Identification of indole inhibitors of human hematopoietic prostaglandin D2 synthase (hH-PGDS)

Bioorganic & Medicinal Chemistry Letters 25 (2015) 2496–2500

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Identification of indole inhibitors of human hematopoietic prostaglandin D2 synthase (hH-PGDS) Fredrik Edfeldt a,⇑, Johan Evenäs b, Matti Lepistö b, Alison Ward b, Jens Petersen a, Lisa Wissler a, Mattias Rohman a, Ulf Sivars b, Karin Svensson b, Matthew Perry b, Isabella Feierberg a, Xiao-Hong Zhou b, Thomas Hansson b, Frank Narjes b a b

Discovery Sciences, Innovative Medicines, AstraZeneca R&D, 431 83 Molndal, Sweden Respiratory, Inflammation and Autoimmunity, Innovative Medicines, AstraZeneca R&D, 431 83 Molndal, Sweden

a r t i c l e

i n f o

Article history: Received 10 March 2015 Revised 19 April 2015 Accepted 20 April 2015 Available online 28 April 2015 Keywords: Prostaglandin D2 synthase PGDS inhibitors Indole Focused screening Hit validation

a b s t r a c t Human H-PGDS has shown promise as a potential target for anti-allergic and anti-inflammatory drugs. Here we describe the discovery of a novel class of indole inhibitors, identified through focused screening of 42,000 compounds and evaluated using a series of hit validation assays that included fluorescence polarization binding, 1D NMR, ITC and chromogenic enzymatic assays. Compounds with low nanomolar potency, favorable physico-chemical properties and inhibitory activity in human mast cells have been identified. In addition, our studies suggest that the active site of hH-PGDS can accommodate larger structural diversity than previously thought, such as the introduction of polar groups in the inner part of the binding pocket. Ó 2015 Elsevier Ltd. All rights reserved.

Prostaglandin D2 (PGD2) is an allergic and inflammatory mediator produced by mast cells and Th2 cells after cross-linking of an allergen with the specific IgE antibody on the cell membrane.1 PGD2 acts on two G-protein-coupled receptors, DP1 and CRTH2 that have been associated with inflammatory conditions.2 Production of PGD2 in the peripheral tissues and in immune and inflammatory cells from the precursor prostaglandin H2 (PGH2) is mainly catalyzed by human hematopoietic prostaglandin D synthase (hH-PGDS). This has led hH-PGDS to be envisioned as a target for the treatment of asthma and inflammatory diseases.3 hH-PGDS is a 26-kDa cytosolic homodimer of the sigma class glutathione-Stransferase (GST) family. It depends on glutathione (GSH) for catalytic activity, which is further increased by divalent metal ions.4 Several inhibitors of hH-PGDS, most of them containing a signature bis-aryl amide motif, have been disclosed in recent years by a number of organizations, such as (1 and 2) Pfizer,5 (3) SanofiAventis,6 (4) Evotec,7 (5) AstraZeneca8 and (6) Taiho9 (Fig. 1). For a recent review of hH-PGDS inhibitors, see Ref. 3. Our previous efforts led to the identification of compound (5) as an hH-PGDS inhibitor. The current study reports our continued work on identifying novel inhibitors of this enzyme applying a fragment based screening approach, aiming to identify smaller ⇑ Corresponding author. Tel.: +46 31 776 1604; fax: +46 31 776 3792. E-mail address: [email protected] (F. Edfeldt). http://dx.doi.org/10.1016/j.bmcl.2015.04.065 0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

and more ligand efficient hits. A fluorescence polarization assay10 was used to screen two library subsets of 25,000 and 17,000 compounds at 125 lM and 250 lM, respectively. IC50 values were determined for compounds showing a >30% displacement effect, resulting in 1040 compounds with IC50 values <100 lM. To further validate the actives a 1D NMR binding assay was deployed. Kd values were determined using the competitive displacement of reporter ligands with known affinity.11 The hits were clustered using structural similarity and 216 cluster representatives were selected for testing in the NMR assay. 187 of these hits were confirmed in the NMR assay, corresponding to a validation rate of 87%. Validated hits were then tested in a GST enzymatic assay using CDNB (1-chloro-2,4-dinitrobenzene) as chromogenic substrate12 and IC50 values were generated. We chose to monitor the GST enzymatic activity of hH-PGDS rather than its PGD2 synthase activity due to technical feasibility. Both enzymatic activities reside in the same binding pocket.4b,13 For a set of four reference compounds (1–3, and 5) activity in the NMR and GST assays, as well as isothermal titration calorimetry (ITC),14 correlated overall well with the inhibition of PGD2 production in megakaryoblast cells15 (Table 1). We therefore felt confident that our screening set-up was fit for purpose in driving hit expansion chemistry in a high throughput manner. We observed excellent correlation between the NMR binding assay and inhibition of GST activity for our screening hits. 168 compounds had an IC50

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O N

N

O N

N

N CF3

1 N

N

O

N

N

HN

N

H N

HN

H N O

N

7

O

8

OH

O

N

4

N

HN

N O

N

S

N

N

HN

O

O

9

O

SO2NH2

Figure 2. Initial screening hits containing the indole motif.

3 O

O

S

5

N O

N

CF3

2

N N

H N

O

N N N

N

N

O

6

Figure 1. Examples of published inhibitors of hH-PDGS.

<10 lM, in good agreement with the NMR Kd values. We subsequently used binding (NMR) and enzymatic activity (GST) in combination to generate compound SAR. Known chemical motifs, such as aryl amides, were well represented among the hits, but a range of more novel structures were also identified. We found compounds containing an indole motif to be of particular interest (7, 8 and 9, Fig. 2). This class of compounds had reasonable physico-chemical properties in terms of calculated log P values (c log P), ligand efficiency (LE), ligand lipophilic efficiency (LLE) and solubility, and was therefore considered a promising starting point (Table 1). Compound 8 was tested for inhibition cellular PGD2 synthase activity and gave an EC50 value of 0.35 lM. We then proceeded to determine the crystal structures with compounds 7, 9, and the reference 2 bound to hH-PGDS, which were obtained by co-crystallization at a resolution of 1.8, 2.0 and 2.1 Å, respectively.18 We were unable to determine the structure with 8, but the binding mode is expected to be similar to 7. Several crystal structures of inhibitors in complex with hHPGDS have been described.5,8,19 The substrate pocket can be subdivided into three different regions: the inner cavity, typically occupied by a phenyl residue as evidenced in structures 1–6; the central cavity, typically occupied by a heterocycle residue, that stacks on Trp104, whereas the amide residue can be involved in interactions with Arg14 and/or the thiolate anion of bound GSH; and the peripheral solvent exposed part of the pocket, where the largest structural variations of inhibitors have been observed.9 The overall binding mode of indoles 7 and 9 resembles that of compound 2, with the indole motif overlapping with the phenyl ring of 2 (Fig. 3). The amide of 7 is reversed relative to 2 and 9. This moiety makes no apparent interactions, but the reverse amide provides a different way to extend compounds. Both the pyridine

Figure 3. Binding mode of indole hits 7 (brown) and 9 (green) overlaid with compound 2 (cyan) in complex with hH-PGDS, showing the conserved water, and key residues Trp104 and Tyr152 (pink). X-ray structure coordinates have been deposited in the RSCB Protein Data Bank (PDB) with accession codes 5AIX (2), 5AIS (7) and 5AIV (9). Crystallization protocol as previously reported.8

(in 7) and oxadiazole (in 9) rings make the critical stacking interaction with Trp104 and the important hydrogen-bonding interaction with the conserved water molecule previously described.5,8,19 In both 7 and 9 the indole binds in the inner cavity, making a hydrogen bond interaction with the hydroxyl group of Tyr152, which to our knowledge has not been observed before. The amine and ether moieties both protrude out of the binding pocket making no critical interactions. These structural features convinced us to further explore the structure activity relationship (SAR) of this series by synthesizing an amide library of related analogs. A set of 20 analogs were synthesized and tested in the NMR binding and GST enzymatic assays; the most relevant examples are shown in Table 2. Several compounds gave significantly improved IC50 values while also improving c log P, LE and LLE. The most potent compound (10) had an IC50 of 0.026 lM, a 7-fold improvement over 7. A large variety of functional R-groups were tolerated, consistent with SAR observed in the literature.5–7,9 This part of the molecule can be utilized to modulate physico-chemical properties, although modest affinity gains were also made. For

Table 1

a b c d e

Compd

FP IC50a (lM)

NMR Kda (lM)

ITC Kd (lM)

GST IC50a (lM)

Cell EC50a (lM)

c Log Pb

Solubilityc (lM)

LEd

LLEe

1 2 3 5 7 8 9

— 0.21 — — 0.82 0.46 2.9

0.0095 0.044 0.023 0.051 0.57 0.34 0.65

0.020 0.012 0.035 0.30 — 0.21 —

0.20 0.060 0.070 0.023 0.18 0.30 0.17

0.035 0.13 0.074 0.11 — 0.35 —

3.0 2.9 1.3 3.2 3.0 1.7 2.1

2.6 4.2 — 1.4 84 95 77

0.34 0.35 0.31 0.65 0.38 0.37 0.42

3.7 4.3 4.9 4.4 3.7 4.8 4.7

Values are the mean of at least two independent experiments. Calculated log P. Measured solubility. Ligand efficiency in kcal per mol per heavy atom.16 Ligand lipophilic efficiency17 defined as pIC50-c log P, calculated using GST IC50.

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Table 2

Table 3

N

H N

HN

Compd Compd

R

7

∗

8

NMR (lM) N

IC50a

b

11 12

13

R

b

LLE

GST (lM)

c Log P

LE

0.57

0.18

3.0

0.38

3.7

0.34

0.30

1.7

0.37

4.8

∗

16

0.026

3.0

0.40

4.6

0.06

0.032

2.9

0.51

4.6

O

0.3

0.035

1.4

0.42

6.1

N

0.2

0.043

1.9

0.42

5.5

1

0.13

3.9

0.36

3.0

∗ ∗

H N

∗

N

N ∗

17

N ∗

18 19

N

N

N ∗

N ∗

20

O

21

HN

N ∗

HN

N ∗

22 15

∗

N

<20

1.2

1.8

0.32

b

GST IC50a (lM)

c Log P

LEb

LLEb

0.076

0.060

2.9

0.35

4.3

0.06

0.031

3.1

0.38

4.5

<0.1

0.048

2.4

0.37

5.0

0.2

0.23

1.8

0.36

4.8

14

—

1.5

—

—

0.4

0.32

1.0

0.34

5.5

13

—

0.95

—

—

1.0

0.59

0.55

0.32

5.7

0.2

0.070

0.5

0.38

6.6

2

—

2.3

—

—

0.4

0.071

0.7

0.39

6.4

10

—

0.5

—

—

O

4.2

N a

∗

O

0.05

N

∗

∗

NMR Kda (lM)

N

∗

N 14

N

O

2

O

10

CF3 HN

O

N

Kda

O

R

R

23

∗

HN O

Values are the mean of at least two independent experiments. Calculated using GST IC50.

∗

N

24 O

instance, the lactam 12 and tetrazole 13 showed significant improvement in LLE. Compound 11 represents the highly ligand efficient minimal scaffold for the series with an IC50 of 0.032 lM and LE of 0.51. This is roughly equipotent to our previously reported compound 5, but it represents a more attractive starting point for further optimization. In parallel to these screening and chemistry efforts we pursued a structure-based design approach to further probe the binding pocket of hH-PGDS. The indole motif suggested that a larger variety of functional groups than previously thought could be tolerated in the inner binding pocket. To further explore this hypothesis a series of compounds related to 1 and 2 were synthesized. A variety of functional groups pointing into the inner cavity were introduced whilst keeping the rest of the molecule constant. We also included polar residues, which we envisioned would be able to engage with Tyr152, and result in overall good lipophilic ligand efficiency. Compounds were pre-screened in the NMR binding assay, and those with a Kd <1 lM were then further profiled in the GST assay (Table 3). All analogs shown in Table 3 proved to be competitive with respect to our reporter ligand. We used 2 for benchmarking, since it gave more consistent data in our hands and we had obtained a co-crystal structure. The methoxyphenyl group in 2 could not only be replaced with a phenoxy group as in 16 (IC50 = 0.031 lM), found also in compound 6, but also with saturated rings such as piperidine (17, IC50 = 0.048 lM). This resulted in a slight increase in potency and an increase in LLE, due to reduction of c Log P. Further SAR around 17 revealed that a pyrrolidine (18, IC50 = 0.23 lM) was still tolerated, whereas further reduction to a dimethylamino group (19) led to a substantial loss in binding. Introduction of a morpholine residue (20) further increased LLE, despite a 10-fold loss in potency with respect to 17, whereas a piperazine (21) was not tolerated, presumably due to the charged amine. Interestingly, the more polar, but neutral piperazinone 22 showed improved binding in the NMR assay and was also active in the GST assay. Further

∗

25

NH O

26 a b

∗

O HN

Values are the mean of at least two independent experiments. Calculated using GST IC50.

investigation around this motif revealed that the related pyridones 23 and 25 showed greatly improved activity, similar to compound 2, and due to the lower c Log P a significant increase in LLE. Regioisomer 26 on the other hand was not active, whilst methoxypyridine 24 resulted in a 10-fold loss in potency compared to 23, pointing to a critical role of the pyridone oxygen for activity. Our attempts to co-crystallize 23 with hH-PGDS were unsuccessful. To better understand the SAR around the novel motifs in the inner pocket, we docked the compounds into the available X-ray structures of hH-PGDS. hH-PGDS functions as a homodimer and the apo-enzyme is activated by Mg2+ and Ca2+ ions, which make multiple cross-linking interactions at the homodimer interface coordinating clusters of acidic residues from both monomers. Structures co-crystallized with inhibitors tend to be asymmetric compared to the Mg2+/Ca2+-complexed apo-structures, often with partial or no occupancy of ligand in the second pocket as well as a more disordered GSH. This asymmetry results in two slightly different kinds of inner cavities, where the Tyr152 OH can act either as an acceptor or as a donor.20 The overlay of pyridones 23 and 25 with indole 7 is shown in Figure 4. Both 23 and 25 appear to form a hydrogen bonding interaction with Tyr152 via the carbonyl oxygen rather than the NH, as seen for 7. The pyridinone 23 may also act as a donor, perhaps explaining its slightly higher potency versus 25. The 25 to 50-fold loss in binding potency of pyridone 26 could be explained by the less favorable positioning of the carbonyl oxygen with respect to 23. Similarly, the loss in potency for the

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2499

Compounds 16–22 were obtained by nucleophilic substitution of the 2-chloro-pyridyl carboxamide 29 with the appropriate phenol or amine. A Suzuki coupling between 29 and the appropriate methoxypyridyl boronate followed by deprotection with boron tribromide gave pyridines 23–26 (Scheme 2). Acknowledgments The authors would like to acknowledge BioDuro (Beijing, China) for assistance in chemical synthesis and Ewa Nilsson for the production of recombinant protein. References and notes

Figure 4. Alternative hydrogen bond network induced by indole versus pyridone substituents. The neighboring dimer subunit is indicated by the light-blue surface. According to modeling the electrostatics of the binding pocket and dimer interface,20 the Mg2+ is coordinated by Asp96 and Asp97 from one subunit with OH /H2O bridging the Mg2+ to Asp97 of the other subunit. This may then induce alternative side chain conformations for Arg14 and Asp96 and hydrogen bond arrangements involving Tyr152, which makes direct interactions with the inhibitors. This allows the phenolic group to act as an acceptor for indoles like compound 7 (light gray; Tyr152 in light blue) or a donor for pyridones 23 (blue; Tyr152 in brown) and 25 (green).

a, b Br

HN

NH 2

HN

27

H N

c HN

N

28

7, 10-15

R1 O

N

Scheme 1. Synthesis of indole-amide library. Reagents: (a) B2(pin)2, (Ph3P)4Pd, KOAc, dioxane, water (10:1), 90 °C, 86%; (b) 3-amino-5-bromopyridine, Pd(dppf)Cl2, Na2CO3, dioxane, water (10:1), 90 °C, 81%; (c) R1CO2H, POCl3, 20 °C, add 27, then to RT; 5-40%.

N

N

O

Cl

CF 3 HN

O

X

a or b

CF 3

R

N

29

HN

N

16 X=O 17-22 X = N

Br

c

B

d 23-26

N

OMe

N

OMe

Scheme 2. Synthetic scheme for inner cavity library. Reagents: (a) ROH, NaH, DMF, microwave, 150 °C; (b) RNH, DMF, K2CO3, 80 °C; (c) B2(pin)2 Pd(dppf)Cl2, KOAc, DMF, 80 °C; (d) 28, Pd(dppf)Cl2, Na2CO3, dioxane, water (10:1), 80 °C; BBr3, DCM, 0 °C.

piperazinone 22 could be explained by less than ideal geometry for the putative hydrogen bond interaction involving the carbonyl. These modeling results could help to rationalize how the structural diversity beyond simple phenyls can be accommodated in the inner part of the substrate binding pocket of hH-PGDS. Both affinity and physico-chemical properties could potentially be improved by manipulating this moiety. In conclusion, we have applied fragment based screening to identify novel inhibitors hH-PGDS containing an indole motif. This in turn led us to the discovery of previously unknown polar head groups for hH-PGDS, which could be used to tune compound properties. The synthesis of the indolyl-pyridine library is laid out in Scheme 1. Commercial 4-bromo-indole 27 was converted to the pinacolato boronate and then coupled with 3-amino-5-bromo pyridine to yield intermediate 28, which was acylated with acid chlorides obtained in situ from the corresponding acid (Scheme 1).

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Corrigendum ibid, 10, 409. 5. (a) Carron, C. P.; Trujillo, J. I.; Olson, K. L.; Huang, W.; Hamper, B. C.; Dice, T.; Neal, B. E.; Pelc, M. J.; Day, J. E.; Rohrer, D. C.; Kiefer, J. R.; Moon, J. B.; Schweitzer, B. A.; Blake, T. D.; Turner, S. R.; Woerndle, R.; Case, B. L.; Bono, C. P.; Dilworth, V. M.; Funckes-Shippy, C. L.; Hood, B. L.; Jerome, G. M.; Kornmeier, C. M.; Radabaugh, M. R.; Williams, M. L.; Davies, M. S.; Wegner, C. D.; Welsch, D. J.; Abraham, W. M.; Warren, C. J.; Dowty, M. E.; Hua, F.; Zutshi, A.; Yang, J. Z.; Thorarensen, A. Med. Chem. Lett. 2010, 1, 59; (b) Blake, T.; Hamper, B. C.; Huang, W.; Kiefer, J. R.; Moon, J. B.; Neal, B. E.; Olson, K. L.; Pelc, M. J.; Schweitzer, B. A.; Thorarensen, A.; Trujillo, J. I.; Turner, S. R. US 2008/0146569. 6. (a) Li, Z.; Xiong, J.; Sabol, J. S. WO 2004/016223; (b) Cao, Y.; Sabol, J. S.; Ayers, S. WO 2006/015195; (c) Aldous, S. C.; Jiang, J. Z.; Lu, J.; Ma, L.; Mu, L.; Munson, H. R.; Sabol, J. S.; Thurairatnam, S.; Vandeusen, C. L. WO 2007/041634; (d) Aldous, S. C.; Fennie, M. W.; Jiang, J. Z.; John, S.; Mu, L.; Pedgrift, B.; Pribish, J. R.; Rauckman, B.; Sabol, J. S.; Stoklosa, G. T.; Thurairatnam, S.; Vandeusen, C. L. WO 2008/121670; (e) Aldous, S. C.; Fennie, M. W.; Jiang, J. Z.; John, S.; Pedgrift, B.; Pribish, J. R.; Rauckman, B.; Sabol, J. S.; Stoklosa, G. T.; Thurairatnam, S.; Vandeusen, C. L. WO 2008/121670; (f) VanDeusen, C. L.; Weiberth, F. J.; Gill, H. S.; Lee, G.; Hillegass, A. WO 2011/044307; (g) Weiberth, F. J.; Yu, Y.; Subotkowski, W.; Pemberton, C. Org. Proc. Res. Dev. 2012, 16, 1967. 7. (a) Hesterkamp, T.; Barker, J.; Davenport, A.; Whittaker, M. Curr. Top. Med. Chem. 2007, 7, 1582; (b) Aicher, B.; Kelter, A. R.; Coulter, T. S.; Taylor, S.; Davenport, A. J.; Hesterkamp, T.; Kirchhoff, C. WO 2008/122787. 8. Hohwy, M.; Spadola, L.; Lundquist, B.; Hawtin, P.; Dahmén, J.; Groth-Clausen, I.; Nilsson, E.; Persdotter, S.; von Wachenfeldt, K.; Folmer, R. H. A.; Edman, K. J. Med. Chem. 2008, 51, 2178. 9. (a) Urade, Y.; Tanaka, Y.; Yamane, K.; Togawa, M. WO 2007/007778; (b) Urade, Y.; Shigeno, K.; Tanaka, Y.; Kuze, J.; Tsuchikawa, M.; Hosoya, T. JP 2007051121; (c) Yamane, K.; Tanaka, Y.; Shigeno, K.; Hosoya, T.; Inoue, S.; Kitade, M.; Harada, T.; Aoyagi, H.; Miyoshi, N.; Mutoh, T.; Togawa, M.; Kiniwa, M.; Yamasaki, Y. Abstracts of Papers, 235th National Meeting of the American Chemical Society, New Orleans, LA, 2008; Abstract MEDI-26. 10. Recombinant wild-type hH-PGDS used for in vitro assays and crystallization was expressed in Escherichia coli and purified using cation exchange chromatography and gel filtration as described in Ref. 8. The fluorescence polarization (FP) assay was adapted from that commercially supplied by Cayman Chemical Inc (Cat. No. 600007). Compound displacement of a potent hH-PGDS inhibitor conjugated to fluorescein (Cayman Chemical Inc Cat. No. 600025) was monitored in dose–response. Fluorescence polarization was measured with excitation and emission wavelengths of 470 nm and 530 nm, respectively, using an Envision reader (Perkin Elmer). Data analysis was performed with the HBase software package developed by AstraZeneca. 11. (a) A ligand-observed 1D NMR T1q binding assay was set-up monitoring the binding and displacement of a reporter molecule, either compound 1phenylpyrazole-4-carboxylic acid or 6-(3-fluorophenyl)pyridine-3carboxamide. Active site binding for the reporter molecules was confirmed by competition with compound 4. Kd values were calculated based on the level of displacement of reporter at two different compound concentrations, taking into account the experimentally determined concentrations of reporter and compound using the exact mathematical expression described in Wang, Z. X. FEBS Lett. 1995, 360, 111. Kd values for 1-phenylpyrazole-4-carboxylic acid and 6-(3-fluorophenyl)pyridine-3-carboxamide were determined to be 140 lM and 0.80 lM using isothermal titration calorimetry (ITC); (b) Dalvit, C.; Flocco, M.; Knapp, S.; Mostardini, M.; Perego, R.; Stockman, B. J.; Veronesi, M.; Varasi, M. J. Am. Chem. Soc. 2002, 124, 7702; (c) Jahnke, W.; Floersheim, P.; Ostermeier,

2500

12.

13. 14.

15.

16. 17. 18.

19.

20.

F. Edfeldt et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2496–2500

C.; Zhang, X.; Hemmig, R.; Hurth, K. Angew. Chem., Int. Ed. 2002, 41, 3420; (d) Holdgate, G. A.; Anderson, M.; Edfeldt, F.; Geschwindner, S. J. Struct. Biol. 2010, 172, 142. Rice, G.; Bump, E.; Shrieve, D.; Lee, W.; Kovacs, M. Quant. Cancer Res. 1986, 46, 6105. 60 nL of compound was added to a 1536 well plate (greiner) using an Echo dispenser, followed by the addition of 2 lL enzyme, 1.5 lg/ml in reaction buffer (50 mM tris, 2 mM MgCl2, 0.1% Chaps) using a Flexdrop dispenser. After incubation for 30 min, 2 lL of 0.4 mM MCBL and 2 lL of glutathione were added using the Flexdrop dispenser. The background was analysed by detecting fluorescence (ex 360, em 460) using a Pherastar reader, followed by incubation for 30 min. Finally, the fluorescence after 30 min was detected using the same settings as for the background. 0% inhibition was defined as the fluorescence for wells containing only DMSO, and 100% inhibition was defined as the fluorescence for a control compound. Urade, Y.; Fujimoto, N.; Ujihara, M.; Hayaishi, O. J. Biol. Chem. 1987, 262, 3820. Isothermal titration calorimetry experiments were performed by titrating 2 ll aliquots of ligand solution at an experimentally determined concentration (typically 500 lM) into a solution of 30–40 lM hH-PGDS. For compounds 1 and 5 reverse titrations, for which protein was titrated into ligand solution, were performed due to low ligand solubility. PGD2 production assays. MEG-01 (ECACC, cat.no.94012401) cells were differentiated with 0.1 lM PMA (Sigma, P1585) for 16 h. Cells were plated in 96-well plates and incubated in the presence of hH-PGDS inhibitor in concentration response, 10 lM–0.15 nM, for 30 min at 37 °C, followed by stimulation with 5 lM Ionomycin (Sigma, I0634-5MG) for 30 min at 37 °C. Secreted PGD2 present in the media was then measured with the PGD2 enzyme immunoassay (EIA) kit, Prostaglandin D2-MOX Express EIA Kit (Cat. No. 500151) according to the manufacturer’s instructions (Cayman Chemical Co.). We obtained an EC50 of 8.1 lM for HQL-79, which is in line with published results. Hopkins, A. L.; Groom, C. R.; Alex, A. Drug Discovery Today 2004, 9, 430. Springthorpe, B.; Leeson, P. D. Nat. Rev. Drug Disc. 2007, 6, 881. Crystallization and structure determination was performed essentially as described in Ref. 8. Compounds were co-crystallized with hH-PGDS. X-ray diffraction data were collected at the European Synchrotron Radiation Facility (ESRF), Grenoble, France. Trujillo, J. I.; Kiefer, J. R.; Huang, W.; Day, J. E.; Moon, J.; Jerome, G. M.; Bono, C. P.; Kornmeier, C. M.; Williams, M. L.; Kuhn, C.; Rennie, G. R.; Wynne, T. Y.; Carron, C. P.; Thorarensen, A. Bioorg. Med. Chem. Lett. 2012, 22, 3795. The metal ions change the hydration and the hydrogen bonding network at the dimer interface, releasing Arg14 from hydrogen bonding to one of the metalcoordinating Asp96 to stabilize glutathione thiolate. This lowers the KM for

GSH four-fold and stabilizes the thiolate nucleophile. The thiolate attacks the substrate endoperoxide and induces the rearrangement reaction. It also makes important hydrogen bonds with amide- or heterocycle-NH’s of many inhibitors including the ones reported here (see Ref. 4c). The inner cavity is separated from the dimer interface by the GSH thiolate, Arg14, Ser94, Asp96 and two water molecules forming what appears to be a flexible hydrogen bonding network. The presence of a hydroxide ion rather than a water molecule at the interface creates asymmetry around the Mg2+ ion coordination for Asp96. In order to evaluate our novel head groups we needed to construct a model which would not deviate from the initial coordinates under non-constrained minimizations. To obtain a stable model with respect to the position of the Mg2+ ions, the bridging water was deprotonated, which seems reasonable, since the Mg2+ ion is a fairly strong Lewis acid. Protonating the GSH amino terminus yielded a model that did not show significant deviations from the initial state, and which was used to evaluate the binding modes and approximate binding enthalpies of our compounds. The electrostatics of the system was assessed at the approximate conditions of crystallization, with the pH at 8–8.5 range. The PROPKA (Maestro/Schrodinger Inc.) routine was used to generate initial states. The refined models stabilities were checked by subjecting them to a 1000 step non-constrained minimization in the OPLS2005 force field (Macromodel/Maestro/Schrodinger Inc.). None of these models showed sufficient stability, especially regarding the position of the Mg2+-ion. The most stable models had one of the GSH N-termini deprotonated, losing interaction with Asp96 and staying fairly symmetrical with a water molecule bridging the Mg2+-ion and Asp97 from both monomers. However, deprotonating the bridging water and protonating the GSH amino terminus yielded a model that did not deviate significantly from the initial state (backbone RMSD 0.53, Mg2+ 0.44 Å). This model was used to evaluate the binding modes and approximate binding enthalpies of our compounds. The presence of a hydroxide ion rather than a water molecule at the interface creates asymmetry around the Mg2+-ion coordination for the Asp96–97 pairs. In one monomer, both Asp96 and Asp97 coordinate the Mg2+ ion and GSHamino and Arg14 OH-groups, respectively. In the other monomer, however, the hydroxide now bridges the Mg2+-ion and Asp97 (in turn bound to GSH-amino), while Asp96 accepts a hydrogen bond from the Tyr152-OH, rendering the latter a better acceptor for the indole-NH’s in the inhibitors 7–15 in Table 2. In effect, this creates two slightly different kinds of bottoms to the binding pockets of the two monomers, with the Tyr152 OH acting as an acceptor in one and as a donor in the other. It is unclear whether this is an artifact of our crystallization conditions or a functional allostery allowing for regulation of activity or reactivity (isomerase vs GSH-transferase).