Article pubs.acs.org/jmc
Identification of Ligand Binding Hot Spots of the Histamine H1 Receptor following Structure-Based Fragment Optimization Sebastiaan Kuhne,† Albert J. Kooistra,† Reggie Bosma,† Andrea Bortolato,‡ Maikel Wijtmans,† Henry F. Vischer,† Jonathan S. Mason,‡ Chris de Graaf,† Iwan J. P. de Esch,† and Rob Leurs*,† †
Amsterdam Institute for Molecules, Medicines and Systems (AIMMS), Division of Medicinal Chemistry, Faculty of Science, Vrije Universiteit Amsterdam, De Boelelaan 1108, 1081 HZ Amsterdam, The Netherlands ‡ Heptares Therapeutics Ltd., BioPark, Broadwater Road, Welwyn Garden City, Herts AL7 3AX, U.K. S Supporting Information *
ABSTRACT: Developments in G protein-coupled receptor (GPCR) structural biology provide insights into GPCR-ligand binding. Compound 1 (4-(2-benzylphenoxy)piperidine) with high ligand efficiency for the histamine H1 receptor (H1R) was used to design derivatives to investigate the roles of (i) the amine-binding region, (ii) the upper and lower aromatic region, and (iii) binding site solvation. SAR analysis showed that the amine-binding region serves as the primary binding hot spot, preferably binding small tertiary amines. In silico prediction of water network energetics and mutagenesis studies indicated that the displacement of a water molecule from the amine-binding region is most likely responsible for the increased affinity of the Nmethylated analog of 1. Deconstruction of 1 showed that the lower aromatic region serves as a secondary binding hot spot. This study demonstrates that an X-ray structure in combination with tool compounds, assessment of water energetics, and mutagenesis studies enables SAR exploration to map GPCR-ligand binding hot spots.
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INTRODUCTION In the past 8 years tremendous progress has been made in the crystallization of GPCRs, leading to more than 150 structures of 33 receptors representing different GPCR families and subfamilies,1 including aminergic GPCRs2 such as the histamine H1 receptor (H1R).3 With the emerging GPCR crystal structures2,4 a new opportunity has emerged to link ligand-based data with (protein) structure-based information. Among all available class A GPCR crystal structures, the H1R has the deepest ligand-binding pocket, with doxepin as the cocrystallized ligand (Figure 1A−C). The H1R antagonist binding site is divided into three regions: (i) the amine-binding region, (ii) the upper aromatic region, and (iii) the lower aromatic region (Figure 1C), and the importance of the residues lining these regions for ligand binding is supported by several site-directed mutagenesis studies.5−10 The aminebinding region is defined by D1073.32, W4286.48, Y4316.51, I4547.39, and Y4587.43 (Figure 1A,C). The amine moiety of doxepin interacts with the hallmark D1073.32 that is present in all aminergic GPCRs.4,11,12 In addition, the H1R has a cluster of © 2016 American Chemical Society
aromatic residues in TM3, -4, -5, and -6 that accommodate the aromatic ring systems of the H1R antagonists (Figure 1A,C) and can be divided into the upper aromatic region (defined by Y1083.33, W1584.56, Y4316.51, F4326.52, and F4356.55, Figure 1A,C) and the lower aromatic region (defined by F1995.47, F4246.44, and W4286.48, positioned deep in the transmembrane helical bundle, Figure 1A,C). Together, both the upper and lower aromatic regions accommodate the butterfly shape of the hydrophobic aromatic rings of the cocrystallized ligand doxepin (Figure 1A,C). It has been proposed that desolvation of these hydrophobic aromatic regions is an important determinant of GPCR ligand binding.13,14 Previously, we used the H1R X-ray structure for a highly successful virtual screening (73% hit rate) for new fragment-like H1R antagonists, resulting in the identification of VUF14544 (4-(2-benzylphenoxy)piperidine, 1) with an H1R affinity of 6 nM.15,16 Docking studies with 1 indicated that the fragment-like Received: July 4, 2016 Published: September 19, 2016 9047
DOI: 10.1021/acs.jmedchem.6b00981 J. Med. Chem. 2016, 59, 9047−9061
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Article
RESULTS
Probing the H1R Binding Site. To explore the three different H1R binding pockets (Figure 1C) we designed and synthesized a variety of analogs of 1. By making subtle chemical changes (Figure 2), we probed each of the three binding pockets in order to define H1R binding hot spots (Figure 3). Targeting the Amine-Binding Region. Many high affinity H1R reference compounds contain a tertiary amine, e.g., doxepin (Figure 1B). To investigate the effect of N-methyl substitution on H1R binding affinity, secondary amine 1, tertiary amine 2, and quaternary ammonium cation 3 were designed and synthesized (Figure 2). Opening of the piperidine ring of 1 allowed the comparison of a primary (4a, 5a), secondary (4b, 5b), and tertiary (4c, 5c) amine. In addition, the steric and hydrophobic fit of the tertiary amine was investigated by amines incorporated in a series of aliphatic rings (4d−g and 5d−g), also seen in H1R reference compounds such as triprolidine.10 In addition, the linear analogs (4a−g and 5a− g) bearing a two or three methylene spacer between the phenyl ether and the basic amine allowed for the investigation of the sensitivity of the distance between the basic amine interacting with D1073.32 and the aromatic pharmacophoric features. The compounds probing the amine-binding region were synthesized as outlined in Scheme 1. Commercially available phenol 13 was used in a Mitsunobu reaction to obtain both the Boc-protected ligand 14 and amine 2 in moderate yield. Compound 14 was deprotected using TFA to afford ligand 1. Methylation of amine 2 with methyl iodide gave quaternary ammonium cation 3. Alkylation of phenol 13 with 1-bromo-2chloroethane and 1-bromo-3-chloropropane afforded 15 and 16, respectively. Subsequent nucleophilic aliphatic substitution using microwave assisted heating afforded 4a−g and 5a,b,d−g in moderate to good yield. Compound 5c was obtained by reacting 16 with dimethylamine at room temperature. Targeting the Upper and Lower Aromatic Region. The relative importance of the two aromatic rings in ligand 1 was investigated by (i) ligand deconstruction (6 and 7), (ii) different vectors (8, 9, and 10), and (iii) chloro substitution (11 and 12). The compounds probing the aromatic region were synthesized as outlined in Scheme 2. Amine 7 was obtained via a reductive amination with secondary ammonium salt 6 and formaldehyde in the presence of acetic acid and sodium triacetoxyborohydride as a reducing agent. Due to the moderate yields and tedious purification in the Mitsunobu reactions described above, a second strategy using mesylate 18, which was synthesized by reacting methanesulfonyl chloride with alcohol 17 in the presence of triethylamine, was used to obtain compound 19. Significant amounts of elimination product were formed during this reaction, but after treatment with TFA and column purification, amine 8 was obtained in sufficient overall yield and good purity. The chloro-substituted analogs 11 and 12 were synthesized using phenol 24, which was synthesized according to a patent procedure.18 First, 2bromophenol (20) was protected using benzyl bromide to provide benzyl ether 21. Bromolithium exchange followed by the addition of 4-chlorobenzaldehyde gave alcohol 22. Reduction of alcohol 22 using BF3·Et2O and triethylsilane afforded benzyl ether 23, which was subsequently deprotected using BF3·Et2O and dimethylsulfide to obtain phenol 24 in good overall yield. The same synthetic strategies used for 19, 8, 7 were applied to obtain the compounds 25, 11, 12, respectively.
Figure 1. (A) H1R crystal structure (PDB code 3RZE) with the cocrystallized ligand doxepin (green carbon atoms)3 and the proposed binding mode of the structure-based virtual screening hit 1 (magenta carbon atoms).15 The yellow ribbons represent parts of the backbone of transmembrane (TM) helices 2, 3, 5, 6, and 7. Selected binding site residues are depicted as ball-and-sticks with light gray carbon atoms. Oxygen, nitrogen, and hydrogen atoms are colored red, blue, and cyan, respectively. Polar hydrogen atoms of the ligands are shown but are omitted for the binding site residues. For clarity residue Y1083.33 is omitted. Throughout this manuscript the Uniprot numbers (first) and the Ballesteros−Weinstein numbering (second in superscript) are reported for each residue.17 (B) Structure of doxepin and H1R affinity as determined in this study. Ligand efficiency LE = (ΔG)/N, where ΔG = −RT ln(Ki), N is the number of non-hydrogen atoms (HA), R = (8.314 472 15 J K−1 mol−1 and 4184 J = 1 kcal), and T = 298.15 K. (C) The H1R binding site surface is shown and colored to indicate the different regions of the binding site, i.e., the amine-binding region and the upper and lower aromatic binding region. (D) Schematic 2D representation of the structure-based virtual screening hit 1 interacting with the three examined regions.
molecule can be accommodated in the doxepin binding site (Figure 1A,C) and offers a good starting point to probe the various main binding pockets in the orthosteric H1R binding site. Ligand 1 benefits from a high ligand efficiency (LE, defined as the binding energy divided by the number of heavy atoms of a ligand) and does not contain stereo- or regioisomers. This ligand is therefore used in the current study as a scaffold to design and synthesize a set of 23 derivatives to systematically investigate the role of (1) the amine-binding region, (2) the upper and lower aromatic binding region, and (3) the impact of (de)solvation of the binding site on the binding of H1R antagonists. WaterFLAP14 analysis of H1R binding site solvation was for the first time applied to guide site-specific protein mutagenesis studies in order to complement the structure-based SAR analysis. 9048
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Figure 2. Scaffold exploration. The different molecules designed to revisit the molecular determinants of H1R binding. The color of the molecules corresponds to the color coding in Figure 6.
Scheme 1. Synthesis of Ligands To Target the AmineBinding Region
Figure 3. Binding hot spots. The H1R crystal structure3 (PDB code 3RZE) with the predicted binding pose of tertiary amine 2 and the binding hot spot map with two views. The color coding of the surface reflects the GE of all ligands described in Table 1 and Table 2 and is projected on the protein atoms within 4 Å distance. The binding pocket surface was colored according to the maximum GE values for each protein atom. Selected binding site residues are depicted as balland-sticks with light gray carbon atoms. Oxygen, nitrogen, and hydrogen atoms are colored red, blue, and cyan, respectively. Polar hydrogen atoms of the ligand are shown but are omitted for the binding site residues. a
Reagents and conditions: (a) 14: tert-butyl 4-hydroxypiperidine-1carboxylate, PPh3, DEAD (40 wt % in toluene), THF, 0 °C to rt, 5 h. 2: 1-methylpiperidin-4-ol, PPh3, DEAD (40 wt % in toluene), THF, 0 °C to rt, 4 h. (b) TFA, DCM, 0 °C to rt, 4 h. (c) MeI, DCM, rt, 5.5 h. (d) 15: Br(CH2) 2Cl, K2 CO3 , (CH3) 2 CO, reflux, 16 h. 16: Br(CH2)3Cl, K2CO3, (CH3)2CO, reflux, 25 h. (e) 4a: NH3 (7 M in MeOH), microwave (130 °C, 6 h). 5a: NH3 (7 M in MeOH), microwave (125 °C, 2 h). 4b and 5b: CH3NH2 (33 wt % in EtOH), microwave (130 °C, 35 min). 4c: (CH3)2NH (5.6 M in EtOH), microwave (150 °C, 25 min). 5c: (CH3)2NH (5.6 M in EtOH), rt, 11 days. 4d and 5d: azetidine, NaHCO3, DMF, microwave (120 °C, 30 min and 140 °C, 30 min). 4e−g and 5e−g: amine, NaHCO3, NaI, MeCN, microwave (120−160 °C, 20−50 min). Compound 1, 2, 4a− d, and 5a−d were obtained as (hemi)fumaric acid salts.
Pharmacological Evaluation. Binding affinities (K i values) for the human H1R proteins (wild type and H1R mutants) of test compounds described in Tables 1 and 2 were evaluated using radioligand displacement experiments, with [3H]mepyramine as a radioligand.19 Receptor proteins were transiently expressed in HEK293T cells as described previously.20 Structure−Activity Relationship for Human H1R Binding. All ligands are evaluated based on their LE21 and group efficiency (GE).22 GE is used to dissect the contributions of different parts of the ligand to its binding affinity,22 e.g., using a ligand deconstruction approach.23 The GE values were furthermore plotted on the protein surface in order to visualize binding hot spots (Figure 3). The SAR of the compounds targeting the amine-binding region and the aromatic regions are discussed below in detail. Efficiency of Ligand Binding in the Amine-Binding Region. An 8-fold increase in affinity upon methylation of secondary amine 1 to obtain tertiary amine 2 was observed (Table 1). Subsequent methylation to obtain quaternary
ammonium salt 3 shows a 12-fold decrease in affinity. Table 1 shows that each subsequent methyl substitution in both linear analog series (4a−c and 5a−c) leads to a roughly 10-fold increase in binding affinity and an increase of 0.1 in LE in total. Thus, going from a primary amine to a tertiary amine, a 100fold increase in H1R affinity is obtained. The affinities for the ligands with a three-methylene spacer (5a−c) are 3- to 5-fold 9049
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Scheme 2. Synthesis of Ligands To Target the Aromatic Region
a Reagents and conditions: (a) CH2O (37 wt % in water), AcOH, Na(CH3COO)3BH, DCM/MeOH 2:1, rt, 6 h. (b) CH3SO2Cl, TEA, DCM, 0 °C to rt. (c) 18, Cs2CO3, DMF, 65 °C, 26 h. (d) TFA, DCM, 0 °C to rt, 16 h. (e) (1) K2CO3, (CH3)2O, rt, 1 h, (2) PhCH2Br, reflux, 2 h. (f) (1) nBuLi, −78 °C, 30 min, (2) 4-Cl-PhCHO, THF, −78 to 0 °C, 2 h. (g) Et3SiH, BF3·Et2O, MeCN, −40 °C to rt, 3 h. (h) Me2S, BF3.Et2O, DCM, 0 °C to rt, 28 h. (i) 18, Cs2CO3, DMF, 65 °C, 26 h. (j) TFA, DCM, 0 °C to rt, 15 h. (k) CH2O (37 wt % in water), AcOH, Na(CH3COO)3BH, DCM/ MeOH 2:1, rt, 2 h. Compound 7, 8, 11, and 12 were obtained as fumaric acid salts.
the H1R crystal structure3 in order to project the GEs of the ligands on the protein surface to visualize the binding hot spots (Figure 3). The most energetically favorable pose that forms an ionic and H-bond interaction with D1073.32 was selected (i.e., the pose with the lowest PLANTS ChemPLP score,24 Supporting Information Table S1), and subsequently, the GEs of all ligands were projected on the protein surface resulting in different hot spots (Figure 3). The most prominent hot spot is in the amine-binding region, followed by the lower aromatic region and the upper aromatic region. All tested ligands described in Tables 1 and 2 have LEs in the range of 0.34 to 0.64 and GEs in the range of −0.14 to 1.64. All ligands, except 9, have a similar or higher LE compared to the mean LE (0.4) reported for ligands targeting aminergic GPCRs.26 The changes in substituents for all compounds, except 3, 9, and 12, have a positive GE value (Tables 1 and 2). Positive GE values indicate a beneficial contribution of a substituent to the binding affinity; i.e. the higher is the GE value, the more efficient is the probing of a certain binding region by that particular substituent. Taking GE values for several case studies22,27,28 into account, we consider our values to be satisfactory. The concentrated primary binding hot spot in the amine-binding
higher compared to the two-methylene spacer series (4a−c). Interestingly, the affinities of ligand 2 and its flexible analog 5c are similar. Further extending the side chain by the introduction of aliphatic rings (4d−g, 5d−g) on the basic amine, and hence increasing the lipophilicity of the ligand, generally shows a decrease in affinity and therefore a drop in LE and GE. This clearly shows that small tertiary amines (2, 4c, and 5c) are optimal. Efficiency of Ligand Binding in the Aromatic Regions. Deconstruction of ligands 1 and 2 leads to compounds 6 and 7, respectively. Both ligands lacking the benzyl moiety show a ∼100-fold decrease in affinity, but the LE is increased due to the reduced heavy atom count (Table 2). Ligands 8 and 9, with the benzyl group on the meta and para positions, show a 100and 500-fold decrease in affinity, respectively, compared to 1. Direct attachment of the phenyl ring at the ortho position (10) shows only a 10-fold decrease in affinity compared to 1. Chloro-substitution on the benzyl ring of secondary amine 1 to afford 11 shows a slight increase in affinity, while chlorosubstitution of tertiary amine 2 shows comparable affinity. H1R Binding Affinity Hot Spot Analysis. All ligands described in Tables 1 and 2 were docked with PLANTS24,25 in 9050
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Table 1. continued
Table 1. Pharmacological Evaluation of Ligands Targeting the Amine-Binding Region
a
Affinity values (pKi) were determined using a competition binding experiment for the H 1R between the respective ligand and [3H]mepyramine. Values represent the mean ± SEM of N ≥ 3 experiments. bLE calculated as described in Figure 1. cGroup efficiency = −ΔΔG/ΔN, where ΔΔG = ΔG(molecule B) − ΔG(molecule A) and ΔN = HA(molecule B) − HA(molecule A). Compound 1 was used as molecule A to calculate the GE for compound 2 and 3. Compound 4a was used to calculate the GEs for compounds 4b−g. Compound 5a was used to calculate the GEs for compounds 5b−g.
Table 2. Pharmacological Evaluation of Ligands Targeting the Aromatic Regions
a
Affinity values (pKi) were determined using a competition binding experiment for the H 1R between the respective ligand and [3H]mepyramine. Values represent the mean ± SEM of N ≥ 3 experiments. bLE calculated as described in Figure 1. cGE calculated as described in Table 1. Compound 6 was used as molecule A to calculate the GE for compounds 1 and 7−10. Compound 7 was used as molecule A to calculate the GE for compound 2. Compound 1 was used as molecule A to calculate the GE for compound 11. Compound 2 was used as molecule A to calculate the GE for compound 12.
region is a result of the small amines 2, 4b,c, and 5b,c, having the highest GEs ranging from 1.36 to 1.64. The secondary, but weaker, binding hot spot in the lower aromatic region is the result of the (substituted) benzyl substituents of the ligands 1, 2, and 11 probing this pocket with GEs in the range of 0.41− 0.55. The upper aromatic region is the weakest binding hot spot (GE is −0.08) due to the fact that the (substituted) benzyl 9051
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that the “unhappy” water molecules in the lower aromatic region are displaced by doxepin (Figure 4B), ligand 1 (Figure 4C), and ligand 2 (Figure 4D). The increase in affinity for ligand 2 compared to ligand 1 results in a binding efficiency hot spot near D1073.32 (Figure 3), but there are no specific interactions made by the additional methyl group. However, an “unhappy” water molecule is trapped between the basic amine of ligand 1 (Figure 4C) and the protein and hence has limited degrees of freedom. The “unhappy” water molecule is displaced upon the introduction of the extra methyl group (i.e., ligand 2, Figure 4D), and this observation could explain the increased affinity for ligand 2. The residues F4316.51, I4547.39, and Y4587.43 form a narrow and hydrophobic subpocket around the “unhappy” water molecule. While F/Y6.51 and Y7.43 are highly conserved among aminergic GPCRs, residue 7.39 is variable in terms of polarity and size. To investigate H1R specific determinants of ligand-dependent binding site desolvation, we therefore focused on the role of the H1R specific residue I4547.39 in trapping the identified “unhappy” water molecules in the amine-binding region. WaterFLAP analysis was for the first time applied to guide prospective protein mutagenesis studies by the systematic in silico substitution of I4547.39 to all natural occurring amino acids and the computational assessment of the water network energetics of the binding sites of the resulting H1R mutants. On the basis of these WaterFLAP calculations, an isoleucine (present in the wild type receptor) and phenylalanine are optimal for a maximal increase in affinity upon Nmethylation. Both the wild type H1R (I4547.39) and the H1R I4547.39F mutant receptor show “unhappy” waters in the immediate vicinity of the basic amine that will be displaced upon methylation of ligand 1 (Figure 4C−F). For the threonine mutant H1R I4547.39T a reduced hydrophobic interaction field as well as an absence of “unhappy” waters was observed in the vicinity of the amine. Consequently, this WaterFLAP analysis suggested that a I4547.39T mutation would reduce the effect of N-methylation (Figure 4E). Site-Directed Mutagenesis. To test the WaterFLAP hypothesis, the binding affinities (Ki) of compounds 1 and 2 were evaluated on the wild type H1R (WT) and the mutants H1R I4547.39T and H1R I4547.39F using radioligand displacement of [3H]mepyramine. The radioligand [3H]mepyramine binds all three receptors with high nanomolar affinity and only shows a slight decrease in affinity for the I4547.39T and I4547.39F mutants (pKD of 8.4 ± 0.1 and 8.5 ± 0.1, respectively) compared to WT (pKD of 8.6 ± 0.1; Supporting Information Table S2). The expression levels of I4547.39T and I4547.39F were high, but compared to wild-type, this was decreased 2- and 10-fold, respectively (Supporting Information Table S2). The total amount of membranes was therefore adjusted to bind similar levels of [3H]mepyramine (Supporting Information Figure S3). As can be seen in Figure 5A and Figure 5C, at both the WT H1R and the H1R I4547.39F, methylation of secondary amine 1 to give tertiary amine 2 results in an almost 10-fold increase in affinity. Moreover, in agreement with the WaterFLAP analysis the difference in affinity between the ligands 1 and 2 is lost upon the I4547.39T mutation (Figure 5B).
substituents are probing the lower aromatic region instead (Supporting Information Figure S1A). WaterFLAP Analysis. To explain the observed SAR, we investigated the role of water molecules in the binding of H1R ligands using the WaterFLAP method.14 This method can be used to build water networks in binding sites to predict the relative free energy of water molecules and to analyze the perturbation of an explicit water network as a consequence of ligand binding. The WaterFLAP method combines a short molecular dynamic simulation to assess the degrees of freedom of the water molecules (entropy) with a molecular interaction field (GRID) analysis of the hydrophobic and apolar interaction field around the water molecules in order to identify energetically favorable (“happy”) and unfavorable (“unhappy”) waters.14,29,30 Previous WaterFLAP analyses of the H1R crystal structure without doxepin in the binding site (apo) predicted that there are “unhappy” water molecules located in the lower aromatic region and near the amine-binding region13 (Figure 4A). Complementary statistical analysis of hydration sites observed in molecular dynamics simulations using WaterMap14,31,32 identified unhappy water molecules in the same binding site regions (Supporting Information Figure S2). WaterFLAP analyses of ligand bound wild-type H1R indicated
Figure 4. WaterFLAP calculations of the binding site of WT H1R (I4547.39), (A) without ligand, in complex with (B) doxepin, (C) ligand 1, and (D) ligand 2, (E) H1R I4547.39T mutant in complex with ligand 1, and (F) H1R I4547.39F mutant in complex with ligand 1. Waters are color coded to show the most “unhappy” as red (>3.5 kcal/ mol), then yellow (2.2−3.5 kcal/mol), bulk solvent as gray (−1 to 2.2 kcal/mol), and “happy” as blue (
