Fragment Library Screening Reveals Remarkable Similarities Between the G Protein-Coupled Receptor Histamine H4 and the Ion Channel Serotonin 5-HT3A Mark H

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5460–5464

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Bioorganic & Medicinal Chemistry Letters

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Fragment library screening reveals remarkable similarities between the G protein-coupled receptor histamine H4 and the ion channel serotonin 5-HT3A Mark H. P. Verheij a, Chris de Graaf a, Gerdien E. de Kloe a, Saskia Nijmeijer a, Henry F. Vischer a, Rogier A. Smits b, Obbe P. Zuiderveld a, Saskia Hulscher a, Linda Silvestri c, Andrew J. Thompson c, ⇑ Jacqueline E. van Muijlwijk-Koezen a, Sarah C. R. Lummis c, Rob Leurs a, Iwan J. P. de Esch a, a Leiden/Amsterdam Center of Drug Research (LACDR), Division of Medicinal Chemistry, Faculty of Sciences, VU University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands b Grifﬁn Discoveries BV. De Boelelaan 1083, Room P-246, 1081 HV Amsterdam, The Netherlands c Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, UK article info abstract

Article history: A fragment library was screened against the G protein-coupled histamine H4 receptor (H4R) and the Received 2 May 2011 ligand-gated ion channel serotonin 5-HT3A (5-HT3AR). Interestingly, signiﬁcant overlap was found Revised 27 June 2011 between H4R and 5-HT3AR hit sets. The data indicates that dual active H4R and 5 HT3AR fragments have Accepted 28 June 2011 a higher complexity than the selective compounds which has important implications for chemical Available online 2 July 2011 genomics approaches. The results of our fragment-based library screening study illustrate similarities

in ligand recognition between H4R and 5-HT3AR and have important consequences for selectivity profiling Keywords: in ongoing drug discovery efforts on H R and 5-HT R. The affinity profiles of our fragment screening Fragment-based lead discovery (FBLD) 4 3A studies furthermore match the chemical properties of the H R and 5-HT R binding sites and can be used Chemogenomics 4 3A to define molecular interaction fingerprints to guide the in silico prediction of protein-ligand interactions Serotonin 5-HT3A receptor

Fragment-based lead discovery (FBLD) uses low molecular a wide array of chemicals on a wide array of biological targets is weight compounds as starting points for hit and lead optimization. investigated.7 The resulting two-dimensional matrix of targets ver- Compared to the drug-like compounds that are screened in typical sus hit compounds is useful for the discovery of ligands for novel high-throughput screening campaigns, fragments are better able to drug targets and to have better control over the selectivity of li- cover the corresponding chemical space. Consequently, typical gands and/or drugs. Furthermore, the data can lead to a better fragment libraries consist of about 1000 small molecules.1 Bio- understanding of ligand-receptor interactions. chemical and biophysical techniques are used to detect the low We have screened our fragment library against the histamine affinity fragment binding. Ligand efficiency (LE), defined as the H4 receptor (H4R) for which we have ongoing drug discovery pro- À1 binding energy of the ligand (DG in kcal mol ) per non-H atom grams. H4R fragment hits were grown into potent H4R ligands and (Heavy Atoms, HA), is used to select the most promising hits and fragment-merging approaches resulted in efficient scaffold hop- 2 8,9 guide the optimization studies. Typical hit rates for a fragment li- ping towards new chemical series. The H4R is considered a very brary screen are considerably higher than for the high throughput promising target for treating inflammatory and allergic disorders screening of drug-like compounds.3 The higher complexity of the as well in the modulation of pain and pruritis.10 latter compounds drastically reduces the chances of perfect com- Meanwhile, the same fragment library is being screened against plementarity with the biological targets. Thus, fragments are par- a rapidly expanding variety of targets. Here, we describe a remark- 4,5 ticularly suited to probe the binding site of receptors, and are able overlap of the fragment hit set of the H4R and the 5-HT3AR. therefore ideal tools in chemogenomic approaches that link chem- This ligand-gated ion channel is a drug target for the treatment ical with biological space.6 In chemogenomics studies the effect of of irritable bowel syndrome (IBS) and chemotherapy-induced nau- 11 sea and vomiting (CINV). Marketed drugs of 5-HT3AR include tropisteron (NavobanÒ) and palonosetron (AloxiÒ). The results of ⇑ Corresponding author. Address: Division of Medicinal Chemistry, Faculty of Sciences, VU University Amsterdam, Room: G-379a, De Boelelaan 1083, 1081 HV our fragment-based library screening indicate similarities in ligand Amsterdam, The Netherlands. Tel.: +31 205987841; fax: +31 205987610. recognition between H4R and 5-HT3AR and potential selectivity is- E-mail address: [email protected] (I.J.P. de Esch). sues when developing H4R or 5-HT3AR drugs. On the other side,

0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2011.06.123 M. H. P. Verheij et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5460–5464 5461 dual activity compounds might also have clinical advantages. Next at a concentration of 100 lM using stably expressed human to the established role of 5HT3AR in IBS, recent findings also sug- 5-HT3AR in HEK293 cells. Binding affinities of hits were determined 3 gest a role of H4R in this disease. It has been found that an in- using radioligand binding studies measuring [ H]granisetron creased innate immune activity in the intestinal mucosa and in binding using membranes of HEK293 cells expressing the human 12 18 blood is found in subpopulations of patients with IBS. Mast cells 5-HT3AR. 15 and monocytes seem to be particularly important and might indi- The SCA plot in Figure 1a shows the distribution of 5-HT3AR cate that the H4R is also involved in this ailment. selective hits, H4R selective hits, and dual 5-HT3AR/H4R hits in the We screened the biological activity of a diverse set of 1010 frag- chemical space covered by the fragment library and demonstrates ment-like molecules against H4R and 5-HT3AR. The compounds in the structural diversity of the fragment hits. Interestingly, signifi- 13 this library obey general fragment library rules : (i) heavy atoms cant overlap between the H4R and 5-HT3AR hit sets occur, for exam- count 6 22; (ii) clogP <3; (iii) number of H-bond donors 6 3; (iv) ple, 24% of the 5-HT3AR hits also bind H4R and 30% of the H4R hits also number of H-bond acceptors 6 3; (v) number of rotatable bind 5-HT3AR(Fig. 1b). This is ca. 10% higher than any other overlap bonds 6 5. The fragments furthermore contain at least one ring between non-related targets that we have screened so far. In Table 1 14 structure and do not contain reactive functional groups. The struc- some selective H4R ligands, selective 5-HT3AR ligands as well as tural diversity of the library was analysed, among others, by means compounds with affinity for both receptors are displayed. Dual hits 15 of a scaffold classification analysis (SCA). In this analysis, frag- 7, 8, 11 have comparable affinities for 5-HT3R and H4R, while dual hit ments are indexed by two parameters, that is, cyclicity and complex- 9 has 500-fold selectivity for 5-HT3R over H4R, and hit 10 has 200- ity. Cyclicity is the ratio between ring atoms and side chain atoms fold for H4R over 5-HT3R(Table 1). (thus, if all the atoms of the molecule belong to the ring structure Many of the dual H4R/5-HT3AR ligands contain a quinazoline, cyclicity equals one). In addition, the complexity was calculated as quinoxaline, aminopyrimidine, imidazole, or benzimidazole scaf- a descriptor of the size and shape of the scaffold, taking into account fold in combination with a positively ionizable ring system the smallest set of smallest rings, the number of heavy atoms, the (Table 1). Figure 1c shows that most of these dual H4R/5-HT3AR number of bonds between the heavy atoms, and the sum of heavy fragments have a higher complexity than the H4R and 5-HT3AR atoms atomic number.15 Chemical diversity of the fragment library selective fragments. The structural complexity of 71% of the dual is furthermore confirmed by the fact that only 1.6% of the pair wise 5-HT3A/H4R fragments is 0.7 or higher, while 79% of the H4R selec- comparisons of the ECFP-4 topological fingerprints of the fragments tive hits and 74% of the 5-HT3AR selective fragments is lower than give Tanimoto similarity values higher than 0.26.16 0.7. While earlier chemoinformatics analyses suggested that

For the H4R fragment screen a radioligand displacement study selective ligands are more complex in terms of pharmacophore fea- was performed at a 10 lM fragment concentration. Hits were as- tures4 and molecular shape19, our fragment-based chemogenomics signed when the fragment displaced 50% or more of the radioli- study suggests a more delicate balance between ligand complexity gand, resulting in 56 hits (hit rate: 6%). Radioligand binding was and target selectivity. Our fragment library screening data indicate measured by displacement of [3H]histamine using membranes of that fragments need to have high enough complexity to hit several 17 HEK293 cells transiently expressing the human H4R. For the hit targets, but low enough complexity to be too specific for a single compounds, affinities were determined by subsequent radioligand site. This is in line with the theoretical model by Hann and displacement studies. co-workers4 that describes probability of finding a hit when con-

For the 5-HT3AR we performed a high throughput functional sidering the complexity of the ligand. The probability of detecting fragment screen18 using a fluorescent readout (Flex Station) apply- a binding event is given by multiplying the probability of matching ing a fluorescent membrane potential dye. With this screening features and the probability of being able to detect low affinity technique we can identify compounds that have affinity for the binders. Our experimental data set shows that indeed the chance receptor and in addition classify the hits as agonists, antagonists of finding fragment hits on two different targets favors higher or inactive. From this fragment screening we identified 70 hits complexity compounds. The relatively high complexity of the for the 5-HT3A receptor (hit rate: 7%). Fragments were screened overlapping H4R and 5-HT3AR fragment hit set is furthermore a

ab11 1 2 1 5 8 3

H4R 5-HT3AR 0.9 4 7 10 9 39 17 53 0.8 c 0.7 100 6 Inactive Cyclicity 80 0.6 H4R Selective % 60 40 0.5 5-HT3AR Selective 20 Dual Binders 0.4 0 0.3 0.4 0.5 0.6 0.7 0.8 0.9 5 0. 0.9 < 0.4 - 0.6 - 0.7 - 0.8 - Complexity 0.4 - 0.5 0.6 0.7 0.8 Complexity

Figure 1. (a) SCA plot showing the hit distribution for the H4R (Red), the 5-HT3AR (blue), the 5-HT3AR and H4R (green) as well as inactives, compounds which do not bind H4R or 5HT3AR (grey). Hits presented in Table 1 are labeled by their corresponding number. (b) Schematic representation of the overlap between H4R and 5-HT3AR ligands. (c)

Distribution of the complexity of H4R selective fragments (red line), 5-HT3AR selective fragments (blue line), dual H4R/5-HT3AR fragments (green line), and inactives (dotted grey line). 5462 M. H. P. Verheij et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5460–5464

Table 1 0.8 Structures of fragments that bind solely H4R (1–3), solely 5-HT3AR (4–6) and both

H4R and 5-HT3AR (7–11)

#H4R/5-HT3AR Structure Afﬁnity (pKi) 0.7 a c H4R 5-HT3AR N N 1 7.0 ± 0.1 n.a. 0.6 NH N NH Inactive 2 N 6.2 ± 0.1 n.a. 0.5 H H4R Selective 2 5-HT3AR Selective 11 N Dual Binders 0.4 3 N 6.7 ± 0.0 n.a. 1 N N HN 0.3

4 n.a. 6.1 ± 0.2

Cl 0.2 Histamine Similarity (ECFP-4 Tc)

5 n.a. 6.0 ± 0.0 NNH 2 0.1 6, 9 NH2 6 N NH n.a. 6.1 ± 0.1 H 7, 8, 10 3, 4, 5 0.0 N 0.0 0.1 0.2 0.3 0.4 NN 7 6.2 ± 0.0 6.6 ± 0.3 Serotonin Similarity (ECFP-4 Tc)

Cl Figure 2. Chemical similarity (ECFP-4 Tc) between dual and selective hits of H4R N and 5-HT3AR and the endogenous ligands of H4R (histamine) and 5-HT3AR (serotonin). NN 8 7.2 ± 0.0 7.9 ± 0.3 N O H binders are histamine-like (including 11 see Table 1). These data

N show that complex H4R/5-HT3AR dual fragments are dissimilar from the (less complex) endogenous ligands of H4R and 5-HT3AR. 9 Cl N 6.1 ± 0.1b 8.8 ± 0.1 The higher complexity of the dual H4R and 5-HT3AR fragments is N further illustrated by the analysis of the physical-chemical distri- H butions of fragment hits (Fig. 3). Whereas most properties are sim- N ilar when comparing the selective and the dual activity fragments N (see Table S1, S2 and Figure S1 for details21), the number of rings 10 8.2 ± 0.1 5.9 ± 0.1 and the heavy atom count (and associated molecular weight) are N N higher for the dual activity hits compared to for H4R and 5-HT3AR

NH2 selective fragments (Fig. 3 and Table S2). N The fragment screening does not only illustrate similarities in 11 6.2 ± 0.1b 5.9 ± 0.3 H4R and 5-HT3AR binding profiles, but also identifies subtle differ- NH ences between the properties of selective receptor ligands. Figure 3 shows that the number of H-bond donor atoms is significantly high- n.a.: Non active. a Measured by displacement of [3H]histamine binding using membranes of er for the H4R selective fragments (on average 1.7 H-bond donors)

HEK293 cells transiently expressing the human H4R. pKi’s are calculated from at than for 5-HT3AR selective fragments (on average 0.8 H-bond least three independent measurements as the mean ± SEM. donors). This can be correlated with the H4R ligand pharmaco- b Determined using membranes of SK-N-MC cells transiently expressing the 22 phore that contains two H-bond donors. In the H4R binding pocket, human H4R. pKi’s are calculated from at least three independent measurements as the mean ± SEM. these features are complementary to two negatively charged c 3 22 pKi: Measured by displacement of [ H]granisetron binding using membranes of residues, D3.32 and E5.46 (Fig. 4). As a result of these strong 23 HEK293 cells expressing the human 5-HT3AR. pKi’s are calculated from at least two non-hydrophobic interactions between ionizable H-bonding independent measurements as the mean ± SEM. partners, many H4R ligands (including the high affinity endogenous ligand histamine) can bind the receptor with a high lipophilic 24 efficiency, explaining the relatively low c log P values of H4R ligand clear indication that the ligand recognition profiles of these recep- (Fig. 3). In the 5-HT3AR binding pocket one essential glutamate tors are similar. Figure 2 shows the chemical similarity of the frag- H-bond interaction partner (E129) has been identified (Fig. 4).25 ment library compared to serotonin and histamine (determined by Ligand binding to 5-HT3AR is furthermore largely determined by Pipeline Pilot ECFP-4 circular fingerprint20 Tanimoto similarity aromatic interactions like p–p stacking and cation-p interactions 25,26 coefficients (Tc)). While some of the H4R selective fragments (W183, W195, Y141, Y143, Y153, Y234, see Fig. 4) , matching (38%) are chemically similar to histamine (i.e., ECFP-4 Tc >0.26 the requirement of a lower number of H-bond donors (and including 2 (Table 1)), none of the 5-HT3AR selective fragments somewhat higher hydrophobicity) for 5-HT3AR ligands compared share chemical similarity serotonin-like, and only two of the dual to H4R ligands. In line with the notion that the H4R binding site M. H. P. Verheij et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5460–5464 5463

100 100 100 Inactive 80 80 80 H4R Selective % 60 % 60 % 60 5-HT3AR Selective 40 40 40 Dual Binders 20 20 20 0 0 0 0123

# H-Bond Donors clogP # Heavy Atoms

Figure 3. Distribution of physical-chemical properties that discriminate H4R selective fragments (red), 5-HT3AR selective fragments (blue line), dual H4R/5-HT3AR fragments (green), and inactives (grey dotted line). Distributions of other physical-chemical properties do not discriminate between the sets as shown in Figure S1

a F7.39 H4R 5-HT3AR Y6.51

E129

Y3.33 Y153 D3.32 E5.46 Y234 HOH2

C3.36 W183 HOH1

H4R

5-HT3AR

1) contact; 2) face-to-face stacking; 3) edge-to-face stacking; 4) HB acc.-HB don.; 5) HB don.-HB acc.; 6) neg.-pos.; 7) pos.-neg. (extra: 8) cation-pi)

Figure 4. Panel A shows the predicted binding modes of the dual H4R/5-HT3AR hit 8 (green carbon atoms, see Table 1 for molecular structure) in structural models of H4R and

5-HT3AR. Parts of the backbone of transmembrane (TM) helices 3, 5, 6 and 7 (the top TM3 is not shown for clarity) in H4R and loops A, B, C and E of the extracellular ligand- binding domain (ECD) of 5-HT3AR are represented by light yellow ribbons. Important binding residues are depicted as ball-and-sticks with grey carbon atoms. Oxygen, nitrogen, and hydrogen atoms are colored red, blue, and cyan, respectively. H-bonds described in the text are depicted by black dots. The molecular interaction fingerprint 27 (IFP) bit strings of 8 in H4R and 5-HT3AR are compared in panel B. contains two essential H-bonding acceptor atoms and the 5-HT R H O 3A NH NH site only one such atom, is the observation that fragment 10 N N possesses affinity for both 5-HT3AR and H4R, whereas the H analogous fragment 3 that lacks an NH2 group, only shows affinity N N N N for the H4R. Figure 4 demonstrates how the affinity profiles from our frag- NH2 NH2 ment screening studies correspond with the chemical properties 12 13 of the H4R and 5-HT3AR binding sites and can be used to derive molecular interaction fingerprints27 and validate structural models H R: pK : 7.1 H R: pK :7.1 of protein-ligand complexes.28 In both H R17 and 5-HT R models 4 i 4 i 4 3A 5-HT R:98%inh.at10µM 5-HT R: 98% inh. at 10µM (see Supplementary data for a description of the protein modeling 3A 3A procedure), the positively ionizable piperazine group of the dual Figure 5. Compounds in preclinical trials by Abbott. H4R/5-HT3AR hit 8 forms a salt bridge (D3.32 in H4R, E129 in 5-HT3AR) and makes cation-p (F7.39 in H4R, Y234 and W183 in 5- HT3AR) and aromatic p–p stacking interactions (Y3.33 and Y6.51 protein-ligand H-bond interaction network in several crystal struc- 29 in H4R, Y153 in 5-HT3AR). Interestingly, while C3.36 and E5.46 are tures of the homologous AChBP ) fulfill the same role in 5-HT3AR. proposed to act as H-bond donor and acceptor to the carboxamide The binding mode modes of 8 presented in Figure 4 do not only group of 10 in H4R, two water molecules (which form a conserved match the fragment-based chemogenomics analysis reported in 5464 M. H. P. Verheij et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5460–5464 the current study, but are also supported by earlier reported site-di- screens) associated with this article can be found, in the online ver- rected mutagenesis studies, underlining the important role of E129, sion, at doi:10.1016/j.bmcl.2011.06.123. 25,26 W183, Y153, and Y234 in ligand binding to 5-HT3AR, and the 22–24 essential role of D3.32 and E5.46 in H4R-ligand interactions. References and notes The binding orientation of 8 is furthermore in line with previously 17 1. de Kloe, G. E.; Bailey, D.; Leurs, R.; de Esch, I. J. Drug Discovery Today 2009, 14, experimentally validated ligand binding modes in H4R. 630. Chemogenomics analyses of inter-gene family ligand promiscu- 2. Hopkins, A. L.; Groom, C. R.; Alex, A. Drug Discovery Today 2004, 9, 430. 30 ity is of growing interest. Although GPCRs and LGICs obviously 3. Schuffenhauer, A.; Ruedisser, S.; Marzinzik, A. L.; Jahnke, W.; Blommers, M.; have a very different protein architecture, their ligand-binding Selzer, P.; Jacoby, E. Curr. Top. Med. Chem. 2005, 5, 751. 4. Hann, M. M.; Leach, A. R.; Harper, G. J. Chem. Inf. Comput. Sci. 2001, 41, 856. sites can obviously bind similar (sub)structures. In this respect, 5. Hajduk, P. J.; Huth, J. R.; Tse, C. Drug Discovery Today 2005, 10, 1675. the special chemical taxonomy of serotonin, that binds to several 6. Chen, I. J.; Hubbard, R. E. J. Comput. Aided Mol. Des. 2009, 23, 603. 31 7. Rognan, D. Br. J. Pharmacol. 2007, 152, 38. GPCRs and one ion-channel (5-HT3A) has been previously noted. Moreover, Mestres and co-workers have recently reported a strik- 8. Smits, R. A.; de Esch, I. J.; Zuiderveld, O. P.; Broeker, J.; Sansuk, K.; Guaita, E.; Coruzzi, G.; Adami, M.; Haaksma, E.; Leurs, R. J. Med. Chem. 2008, 51, 7855. ing cross-pharmacology between aminergic GPCRs and the 5-HT3 9. Smits, R. A.; Lim, H. D.; Hanzer, A.; Zuiderveld, O. P.; Guaita, E.; Adami, M.; receptors in their in silico target profiling platform.32 Our fragment Coruzzi, G.; Leurs, R.; de Esch, I. J. J. Med. Chem. 2008, 51, 2457. screening studies complement these findings by identifying rela- 10. Westly, E. Nat. Med. 2010, 16, 1063. 11. Thompson, A. J.; Lummis, S. C. Expert Opin. Ther. Targets 2007, 11, 527. tively high fragment cross-reactivity between H4R and 5-HT3AR 12. Ohman, L.; Simren, M. Nat. Rev. Gastroenterol. Hepatol. 2010, 7, 163. ( Fig. 1b) and demonstrate that fragments are ideally suited to 13. Siegal, G.; Ab, E.; Schultz, J. Drug Discovery Today 2007, 12, 1032. interrogate ligand binding sites. In the hit optimization phase, 14. Oprea, T. I. J. Comput. Aided Mol. Des. 2000, 14, 251. 15. Xu, J. J. Med. Chem. 2002, 45, 5311. selectivity for either H4R or 5-HT3AR can be achieved, although in 16. Steffen, A.; Kogej, T.; Tyrchan, C.; Engkvist, O. J. Chem. Inf. Model 2009, 49, 338. some cases this might proof complicated. This is illustrated by re- 17. Lim, H. D.; de Graaf, C.; Jiang, W.; Sadek, P.; McGovern, P. M.; Istyastono, E. P.; cent publications from Abbott Laboratories.33,34 These studies, that Bakker, R. A.; de Esch, I. J.; Thurmond, R. L.; Leurs, R. Mol. Pharmacol. 2010, 77, 734. are part of their H4R drug development program, describe the 18. Thompson, A. J.; Verheij, M. H. P.; Leurs, R.; De Esch, I. J. P.; Lummis, S. C. R. in vitro and in vivo characterization of the H4R ligands 12 and 13 Biotechniques 2010, 49,8. (A-940894). Intriguingly, both these compounds ( Fig. 5) show 19. Clemons, P. A.; Bodycombe, N. E.; Carrinski, H. A.; Wilson, J. A.; Shamji, A. F.; Wagner, B. K.; Koehler, A. N.; Schreiber, S. L. Proc. Natl. Acad. Sci. U.S.A. 2010, strong inhibition at the 5-HT3AR receptor (98% inhibition at 107, 18787. 10 lM). It is noted that these compounds contain the 2-amino-4- 20. Accelrys Software Inc. S. D., C., USA. piperazine-pyrimidine scaffold that was also identified as binding 21. Distributions of physical-chemical properties of fragment hits and to both H R and 5-HT R in our fragment-screening (fragment experimental details of H4R and 5-HT3AR screens are available in the 4 3A Supplementary data 10, table 1). 22. Jongejan, A.; Lim, H. D.; Smits, R. A.; de Esch, I. J.; Haaksma, E.; Leurs, R. J. Chem. In conclusion, the present study identifies a significant overlap Inf. Model 2008, 48, 1455. between the hit fragment set for H R and 5-HT R, illustrating sim- 23. Shamovsky, I.; de Graaf, C.; Alderin, L.; Bengtsson, M.; Bladh, H.; Borjesson, L.; 4 3A Connolly, S.; Dyke, H. J.; van den Heuvel, M.; Johansson, H.; Josefsson, B. G.; ilarities in ligand recognition and suggests that fragment-based Kristoffersson, A.; Linnanen, T.; Lisius, A.; Mannikko, R.; Norden, B.; Price, S.; chemogenomics analysis and molecular modeling building can be Ripa, L.; Rognan, D.; Rosendahl, A.; Skrinjar, M.; Urbahns, K. J. Med. Chem. 2009, used to efficiently navigate chemical space during hit optimization 52, 7706. 24. Leeson, P. D.; Springthorpe, B. Nat. Rev. Drug Disc. 2007, 6, 881. in programs aimed to develop selective leads or compounds with a 25. Price, K. L.; Bower, K. S.; Thompson, A. J.; Lester, H. A.; Dougherty, D.; Lummis, dual activity profile. S. Biochemistry 2008, 47, 6370. 26. Price, K. L.; Lummis, S. C. J. Biol. Chem. 2004, 279, 23294. 27. Marcou, G.; Rognan, D. J. Chem. Inf. Model 2007, 47, 195. Acknowledgments 28. de Graaf, C.; Rognan, D. Curr. Pharm. Des. 2009, 15, 4026. 29. Hibbs, R. E.; Sulzenbacher, G.; Shi, J.; Talley, T. T.; Conrod, S.; Kem, W. R.; Taylor, M.V. and I.dE. would like to acknowledge EEC grant (Neurocy- P.; Marchot, P.; Bourne, Y. EMBO J. 2009, 28, 3040. 30. Keiser, M. J.; Setola, V.; Irwin, J. J.; Laggner, C.; Abbas, A. I.; Hufeisen, S. J.; pres) for financial support. C.dG. acknowledges The Netherlands Jensen, N. H.; Kuijer, M. B.; Matos, R. C.; Tran, T. B.; Whaley, R.; Glennon, R. A.; Organization for Scientific Research (VENI Grant 700.59.408) for Hert, J.; Thomas, K. L.; Edwards, D. D.; Shoichet, B. K.; Roth, B. L. Nature 2009, financial support. This work was furthermore supported by COST 462, 175. Action BM0806COST. SCRL and AJT are Wellcome Trust funded 31. Keiser, M. J.; Irwin, J. J.; Shoichet, B. K. Biochemistry 2010, 49, 10267. 32. Gregori-Puigjane, E.; Mestres, J. Comb. Chem. High Throughput Screening 2008, (081925). There are no competing interests. 11, 669. 33. Liu, H.; Altenbach, R. J.; Carr, T. L.; Chandran, P.; Hsieh, G. C.; Lewis, L. G.; Manelli, A. M.; Milicic, I.; Marsh, K. C.; Miller, T. R.; Strakhova, M. I.; Vortherms, Supplementary data T. A.; Wakefield, B. D.; Wetter, J. M.; Witte, D. G.; Honore, P.; Esbenshade, T. A.; Brioni, J. D.; Cowart, M. D. J. Med. Chem. 2008, 51, 7094. Supplementary data (Distributions of physical-chemical prop- 34. Strakhova, M. I.; Cuff, C. A.; Manelli, A. M.; Carr, T. L.; Witte, D. G.; Baranowski, J. L.; Vortherms, T. A.; Miller, T. R.; Rundell, L.; McPherson, M. J.; Adair, R. M.; erties of fragment hits, descriptions of H4R and 5-HT3AR protein Brito, A. A.; Bettencourt, B. M.; Yao, B. B.; Wetter, J. M.; Marsh, K. C.; Liu, H.; modeling procedures and experimental details of H4R and 5-HT3AR Cowart, M. D.; Brioni, J. D.; Esbenshade, T. A. Br. J. Pharmacol. 2009, 157, 44.