Bioorganic & Medicinal Chemistry 23 (2015) 2310–2317

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Identification of N-ethylmethylamine as a novel scaffold for inhibitors of soluble epoxide hydrolase by crystallographic fragment screening Yasushi Amano ⇑, Eiki Tanabe, Tomohiko Yamaguchi Drug Discovery Research, Astellas Pharma Inc., 21, Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan

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Article history: Received 10 February 2015 Revised 30 March 2015 Accepted 31 March 2015 Available online 6 April 2015 Keywords: Soluble epoxide hydrolase Fragment-based drug discovery Protein crystallography Arachidonic acid cascade Hypertension Inflammation Structure-based drug design

a b s t r a c t Soluble epoxide hydrolase (sEH) is a potential target for the treatment of inflammation and hypertension. X-ray crystallographic fragment screening was used to identify fragment hits and their binding modes. Eight fragment hits were identified via soaking of sEH crystals with fragment cocktails, and the co-crystal structures of these hits were determined via individual soaking. Based on the binding mode, N-ethylmethylamine was identified as a promising scaffold that forms hydrogen bonds with the catalytic residues of sEH, Asp335, Tyr383, and Tyr466. Compounds containing this scaffold were selected from an in-house chemical library and assayed. Although the starting fragment had a weak inhibitory activity 800 lM), we identified potent inhibitors including 2-({[2-(adamantan-1(IC50: yl)ethyl]amino}methyl)phenol exhibiting the highest inhibitory activity (IC50: 0.51 lM). This corresponded to a more than 1500-fold increase in inhibitory activity compared to the starting fragment. Co-crystal structures of the hit compounds demonstrate that the binding of N-ethylmethylamine to catalytic residues is similar to that of the starting fragment. We therefore consider crystallographic fragment screening to be appropriate for the identification of weak but promising fragment hits. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Fragment-based drug discovery (FBDD) has been developed over the last decade and applied to a number of drug-target proteins.1,2 FBDD has been applied to a number of drug-target proteins, and identified several drugs which are currently under clinical study.3 However, due to the low affinity of fragments for target proteins in FBDD, fragment screening is conducted using biophysical assays such as NMR, SPR, TSA, and X-ray crystallography or biochemical assays with a high-concentration of fragments.1 Soluble epoxide hydrolase (sEH) converts epoxyeicosatrienoic acids (EETs) to dihydroxyepoxyeicosatrienoic acids (DHETs). EETs are known to possess anti-inflammatory and anti-hypertensive properties. Thus, sEH is a promising drug target for the treatment of inflammation and hypertension.4 To date, various sEH inhibitors have been developed.5 Figure 1 shows representatives of potent sEH inhibitors: N,N0 -dicyclohexylurea (DCU); trans-4-[4-(3adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB); AR9281; and GSK2256294.6–9 Of these inhibitors, AR9281 showed ⇑ Corresponding author. Tel.: +81 29 863 7054; fax: +81 29 854 1519. E-mail address: [email protected] (Y. Amano). http://dx.doi.org/10.1016/j.bmc.2015.03.083 0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

efficacy in animal models of hypertension and dysglycemia.8 In addition, GSK2256294 showed protective effect against cigarette smoke-induced pulmonary inflammation in the mouse.10 These two inhibitors are now under clinical study. The majority of sEH inhibitors identified thus far are urea or amide derivatives. X-ray crystallography has demonstrated that these derivatives form a strong network of hydrogen bonds between urea or amide moieties and the catalytic residues of sEH.6,11–18 Figure 2 shows the co-crystal structure of sEH with a urea-based inhibitor, t-AUCB.19 Briefly, the carbonyl oxygen forms hydrogen bonds with the Tyr residues Tyr383 and Tyr466, and the NH forms hydrogen bonds with Asp335. However, novel scaffolds other than urea or amide have also been reported, including benzoxazole, aminopyrimidine, sulfoxide, and aminothiazole.16,20–25 We previously conducted fragment screening against sEH using biochemical assays and X-ray crystallography.19 Despite attempting to resolve crystal structures of complexes with over 300 fragments, we only identified 2 new scaffolds that bind to the catalytic triad: dihydrothiazole and benzimidazole. However, this study demonstrated that X-ray crystallography is a powerful approach to identifying fragment hits for sEH and specific interactions between fragments and sEH. We therefore consider

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Figure 1. Representatives of known sEH inhibitors.

fragment was individually soaked into the crystal to confirm binding. Figure 4 shows omitted F0  Fc maps of fragment hits. As one fragment might have prevented others in the same cocktail from binding, sEH crystals were soaked with cocktails of the other nine fragments in the hit cocktails, with no fragment binding to sEH. Ultimately, 8 fragment hits were identified from a fragment library of 800 (1.0% hit rate) using crystallographic screening. To confirm the inhibitory activity of these fragment hits, enzymatic assays were conducted. The IC50 values of 7 fragment hits ranged from 52 to 2200 lM. However, Fragment 4 might influence the fluorescence of the substrate and the IC50 value could not be determined. Fragment screening is summarized in the flow chart of Figure 3. 2.2. Binding modes of fragment hits

Figure 2. Crystal structure of t-AUCB bound to sEH (PDB ID code: 3WKE). Light blue ribbon diagram and stick models indicate the protein structure and the residues of the catalytic triad, respectively. Green ball and stick model indicates t-AUCB.

crystallographic fragment screening suitable for use in identifying fragment hits of sEH. Further, crystallographic screening simultaneously provides validated hits and structural information regarding the binding mode.26–31 In this method, protein crystals are soaked with individual fragments or fragment cocktails. Although cocktail soaking is more efficient for the screening of a fragment library, soaking requires robust crystals that diffract to a high resolution. In this regard, the sEH crystals identified in our previous study diffract up to a resolution of 2.0 Å and are highly durable against soaking with high-concentration compounds.19 We therefore conducted crystallographic screening against sEH to identify promising and novel fragments as starting points of FBDD. Based on the binding modes of fragments, we also searched for novel inhibitors from our in-house chemical library. Here, we discuss the findings for crystallographic screening and identification of crystal structures of sEH in complex with compounds.

sEH has a large L-shaped pocket,32 with the catalytic residues Asp335, Tyr383 and Tyr466 at the corner of this pocket forming the catalytic triad.33,34 The short branches of sEH measure 10 Å and the long branches 15 Å. The majority of known inhibitors contain a urea or amide moiety that binds to the catalytic triad.6,11–18 Fragment 1 also forms hydrogen bonds with the catalytic triad via its secondary amine (Fig. 5). The distances between the nitrogen and oxygen atoms are as follows: Asp335, 2.81 Å; Tyr383, 3.09 Å; and Tyr466, 2.45 Å. According to the position of atoms corresponding to these hydrogen bonds, the nitrogen atom likely acts as a hydrogen acceptor for Asp335 or Tyr383 and as a hydrogen donor for Tyr466. The secondary amine is a unique moiety that binds the catalytic triad of sEH and forms hydrogen bonds via a single nitrogen atom. In contrast, the amide or urea moieties that bind the catalytic triad form hydrogen bonds via an oxygen atom and one or two nitrogen atoms.6,11–18 The phenyl moiety of Fragment 1 binds to the short branch of the pocket and forms van der Waals

2. Results and discussion 2.1. Crystallographic fragment screening A fragment library of 800 fragments was selected from commercially available compounds. To identify fragments with novel structures, compounds containing the potent sEH inhibitors urea or amide moieties were omitted. For cocktail soaking, 80 fragment cocktails were generated to ensure structural diversity within the cocktail. Following soaking with fragment cocktails, our highly durable sEH crystals diffracted from 2.0 to 2.7 Å. Analysis of X-ray diffraction data of soaked crystals identified eight fragments that bound to the catalytic pocket of sEH. To verify these fragment hits, each

Figure 3. Flowchart of crystallographic fragment screening.

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Figure 4. Omitted F0  Fc maps of Fragments 1 to 8 bound to sEH (contoured at 2.5 r).

interactions and hydrophobic interactions with Phe267, Leu408, Met419, and Leu428. The methylpyrazole moiety shallowly binds the long branch of the pocket and forms van der Waals interactions with Trp336. Fragment 1 exhibits weak inhibitory activity with an IC50 value of 800 lM and low ligand efficiency (LE) of 0.26. Fragment 2 also has a secondary amine that binds to the catalytic triad in a similar manner to Fragment 1 (Fig. 5). However, the distance between the nitrogen atom of the amine and the oxygen of Tyr383 prevents hydrogen bond formation (3.67 Å). Fragment 2 forms hydrogen bonds with Asp335 by acting as a hydrogen acceptor and with Tyr466 by acting as a hydrogen donor. The phenyl moiety binds to the short branch in a similar manner to that of Fragment 1. The nitrogen atom of the pyridine ring forms a hydrogen bond with a water molecule that binds to the main chain NH of Phe497. Although the aromatic rings of pyridine and benzene occupy the short branch and form a hydrogen bond, the inhibitory activity of Fragment 2 is less than that of Fragment 1 (IC50: 2.2 mM and LE: 0.24). These findings suggest that an increase in inhibitory activity requires hydrogen bonds with all of the catalytic residues and interactions with Trp336. Fragments 3 and 4 have a similar chemical structure, as shown by Figure 5. The pyridinylpiperazine moiety is common between the two, and the ortho-position of the pyridine ring is substituted by a trifluoromethyl or nitrile group in 3 or 4, respectively. Both the trifluoromethyl and nitrile groups are hydrophobic and bulky and do not exhibit any critical differences. Indeed, Fragments 3 and 4 exhibit closely similar binding modes, as shown in Figure 5. The NH of piperazine ring forms hydrogen bonds with Asp335 and Tyr466 in a manner similar to that of Fragment 2, but the distance to Tyr383 prevents hydrogen bond formation. The ortho-substituted pyridine ring binds to the short branch and forms hydrophobic interactions and van der Waals interactions. As the position of the pyridine ring slightly differs from that of Fragment 2, a hydrogen bond via a water molecule does not form. Fragment 3 exhibits an IC50 value of 1.2 mM and an LE value of 0.25. Although the inhibitory activity of Fragment 4 was not elucidated by the enzyme assay, the binding mode suggests a level of activity similar to Fragment 3. Fragment 5 does not form hydrogen bonds with the catalytic triad, but the hydroxyl group of this fragment forms hydrogen bonds with the main chain NH of Phe497 and a water molecule that binds to the main chain oxygen of Lys495 (Fig. 6). The trifluoromethyl group forms hydrophobic interactions with Phe267, Leu408, Met419, and Trp525. In addition, the benzene ring forms p–p stacking with His524. Despite the lack of hydrogen bonds with

the catalytic triad, Fragment 5 exhibits moderate inhibitory activity (IC50: 52 lM and LE: 0.36). The hydrophobic interactions of the trifluoromethyl group are also observed in the binding mode of Fragment 3, thus the moderate inhibitory activity of Fragment 5 is possibly due to the p–p stacking with His524. Fragment 6 binds the long branch of the catalytic pocket, and the hydroxyl group of this fragment forms hydrogen bonds with the catalytic triad (Fig. 6). The central benzene ring forms a van der Waals interaction with Gln384 and a hydrophobic interaction with Trp336. The terminal benzene ring forms a hydrophobic interaction with Ile375. As the hydroxyl group and two benzene rings are connected by flexible linkers, the position of the benzene rings might be preferable regions for ligand binding. Given that the size of the long-branch exceeds this space, this type of structural information is required for the optimization of compounds. Fragment 7 forms hydrogen bonds with the catalytic triad (Fig. 6). The carbonyl oxygen of the carbamate interacts with Tyr383 and Tyr466 as a hydrogen acceptor, and the nitrogen interacts with Asp335 as a hydrogen donor. These interactions are similar to those between the catalytic triad and known amide or ureabased inhibitors. In addition, the nitrogen of the tetrahydroquinoline forms a hydrogen bond with the main chain oxygen of Phe267. Fragment 8 binds to the short branch of the catalytic pocket (Fig. 6) but does not form any hydrogen bonds. The bromide occupies the hydrophobic region formed by Phe267, Phe387, and Leu428. The pyrazole ring is positioned close to His524 and may form a weak p–p interaction. Despite the lack of hydrogen bonds, Fragment 8 exhibits moderate inhibitory activity (IC50: 91 lM) and high LE. Based on the binding modes of fragment hits, three functional groups were identified that form hydrogen bonds with the catalytic triad, secondary amine, hydroxyl group, and carbamate. Notably, Fragments 1 to 4 contain the secondary amine despite the library being structurally diverse and not biased toward amine compounds. The binding modes of the secondary amines are also of interest. Regarding the crystal structure of sEH in complex with amide or urea inhibitors, the NH of amide or urea acts as a hydrogen donor for Asp335.6,11–19 For example, t-AUCB forms hydrogen bonds with the catalytic triad via its NH as a hydrogen donor and via carboxy oxygen of the amide as a hydrogen acceptor (Fig. 7A).19 In contrast, the amine NH of Fragments 1 to 4 do not appear to donate a hydrogen atom to Asp335. Figure 7B shows the predicted hydrogen-bonding pattern of Fragment 1. The side chain carboxylate of Asp335 donates the hydrogen to the unshared electron pair of the nitrogen, and the amine NH of Fragment 1

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Figure 5. Crystal structures of Fragments 1 to 4 bound to sEH. Green ball and stick models indicate compounds. Light blue ribbon diagrams and stick models indicate protein structures. Red balls indicate water molecules. Dashed lines indicate hydrogen bonds.

donates the hydrogen to Tyr466. The unique interaction of the secondary amine was investigated, but the LE values of Fragments 1 to 4 were insufficient as a starting point of FBDD. Compounds that more efficiently inhibited sEH were therefore investigated. 2.3. Structure-based exploration Particular focus was paid to the novel scaffold N-ethylmethylamine of Fragment 1. Of Fragments 1 to 4, this is the only fragment to reach both the sub-pockets of the short and long branches. To increase the inhibitory activity of Fragment 1, the benzyl group was substituted for a more bulky or larger functional group to

occupy the hydrophobic region surrounded by Phe267, Phe387, Leu408, Met419, and Leu428. Binding modes of fragment hits in our present and previous study revealed that hydrophobic and bulky functional groups such as the trifluoromethyl, bromo, or cyano groups are optimal for this region.19 In addition, substitution of 1-methylpyrazole for a more hydrophobic functional group might result in a stronger interaction with Trp336. Based on this structural information, 14 compounds were selected from our in-house chemical library and assays conducted, resulting in identification of compound 9 with an IC50 value of 0.51 and compound 10 with an IC50 value of 9.0 lM. The LE value of compound 9 was 0.41, while that of 10 was 0.40, which are

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Figure 6. Crystal structures of Fragments 5 to 8 bound to sEH. Green ball and stick models indicate compounds. Light blue ribbon diagrams and stick models indicate protein structures.

sufficient starting points for FBDD. To determine the binding modes of compounds 9 and 10, the crystal structures of sEH when in complex with the compounds were solved. Compound 9 binds the catalytic triad via its NH in a manner similar to that of Fragment 1, as expected (Fig. 8). NH forms hydrogen bonds with Asp335 (2.90 Å) and Tyr383 (3.01 Å); however, the distance between NH and the oxygen of Tyr466 prevents hydrogen bond formation (3.36 Å). Instead of NH, the OH of the phenol ring forms a hydrogen bond with Tyr466 (3.11 Å). In addition, the OH forms hydrogen bonds with Tyr383 (2.91 Å) and Gln384 (2.79 Å). Thus,

five hydrogen bonds formed via the amine and the phenol ring to the catalytic triad and Gln384. In addition, the adamantyl group occupies the hydrophobic region surrounded by Phe267, Phe387, Leu408, Met419, and Leu428, as expected. These strong hydrogen bond networks and hydrophobic interactions might contribute to the potent inhibitory activity of compound 9. Compound 10 also binds to the catalytic triad via its NH (Fig. 8). NH forms hydrogen bonds with Asp335 (2.68 Å) and Tyr466 (2.59 Å); however, the oxygen of Tyr383 prevents hydrogen bond formation (3.42 Å). Methylthiophene forms hydrophobic and van

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Figure 7. Comparison of hydrogen bonds with catalytic triads. (A) t-AUCB (PDB ID code: 3WKE). (B) Fragment 1. Arrows indicate predicted directions of hydrogen donation.

Figure 8. Crystal structures of compounds 9 and 10 bound to sEH. Magenta ball and stick models indicate compounds. Light blue ribbon diagrams and stick models indicate protein structures. Omitted F0  Fc maps are shown (contoured at 2.5 r).

der Waals interactions with Trp336 and Met339. The fluorophenyl moiety forms hydrophobic and van der Waals interactions with Phe267, Leu408, His524 and Trp525. However, this moiety does not completely occupy the hydrophobic region. Substitution of fluorophenyl with more bulky functional groups such as adamantyl might therefore increase the potency of compound 10.

N-ethylmethylamine derivatives will help facilitate the identification of novel and potent sEH inhibitors.

3. Conclusions

Fragments 1 to 8 were purchased from Kishida Chemical (Osaka, Japan). Compound 9 was purchased from ChemBridge (San Diego, CA, USA) and compound 10 from TimTec (Newark, DE, USA).

Crystallographic fragment screening can detect fragment hits that, although weak, bind specifically to their target. Structural analysis of these hits identified new scaffolds that bind to the catalytic triad of sEH. In particular, N-ethylmethylamine is a promising scaffold for the optimization of compounds to fit the unique catalytic pocket of sEH, which is separated into two sub-pockets via a catalytic triad. The potential of N-ethylmethylamine was demonstrated by the IC50 values of compound 9 (0.51 lM) and 10 (9.0 lM). Structural analysis of compound 9 also revealed a hydroxyl group as a new pharmacophore. Further optimization of

4. Experimental procedure 4.1. Materials

4.2. Expression, purification and crystallization of sEH Expression, purification and crystallization of sEH were performed as previously described.19 The full-length cDNA of human sEH (GeneBank accession number NM_001979) was subcloned into the pET-30b vector (Merck Millipore, Billerica, MA, USA) containing a C-terminal His6-tag. The construct was then transformed

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into Escherichia coli BL21(DE3) cells for expression. The expressed protein was purified using a Ni-NTA Superflow column (QIAGEN, Hamburg, Germany) followed by ion-exchange chromatography (Q-Sepharose Fast Flow, GE Healthcare, Waukesha, WI, USA) and gel filtration (HiLoad 16/60 Superdex 200, GE Healthcare). For crystallization, the purified protein was concentrated from 15 to 20 mg/ml. Apo-crystals of sEH were obtained using the sittingdrop vapor diffusion method with a reservoir solution containing 0.1 M potassium phosphate (pH 7.5), 0.2 M ammonium dihydrogen phosphate, and 22% (w/v) polyethylene glycol PEG3350. Hexagonal-prism shaped crystals with dimensions of 50 to 100  300 lm were observed from 3 to 4 days 4.3. Fragment library A fragment library of 800 compounds was selected from our inhouse chemical library based on structural diversity, molecular mass, and predicted solubility. Molecular mass of compounds ranged from 151 to 250 Da, and mean molecular mass was 221 Da. The library was divided into 80 cocktails of 10 fragments each for X-ray crystallographic screening. Each fragment was dissolved in DMSO to a final concentration of 100 mM, and the 10 solutions were mixed (10 mM for each fragment).

LE ¼ DG=HAC  RTlnðIC50 Þ=HAC where HAC denotes heavy atom count. Acknowledgements The authors thank Dr. Kazuhiro Yokoyama for assembling the fragment library. The authors thank Dr. Tatsuya Niimi and Dr. Hitoshi Sakashita for their helpful discussions. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmc.2015.03.083. References and notes 1. 2. 3. 4. 5. 6. 7. 8.

4.4. Soaking, data collection and refinement For soaking with cocktails, a 1-ll aliquot of each fragment cocktail was added to a 9-ll aliquot of mother liquor containing 0.1 M potassium phosphate (pH 7.5), 0.2 M ammonium dihydrogen phosphate, and 25% (w/v) polyethylene glycol PEG3350. For soaking with single compound, each compound was added to the mother liquor at a final concentration of 5 mM. Apo-crystals of sEH were transferred to the mother liquor-containing compound and incubated for 4 h at room temperature. Crystals were flash-frozen with glycerol for X-ray data collection. X-ray diffraction data were collected in BL5A or AR-NW12A beamlines at the Photon Factory in the National Laboratory for High Energy Physics (KEK) and on a Rigaku FR-E+ Superbright X-ray generator equipped with a Rigaku R-AXIS VII detector (Rigaku, Tokyo, Japan). Diffraction data were processed using HKL-200035 or CrystalClear (Rigaku). Crystal structures of sEH were determined by the molecular replacement method using AMoRe with 1S8O as a search model.36 Initial refinement was performed using REFMAC37 from the CCP4 suite.38 Compounds were fitted into electron densities observed in initial F0  Fc maps using AFITT (OpenEye Scientific Software, Santa Fe, NM, USA). Water placements and further refinements were performed using Coot39 and REFMAC, and the final models were determined. Coordinates were deposited in the Protein Data Bank (PDB ID code: 4Y2J, 4Y2P, 4Y2Q, 4Y2R, 4Y2S, 4Y2T, 4Y2U, 4Y2V, 4Y2X, and 4Y2Y). 4.5. Enzyme assay sEH assays were conducted as previously described.19 Substrate PHOME was purchased from Cayman Chemical Company (Ann Arbor, MI, USA). The reaction buffer contained 25 mM bis–Tris HCl, pH 7.0 and 0.02% (v/v) Triton X-100. Before initiating the reaction, sEH was incubated with inhibitor for 15 min at room temperature. Reactions were started by adding PHOME (50 lM, final concentration). After incubation for 50 min at room temperature, fluorescence was measured using an ARVO microplate reader (PerkinElmer, Billerica MA, USA). A dose–response curve was plotted, and the IC50 value was calculated. Assays were repeated twice and the final IC50 value was a mean of two assays. LE was calculated using the following equation:

9.

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Identification of N-ethylmethylamine as a novel scaffold for inhibitors of soluble epoxide hydrolase by crystallographic fragment screening.

Soluble epoxide hydrolase (sEH) is a potential target for the treatment of inflammation and hypertension. X-ray crystallographic fragment screening wa...
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