) and is bounded on the opposite edge by the I32 side chain

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(monomer 2).

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Replacement of the 30 carboxylate with either an ester or an amide resulted in a dramatic

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loss in potency.35 This can be explained through a disruption of the salt bridge between the

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inhibitor carboxylate and the R52 guanidinium group. A variety of substitutions at the R3

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position at the C-3 position of the indole ring were tolerated in the previously published SAR

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study.35 In the crystal structure of mPGES-1 bound to 30, the methyl at this position extends

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towards the V128 side chain (monomer 1), and it appears that there is sufficient space in this

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region of the pocket to accommodate a diverse set of substitutions, which may project into the

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same hydrophobic groove between V128 and T131 that the alkyne of 5 traverses.


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It was additionally observed in the previous study that there was a strong preference for

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ortho substituents on both rings of the biaryl. The plane of the second substituent of the

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fluorinated biaryl, the toluene with the ortho methyl, is in a ground-state orientation, orthogonal

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to the plane of the first. The toluene forms hydrophobic and van der Waals interactions with the

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Q134, L135, and A138 side chains (monomer 1) and the Y28 and I32 side chains (monomer 2).

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The ortho-methyl moiety extends partially towards the solvent, but as alluded to above, also

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forms a van der Waals contact with the L135 side chain, which likely accounts for the reported 2

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fold potency boost for the 2-Me-Phenyl group over the unsubstituted benzene.

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The Crystal Structure of mPGES-1 bound to 3

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Compound 336 interacts extensively with α-4 of monomer 1 (Figure 7). Interactions with

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monomer 2 are limited to the head group where the isopropyl group of the 4-isopropoxy-phenyl

284

inserts into a cavity at the top of the binding groove formed by S127 (monomer 1) and R38, L39,

285

F44, D49, H53 (monomer 2) and the bound glutathione molecule. The carboxylate of the indole

286

core forms a salt bridge with the R52 side chain guanidinium, similar to the salt bridge observed

287

between compound 30 and the same residue. A bridging water is observed between the ligand

288

carboxylate and the side chain imidazole of H53. The core indole is packed against P124 and

289

V128 from monomer 1. The 4-tert-butylphenyl is bound in a shallow groove formed by the

290

V128, T131, L132, and L135 side chains of monomer 1 (monomer 1) and projects towards the

291

solvent. The core indole and 4-tert-butylphenyl interact solely with residues from α-4 of the first

292

monomer. The only substituents of the ligand which interact with the second monomer are the 4-

293

isopropoxy-phenyl and the carboxylate. The substitutions R52K and H53R in rat mPGES-1

294

imply a significant species-dependent potency. Other important species differences are the 13 ACS Paragon Plus Environment


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295

substitutions P124R and V128G. These amino acid substitutions likely result in greatly reduced 3

296

potency in the rat versus the human enzyme.

297

Discussion and Conclusions

Page 14 of 41

298

Although considerable progress has been made in the generation of integral membrane

299

protein crystal structures, their numbers are still rather limited. Aside from the low throughput of

300

determining additional complexes with bound ligand for any given integral membrane protein,

301

the resolution of these structures generally makes them less than ideal for supporting a structure-

302

based drug discovery effort. By combining biochemical screening with a high-throughput DSLS-

303

based orthogonal biophysical screen, we were able to efficiently generate high resolution crystal

304

structures and provide robust support to our medicinal chemistry effort. Over a five year period,

305

we produced over 100 high-resolution crystal structures of mPGES-1/inhibitor complexes.

306

Critical to intelligently focusing our crystallization efforts on the complexes most amenable to

307

crystallization was recognizing significant scaffold-dependent variation in the IC50s of inhibitors

308

depending on whether mPGES-1 containing microsomes or detergent-solubilized protein were

309

assayed. Based on this observation, we sought to implement a simple efficient high-throughput

310

biophysical screen to identify mPGES-1/inhibitor complexes most likely to crystallize. Pilot

311

studies demonstrated a strong correlation between crystallizability and induction of an increased

312

aggregation temperature as measured by DSLS. Crystal structures were determined for all

313

biochemical inhibitors that increased the aggregation temperature of mPGES-1 by at least 2oC.

314

No crystal structures were determined for biochemical inhibitors that failed to stabilize mPGES-

315

1 at all compound concentrations tested. This strategy proved successful for a variety of

316

scaffolds.


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317

The crystal structures described in this paper provide an explanation for the SAR

318

described in previous studies and also provide a rational basis for expanding on the SAR and

319

improving potency and overall drug like properties. A general binding mode is observed in

320

which inhibitors pack against the fourth helix of the first monomer while placing head groups

321

into a critical pocket formed above the GSH. While a strong tendency to interact with α-4 of

322

monomer 1 is observed, there is clear potential to access contacts with α-1 of monomer 2 in a

323

possible alternative binding mode.

324

isoleucine at position 32 (Figure 5c), the residues of α-1 that could potentially contribute to

325

ligand binding are conserved between the rat and human sequences, implying that inhibitors

326

utilizing such interactions might be less species-dependent with respect to potency. The tail end

327

of the inhibitors, opposite the head group which binds in the pocket above GSH, is largely

328

exposed to solvent in the crystal structures, implying that modifications of the inhibitor tails

329

likely improve potency through non-specific means. All four inhibitors form critical interactions

330

with mPGES-1 residues that are not conserved between human and rat/mouse primary

331

sequences, highlighting the complexities of using these animal models for pharmacological

332

investigation of mPGES-1 inhibition. Furthermore, the packing of the monomer 1 loop

333

connecting α-3 and α-4 against the monomer 2 accessory helix between α-1 and α-2 forms a

334

critical aspect of the overall architecture of the inhibitor binding site for all four inhibitors. The

335

α-3/α-4 loop sequence that forms the critical packing interface with the ligand-binding accessory

336

helix in human mPGES-1 is LRAP, while in rat the sequence is MNPR. The nature of the

337

substitutions in the rat sequence implies that the packing of these entities leads to significant

338

differences in the shape of the binding pocket. Interestingly, the aforementioned critical amino

339

acids are conserved between predicted guinea pig and human sequences which is the likely

Aside from the conservative substitution of valine for



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Page 16 of 41

340

explanation as to why guinea pig proved to be a more effective animal model in the

341

pharmacological characterization of the mPGES-1 inhibitor PF-4693627.24 As such, the crystal

342

structures presented in this study significantly expand on current knowledge with respect to

343

designing and pharmacologically validating inhibitors of mPGES-1 and ultimately could

344

facilitate the design of clinical molecules for the treatment of pain, inflammation, and cancer.

345

Experimental Section

346

Expression, Purification, and Crystallization. The mPGES-1 cDNA (residues 1-152) was

347

expressed using the Bac-to-Bac baculovirus expression system (Life Technologies) without an

348

N- or C- terminal purification tag. The resulting recombinant baculovirus was used to infect 0.5

349

L suspension cultures of Sf-9 cells. Cells were harvested after 48 hours, pelleted, and stored at -

350

80oC. Membrane extracts were prepared by resuspending cell pellets in 15 mM TRIS-HCL pH

351

8.0, 0.25 M sucrose, 1 mM reduced glutathione (GSH) with stirring at 4oC followed by dounce

352

homogenization on ice and ultracentrifugation at 44k RPM (4oC, Ti45 rotor, Beckman LE80

353

ultracentrifuge). Resulting pellets were resuspended in buffer containing 25 mM HEPES pH 7.5,

354

10% glycerol, 1mM GSH, 1.0% 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC) and

355

dounce homogenized on ice followed by centrifugation at 44k RPM for 1 hour (4oC, Ti45 rotor,

356

Beckman, LE80 ultracentrifuge). Supernatants were injected onto Mono S ion exchange column

357

(GE Healthcare), and protein was eluted using a gradient 0-0.5 M NaCl gradient in 0.02 M

358

HEPES pH 7.6, 1.0% β-octylglucoside (β-OG), 1 mM GSH. Fractions containing mPGES-1

359

were pooled and applied to a gel filtration column (Superdex 200, GE Healthcare) equilibrated

360

with 0.02 M Hepes pH 7.6, 0.05 M NaCl, 1.0% β-OG, 1 mM GSH (GF). Fractions containing

361

mPGES-1 were pooled and concentrated to 10-15 mg/ml. Crystals (space group H3) with one 16 ACS Paragon Plus Environment

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362

mPGES-1 monomer per asymmetric unit were grown by vapor diffusion (1:1 drop ratio) at 21oC

363

with well solutions consisting of 0.1 M TRIS-HCl pH 8.5-9.5 and 25-35% PEG 1K. Crystals

364

were harvested and cryo-cooled directly without the use of additional cryoprotectant.

365

mPGES-1 Enzyme Inhibition Assays. Human or rat mPGES-1 (Invitrogen™ (Cat #97002RG,

366

clone ID 6374722)) was subcloned into pcDNA™3.1 and transiently expressed in 293E cells.

367

Microsomes were prepared from cell pellets based on published methods.40,

368

brought up in homogenization buffer (15 mM TRIS-HCl, pH 8.0; 0.25 M sucrose; 0.1 mM

369

EDTA; 1 mM glutathione) and sonicated 5 × 30 seconds on ice. Homogenate was centrifuged at

370

5000g for 10 minutes at 4°C. The supernatant fraction was decanted, loaded into Beckman

371

Quick-Seal® tubes, and centrifuged at 150000g for 90 minutes at 4°C. The supernatant fraction

372

was discarded by decantation, and the pellets were resuspended in assay buffer (10 mM sodium

373

phosphate (pH 7.0), 10% glycerol, 2.5 mM glutathione, Complete Protese Inhibitor Cocktail

374

(Roche). Protein concentration was determined using the Pierce Coomassie Plus™ reagent. For

375

the enzyme assay, the microsomes were diluted into assay buffer and 14 µL/well was added to

376

384 well plates. Compound dilution plates (Nunc® Catalog #249944) were generated on a Tecan

377

Evo™, and 4 µL/well was added to the assay plates. PGH2 was diluted into assay buffer

378

immediately prior to use, and 7 µL/well was added. Final concentrations were 6.52 µg/mL of

379

microsomes and 1.67 µM PGH2. After a 2.5 minute incubation at room temperature, 5 µL/well

380

of (1 mg/mL of SnCl2 in 0.5 N HCl(aq)) was added to stop the reaction. 5 µL of the reaction was

381

transferred to a 384 well plate containing 0.1% aqueous formic acid (45 µL) for Mass Spec

382

dilution, then the plates were stored at –20°C. The plates were analyzed for PGE2 using standard

383

LC/MS analysis (Biocius Lifesciences, Wakefield, MA, USA). Purified mPGES-1 as prepared in


41

Pellets were


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Page 18 of 41

384

the Expression, Purification, and Crystallization section was assayed under the same conditions.

385

Values are the average of two measurements,

386

Differential Static Light Scattering. mPGES-1 was diluted to 0.2 mg/mL in GF buffer. The

387

aggregation temperature was determined using DSLS (Stargazer-384TM, Harbinger Biotech).

388

Samples were analyzed in a 384-well clear bottom plate (Nunc) with 35 µL sample volume using

389

a temperature gradient from 25.0oC to 85.0oC at a ramp rate of 1.0oC/min. Aggregation

390

temperatures were calculated using BioactiveTM software (Harbinger Biotech). Values are the

391

average of three measurements.

392

Structure Determination. Datasets were collected at LRL-CAT beam line at the Advanced

393

Photon Source, Argonne, IL. Crystals of the mPGES-1/5 complex diffracted to 1.41 Å, and

394

belonged to Space Group R3, with cell parameters a=b=76.4 Å, c=123.3 Å. As no x-ray

395

coordinates for mPGES-1 had been publicly disclosed at the time we obtained diffraction data,

396

the structure was determined by SAD using the anomalous signal from the bromine atom of the

397

bound ligand, compound 5. Phasing was performed with SHELX,42 and density modification and

398

initial model building was implemented within ARP/wARP.43 Following numerous cycles of

399

refinement with REFMAC544 and model building with COOT,45 the model was refined to an

400

Rwork of 17.1% and an Rfree of 18.9%. Subsequent structures (see Table 1) were refined in

401

REFMAC5 by isomorphous replacement using the initial coordinates as a model.

402

Chemical Purity. Purities of synthesized compounds were all found to be >95% by LC/MS:

403

Mass spectra were recorded using an Agilent MSD LC/MS System with UV detection at λ 300

404

nm and 214 nm and an ionization potential of 80 eV. [C18 2.1 mm × 50 mm Phenomenex


Page 19 of 41

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405

Gemini column (particle size 3 µm, pore size 110 Ắ); gradient, 5% to 100% CH3CN in

406

water/0.1% formic acid over 3.75 minutes; column temperature, 50 °C]

407

PDB ID codes. Structure factors and atomic coordinates have been deposited in the RSCB

408

Protein Data Bank with the following coordinates 4YK5, 4YL0, 4YL1, 4YL3. Files may be

409

retrieved online at http://www.rcsb.org/pdb/home/home.do.

410

Acknowledgements. Use of the Advanced Photon Source, an Office of Science User Facility

411

operated for the U.S. Department of Energy (DOE) Office of Science by Argonne National

412

Laboratory, was supported by the U.S. DOE under Contract No. DE-AC02-06CH11357. The

413

beamline staff, Stephen Wasserman, John W. Koss, David Smith, and Laura Morisco are

414

gratefully acknowledged. Spencer Emtage and Stephen K. Burley are acknowledged for helpful

415

discussions.

416

Abbreviations Used. COX, cyclooxygenase; DSLS, differential static light scattering; EDTA,

417

ethylenediaminetetraacetic acid; FLAP, 5-lipoxygenase activating protein; GSH, glutathione;

418

LC/MS, liquid chromatography mass spectrometry; LTC4, leukotriene C-4 synthase; MAPEG,

419

membrane-associated proteins involved in eicosanoid and glutathione metabolism; MGST,

420

microsomal glutathione S-transferase; MMGB, Molecular Mechanics generalized Born;

421

mPGES-1, microsomal prostaglandin E synthase-1; NSAIDS, nonsteroidal anti-inflammatory

422

drug; PGE2, prostaglandin E2; PGH2, Prostaglandin H2; SAD, single wavelength anomalous

423

dispersion.

424



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425

426

427


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prostaglandin E2 synthase (mPGES-1) derived from MK-886. Bioorg. Med. Chem. Lett. 2005,

542

15, 3352-3355.

543

(36) Olofsson K.; Suna, E; Pelcman B,; Ozola, V.; Katkevics M.; Kalvins I. International Patent

544

Publication 2005, Number: WO 2005/123673 A1.

545

(37) Senisterra G.A.; Ghanei H.; Khutoreskaya G.; Dobrovetsky E.; Edwards A.M.; Privé G.G.;

546

Vedadi M. Assessing the stability of membrane proteins to detect ligand binding using

547

differential static light scattering. J. Biomol. Screen. 2010, 15, 314-320.

548

(38) Halgren T.A. Merck molecular force field. I. Basis, form, scope, parameterization, and

549

performance of MMFF94. J. Comp. Chem. 1996, 17, 490-519.

550

(39) Giroux A.; Boulet L.; Brideau C.; Chau A.; Claveau D.; Côté B.; Ethier D.; Frenette R.;

551

Gagnon M.; Guay J.; Guiral S.; Mancini J.; Martins E.; Massé F.; Méthot N.; Riendeau D.;

552

Rubin J.; Xu D.; Yu H.; Ducharme Y.; Friesen R.W. Discovery of disubstituted phenanthrene

553

imidazoles as potent, selective and orally active mPGES-1 inhibitors. Bioorg. Med. Chem. Lett.

554

2009, 19, 5837-5841.

555

(40) Ouellet, M.; Falgueyret, J.-P.; Ear, P. H.; Pen, A.; Mancini, J. A.; Riendeau, D.; Percival, M.

556

D. Purification and characterization of recombinant microsomal prostaglandin E synthase-1.

557

Protein Exp. Purif. 2002, 26, 489–495



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(41) Thorén, S.; Weinander, R.; Saha, S.; Jegerschöld, C.; Pettersson, P. L.; Samuelsson, B.;

559

Hebert, H.; Hamberg, M.; Morgenstern, R.; Jakobsson, P.J. Human Microsomal Prostaglandin E

560

Synthase-1: Purification, functional characterization, and projection structure determination. J.

561

Biol. Chem. 2003, 287, 22199–22209.

562

(42) Sheldrick G.M. A short history of SHELX Acta Cryst. Sec. A 2008, 64, 112-122.

563

(43) Langer G.; Cohen S.X.; Lamzin V.S.; Perrakis A. Automated macromolecular model

564

building for X-ray crystallography using ARP/wARP version 7. Nat. Protoc. 2008, 3, 1171-

565

1179.

566

(44) Murshudov G.N.; Skubak P.; Lebedev A.A.; Pannu N.S.; Steiner R.A.; Nicholls R.A.; Winn

567

M.D.; Long F.; Vagin A.A. REFMAC5 for the refinement of macromolecular crystal structures.

568

Acta Crystallogr. Sec. D 2011, 67, 355-367.

569

(45) Emsley P.; Lohkamp, B.; Scott W.G.; Cowtan K.; Features and development of coot. Acta

570

Crystallogr. Sec. D. 2010, 66, 486-501.

571 572 573


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

574


Figure Legends

575 576

Figure 1. mPGES-1 inhibitors (a) Schematic depiction of the chemical structure of compound 5

577

(b) 63 (c) 30 (d) 3

578

579

Figure 2. Sequence alignment of mPGES-1 amino acids sequences by species. Conserved

580

residues are green. Helical regions are defined by blue cylinders above and numbered by order of

581

transmembrane pass. Residues which form contacts with at least one inhibitor but are not

582

conserved are highlighted in yellow. Sequence of α-3/α-4 loop which packs against accessory

583

helix is boxed in red and not conserved in rat. This region of mPGES-1 is critical to the shape of

584

the inhibitor binding site. (# contacts with all four inhibitors; + contacts with 5, 30, and 3; %

585

contacts with 5 and 63; ^contacts with only 3; *predicted sequence)

586

587

Figure 3. Binding Mode of Compound 5 to mPGES-1. (a) The crystal structure of 5 bound to

588

mPGES-1 is depicted. Monomer 1 is colored cyan and monomer 2 is colored orange. Side

589

chains, GSH, and 5 are shown as sticks and colored by atom (oxygen, red; nitrogen, blue; sulfur,

590

sienna; 5 carbon, yellow; GSH carbon, magenta; monomer 1 carbon, cyan; monomer 2, orange;

591

fluorine, green; chlorine, dark green; bromine, brick). The 2,6-chlorofluorophenyl is buried in a

592

pocket above the glutathione extending inward from the orthogonally oriented bromo-imidazole.

593

The remainder of the scaffold extends downwards tracing along the α-4 helix of monomer 1 with

594

the terminal trifluoro group (two conformations) extending towards the solvent. S127 is observed

595

in a dual conformation. (b) Surface representation of same. 29 ACS Paragon Plus Environment


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Page 30 of 41

596

Figure 4. Comparison of H-bonds in Computational Model of Compound 5 Binding. Ball

597

and stick model of Compound 5 bound to mPGES-1. Color scheme as in Figure 3. (a) 5A

598

imidazole position 3 hydrogen directed towards S127 and the structured water W98. (b) 5B

599

imidazole position 1 hydrogen directed towards H53, respectively. The solvated complex

600

adjacent to position 1 appears equally satisfied in both 5A and 5B. W75 is better satisfied in 5A

601

than in 5B.

602

603

Figure 5. Binding Mode of 63 to mPGES-1. (a) The crystal structure of 63 bound to mPGES-1

604

is depicted. Color scheme is as in Figure 3.. The 2,6-dicyano-phenyl is buried within the binding

605

pocket in a similar manner as the 2,6-chlorofluorophenyl of 5. The imidazole is positioned

606

orthogonally and forms hydrogen bonds with H53 and a water molecule in the same manner as

607

the 5 imidazole. Preferential packing interactions are formed with P124 of monomer 1. (b)

608

Surface representation of same. (c) Surface representation of human to rat sequence conservation

609

in the ligand binding site. Monomer 1, cyan; monomer 2, orange; nonconservative substitutions,

610

red; conservative substitutions blue. Human to rat amino acid substitutions are labeled in white

611

text. The left hand surface of the groove is less conserved.

612

613

Figure 6. Binding Mode of Compound 30 to mPGES-1. (a) The crystal structure of 30 bound

614

to mPGES-1 is depicted. Color scheme is as in Figure 3. The 4-chlorophenyl extends into the

615

same pocket as the 2,6-chlorofluorophenyl of 5 and the 2,6-dicyano-phenyl of 63 with a similar

616

tilt to the ring. A salt bridge is formed between the carboxylate of 30 and the R52 side chain

617

guanidinium. The 5-fluorophenyl of the central indole is seen in two conformations. The overall 30 ACS Paragon Plus Environment

Page 31 of 41

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618

binding mode of 30 is more central relative to the α-4 helix of monomer 1 and the α-1 helix of

619

monomer 2. (b) Surface representation of same.

620

621

Figure 7. Binding Mode of Compound 3 to mPGES-1. (a) The crystal structure of 3 bound to

622

mPGES-1 is depicted. Color scheme is as in Figure 3.. 4-isopropoxy-phenyl protrudes into the

623

cavity above GSH which is occupied by aromatic rings in 5, 63, and 30. A salt bridge is formed

624

between the carboxylate of 3 and the R52 side chain guanidinium analogous to that observed in

625

the mPGES-1/30 complex. The central indole and 4-tert-butyl-phenyl lie in a groove formed by

626

side chains of α-4 monomer 1. Contacts with monomer 2 are limited. (b) Surface representation

627

of same.

628

629

630



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

631

Table 1. Data Collection and Refinement Statistics. Data Statistics Spacegroup Cell Dimensions (a/b, c)(Å) o Angles (α,β, γ)( ) Resolution (Å) Completeness (%) Rsym (%)

Wavelength (Å)

30 H3

MF63 H3

3 H3

76.4, 123.3 90, 90, 120

76.6, 123.7

76.6, 123.0

76.6, 123.6

90, 90, 120

90, 90, 120

90, 90, 120

32.0-1.42 (1.49-1.42) 99.3 (96.5) 4.0 (23.0)

41.0-1.52 (1.60-1.52) 99.0 (95.1) 6.3 (38.3)

32.0-1.41 (1.49-1.41) 99.4 (98.0) 4.4 (31.9)

11.0 (6.1) 5.6 (5.1)

14.9 (8.8) 5.4 (4.9)

7.1 (4.8) 5.3 (4.7)

11.8 (6.0) 5.6 (5.1)

0.91986

0.97929

0.91986

0.91986

a

Refinement Statistics

5

30

MF63

3

Resolution Range (Å) Reflections Rwork (%) Rfree (%) R.m.s. deviations

41.1 - 1.41 50730 17.1 18.9

32.0 - 1.42 50979 17.5 17.8

41.0 - 1.52 41078 20.2 22.3

32.0 - 1.41 51297 17.8 18.6

Bond lengths (Å) o Bond angles ( ) Residues No. atoms mPGES1 Ligand GTT β-OG PEG Water Average B-factors mPGES1 Ligand GTT β-OG PEG

634

5 H3

41.1-1.41 (1.49-1.41) 98.9 (97.2) 5.2 (31.5)

Mean I/σ(I) Redundancy

632 633

Page 32 of 41

Water

0.014

0.011

0.006

0.012

1.28 143

1.18 143

1.00 148

1.21 143

1189 36 20 40 13 159

1172 46 20 38 13 161

1233 28 20 40 20 126

1169 32 20 36 13 170

17.6 23.9 18.0 45.7

14.2 19.4 6.2 39.7

25.0 42.5 26.5 67.8

17.3 34.0 13.4 41.8

55.2 44.1

71.2 39.5

72.4 47.4

73.5 42.6

a, highest resolution shell

635


Page 33 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

636

637


Table 2. Biochemical and Differential Light Scattering Assay Data for mPGES-1 Compound

IC50 microsomea (µM)

5 MF63 30 3

0.007 ± 0.002 0.006 ± 0.003 0.023 ± 0.017 0.216 ± 0.139

o IC50 purifiedb (µM) ∆Tagg ( ) (0.1 mM)

3.94 ± 3.30 1.59 ± 0.92 0.96 ± 0.82 2.58 ± 2.46

7.8 ± 0.3 10.4 ± 0.3 5.4 ± 0.3 7.3 ± 0.3

638

a, isolated microsomes containing recombinantly expressed human mPGES-1; b, detergent-

639

solubilized membrane-extracted mPGES-1 purified by chromatography

640 641



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642

Figure 1

643

644


Page 34 of 41

Page 35 of 41

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645


Figure 2

646



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

647

Figure 3

648

A

B 649 650 651 652 653 654 655


Page 36 of 41

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656

Figure 4

657

A

B

658 659 660 661 662



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

663

Figure 5

664

A

B 665 666 667 668 669 670 671 672 673

674 675

C 676 677 678


Page 38 of 41

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679

Figure 6

680

A

B



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

685

Figure 7

686

A

B 687 688


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689


Table of contents graphic

690

691 692 693


Crystal Structures of mPGES-1 Inhibitor ... - ACS Publications

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