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CELLULAR AND MOLECULAR

Pharmacological Profile of Histaprodifens at Four Recombinant Histamine H₁ Receptor Species Isoforms

Department of Pharmaceutical/Medicinal Chemistry I, School of Pharmacy, University of Regensburg, Regensburg, Germany (A.S., B.S.); Faculty of Chemistry and Pharmacy, University of Regensburg, Regensburg, Germany (H.-J.W.); and Department of Pharmacology and Toxicology, School of Pharmacy, University of Regensburg, Regensburg, Germany (R.S.)

Received August 1, 2007; accepted October 9, 2007.

	Abstract

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There are differences in the pharmacological properties of phenylhistamines and histaprodifens between guinea pig histamine H₁ receptor (gpH₁R) and human histamine H₁ receptor (hH₁R). The aim of this study was to analyze species differences in more detail, focusing on histaprodifen derivatives and including the bovine histamine H₁ receptor (bH₁R) and rat histamine H₁ receptor (rH₁R). H₁R species isoforms were coexpressed with the regulator of G protein signaling RGS4 in Sf9 insect cells. We performed [³H]mepyramine binding assays and steady-state GTPase assays. For a novel class of histaprodifens, the chiral histaprodifens, unique species differences between hH₁R, bH₁R, rH₁R, and gpH₁R were observed. The chiral histaprodifens 8R and 8S were both partial agonists at gpH₁R, but only 8R was a partial agonist at the other H₁R species isoforms. An additional phenyl group in chiral histaprodifens 10R and 10S, respectively, resulted in a switch from agonism at gpH₁Rto antagonism at hH₁R, bH₁R, and rH₁R. In general, histaprodifens showed the order of potency hH₁R < bH₁R < rH₁R < gpH₁R. An active-state model of gpH₁R was generated with molecular dynamics simulations. Dimeric histaprodifen was docked into the binding pocket of gpH₁R. Hydrogen bonds and electrostatic interactions were detected between dimeric histaprodifen and Asp-116, Ser-120, Lys-187, Glu-190, and Tyr-432. We conclude the following: 1) chiral histaprodifens interact differentially with H₁R species isoforms; 2) gpH₁R and rH₁R, on one hand, and hH₁R and bH₁R, on the other hand, resemble each other structurally and pharmacologically; and 3) histaprodifens interact with H₁R at multiple sites.

The highly conserved Asp^3.32 in transmembrane domain (TM) helix 3 (Ohta et al., 1994; Nonaka et al., 1998), Lys^5.39 (Leurs et al., 1995; Bruysters et al., 2004; Jongejan and Leurs, 2005), Thr^5.42 (Leurs et al., 1994; Ohta et al., 1994), Asn^5.46 (Leurs et al., 1994; Ohta et al., 1994), and Phe^6.55 (Bruysters et al., 2004) are involved in histamine binding. Ser^3.36 and Asn^7.45 are involved in hH₁R activation (Jongejan and Leurs, 2005). Trp^4.56, Lys^5.39, Phe^6.52, Phe^6.55, and Lys^5.39 interact with H₁R antagonists (Wieland et al., 1999; Gillard et al., 2002).

Phenylhistamines and histaprodifens (Seifert et al., 2003) and ergolines (Pertz et al., 2006) were studied at the recombinant guinea pig H₁ receptor (gpH₁R) and human H₁ receptor (hH₁R) expressed in Sf9 insect cell membranes by [³H]mepyramine competition binding and G_q protein-catalyzed GTP hydrolysis. Those studies revealed substantial species differences between gpH₁R and hH₁ R. Asn^2.61 acts as a selectivity switch between hH₁R and gpH₁R (Bruysters et al., 2005).

To date, the cDNAs of 12 mammalian H₁R species isoforms are known. A phylogenetic tree of H₁R species isoforms is shown in Fig. 1. The cDNAs of gpH₁R (Traiffort et al., 1994), hH₁R (Fukui et al., 1994), bovine H₁R (bH₁R) (Yamashita et al., 1991), and rat H₁ receptor (rH₁R) (Fujimoto et al., 1993) are available in our laboratory. Figure 2 shows a sequence comparison of hH₁R, bH₁R, rH₁R, and gpH₁R. Table 1 provides a summary on the percentage of identical amino acids for the N terminus, C terminus, each extra- and intracellular loop, each transmembrane domain, and for the complete sequence, respectively, among two H₁R species isoforms. For the complete sequences, similarities range from 66.73% (bH₁R versus gpH₁R) to 81.87% (hH₁R versus bH₁R). In general, amino acids in the transmembrane domains show >80% identity. TM3 shows no species differences. The similarities of the N terminus, C terminus, and the loops are in a range from 100% (I1) to only 26% (N terminus). Collectively, there are only moderate similarities between H₁R species isoforms. Thus, we hypothesized that there are significant differences in pharmacological properties not only between hH₁R and gpH₁R but also rH₁R and bH₁R. To test this hypothesis, we coexpressed hH₁R, bH₁R, rH₁R, and gpH₁R with the regulator of G protein signaling RGS4 in Sf9 insect cells, and we characterized known and novel histaprodifens (Fig. 3) in [³H]mepyramine competition binding and GTPase assays. Moreover, an active state model of gpH₁R was generated.

View larger version (12K): [in this window] [in a new window]   Fig. 1. Phylogenetic tree of cloned mammalian H1Rs. Based on H1R amino acid sequences (Swiss Prot; National Center for Biotechnology Information, Bethesda, MD), we performed their alignment with the software ClustalW 1.83 (European Molecular Biology Laboratory's European Bioinformatics Institute, Cambridge, UK) and generated the phylogenetic tree with Phylo-Draw 0.8 (Graphics Application Lab, Pusan National University, 1999). The H1R species isoforms analyzed in this study are highlighted in bold. In parentheses, the accession codes in Swiss Prot (*) or National Center for Biotechnology Information (**) are given.

View larger version (50K): [in this window] [in a new window]   Fig. 2. Alignment of the amino acid sequences of hH1R, bH1R, rH1R, and gpH1R. The amino acid sequences of hH1R, bH1R, rH1R, and gpH1R are given in the one-letter code. Dots in the sequences of bH1R, rH1R, and gpH1R indicate amino acids identical to hH1R. Hyphens indicate missing amino acids. Bold letters indicate glycosylation sites in the N terminus and E2-loop. Amino acids with gray shading are the most conserved amino acids (X.50, with X being the number of the TM domain), according to the numbering scheme in Ballesteros et al. (2001). Amino acids in white with black shading indicate the amino acids that are proposed to interact with the dimeric histaprodifen in the binding pocket of gpH1R. The alignment was performed manually.

View larger version (14K): [in this window] [in a new window]   Fig. 3. Structures of histamine, histaprodifens and mepyramine. Histamine 1, small histaprodifens 2 to 5, large histaprodifens 6 to 17 and mepyramine 18.

	Materials and Methods

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Materials. The cDNAs from bH₁R and rH₁R were kindly provided by Dr. H. Fukui (Department of Pharmacology, University of Tokushima, Tokushima, Japan), cloned into the pEF-BOS plasmid (bH₁R) and pUC-18 plasmid (rH₁R), respectively. Phusion high-fidelity polymerase, all restriction enzymes, and T4 DNA ligase were from New England Biolabs (Beverly, MA). The anti-FLAG IgG (M1 monoclonal antibody) was from Sigma-Aldrich (St. Louis, MO), and the anti-RGS4 was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). [-³²P]GTP (6000 Ci/mmol) and [³H]mepyramine (30.0 Ci/mmol) were from PerkinElmer Life and Analytical Sciences (Boston, MA). As liquid scintillation cocktail, we used Rotiszint ecoplus from Roth (Karlsruhe, Germany). Histaprodifens were synthesized as described by Elz et al. (2000) and Striegl (2006).

Construction of pGEMbH₁R, pGEMrH₁R, pVLbH₁R, and pVLrH₁R. bH₁R and rH₁R were cloned into the pGEM-3Z-SF-plasmid by overlap-extension PCR (PCR Ia, PCR Ib, and PCR II) with the pGEM-3Z-SF-gpH₁R-plasmid and the pEF-BOS-bH₁R (Yamashita et al., 1991) and pUC-18-rH₁R (Fujimoto et al., 1993) as template. In PCR Ia, the DNA fragment with the signal peptide (S), the FLAG epitope (F), and the N terminus of the bH₁R and rH₁R, respectively, was amplified. In PCR Ib, the DNA fragments encoding bH₁R and rH₁R, respectively, and the His₆ tag (CACCATCATCACCATCAC) were generated. In PCR II, the products of PCR Ia and PCR Ib were annealed in the SF encoding region, resulting in PCR fragments encoding the SF, bH₁R and rH₁R, respectively, the His₆ tag, the stop codon and an XbaI site. For PCRs, the following primers were used: 5'-GCTCACTCATTAGGCACC-3' (bH₁R, forward, PCR Ia, PCR II), 5'-GGACAGGTCATGGCGTCATCATCGTCCTTG-3' (bH₁R, reverse, PCR Ia), 5'-GACGATGATGACGCCATGACCTGTCCCAACTCC-3' (bH₁R, forward, PCR Ib), and 5'-ATCCTCTAGATTAGTGATGGTGATGATGGTGGGAACGAATGTGCAGAATT-3' (bH₁R, reverse, PCR Ib, PCR II); 5'-GCTCACTCATTAGGCACC-3' (rH₁R, forward, PCR Ia, PCR II), 5'-GGCAAAGCTCATGGCGTCATCATCGTCCTTG-3' (rH₁R, reverse, PCR Ia), 5'-CAAGGACGATGATGACGCCATGAGCTTTGCCAATACC-3' (rH₁R, forward, PCR Ib), and 5'-ATCCTCTAGATTAGTGATGGTGATGATGGTGGGAACGAATGTGCAGAATC-3' (rH₁R, reverse PCR Ib, PCR II). The resulting PCR fragments with bH₁R and rH₁R were double-digested with HindIII and XbaI and cloned into the pGEM-3Z-plasmid using the pGEM-3Z-SF-gpH₁R as template. The sequences of bH₁R and rH₁R, cloned into the pGEM-3Z plasmid were checked for their correctness by sequencing (Entelechon, Regensburg, Germany). Using pGEM-3Z-SF-bH₁R and pGEM-3Z-SF-rH₁Rastemplate, bH₁R and rH₁R were cloned into the pVL1392 baculovirus transfer vector using the restriction sites BssHII and XbaI for bH₁R and SacI and XbaI for rH₁R. Both species isoforms were N-terminally tagged with the signal-peptide and FLAG-epitope 5'-ATGAAGACGATCATCGCCCTGAGCTACATCTTCTGCCTGGTATTCGCCGACTACAAGGACGATGATGACGCC-3' and C-terminally tagged with the His₆-tag 5'-CACCATCATCACCATCAC-3'.

Cell Culture, Generation of Recombinant Baculoviruses, and Membrane Preparation. Sf9 insect cells were cultured using SF 900 II medium (Invitrogen, Carlsbad, CA), containing 5% (v/v) fetal calf serum (Sigma-Aldrich, Steinheim, Germany), and 0.1 mg/ml gentamicin (Lonza Walkersville, Walkersville, MD) in 250-ml Erlenmeyer flasks at 28°C and shaking at 125 rpm. Recombinant baculoviruses encoding H₁R species isoforms were produced with Sf9 cells using the BaculoGOLD transfection kit (BD Biosciences PharMingen, San Diego, CA). After initial infection, two sequential virus amplifications followed to generate high-titer virus stocks. For final infection, yielding in membranes, Sf9 cells were sedimented by centrifugation and suspended in fresh medium, yielding in a final concentration of 3.0 x 10⁶ cells/ml. Cells were cotransfected with a 1:100 dilution of higher titer baculovirus stock encoding one of the H₁Rs and RGS4. After 48 h of incubation, membranes were prepared as described previously (Kelley et al., 2001; Houston et al., 2002).

Determination of Protein Concentration. Protein concentration was determined using the Bio-Rad DC protein assay kit (Bio-Rad, Hercules, CA).

SDS-Polyacrylamide Gel Electrophoresis and Immunoblot Analysis. Membrane proteins were separated on SDS polyacrylamide gels containing 12% (w/v) polyacrylamide. After transfer of the proteins onto Immobilon-P transfer membranes (Millipore Corporation, Bedford, MA), membranes were reacted with the M1 antibody or anti-RGS4 IgG (1:1000 each). Immunoreactive bands were visualized by sheep anti-mouse IgG (1:1000) (for the M1 antibody) or donkey anti-goat IgG (for the RGS4 antibody) as described previously (Seifert et al., 2003).

Preparation of Compound Stock Solutions. Chemical structures of the analyzed compounds are given in Fig. 3. Compounds 1 and 18 (10 mM each) were dissolved in double-distilled water. Compounds 2 to 16 (5 mM each) were dissolved in a solvent containing 30% (v/v) DMSO, 30% (v/v) Tris-HCl, pH 7.4 (10 mM), and 40% (v/v) double-distilled water. Compound 17 (1 mM) was dissolved in 50% (v/v) DMSO and 50% (v/v) Tris-HCl, pH 7.4 (10 mM). The final DMSO concentration in all assays was adjusted to 3 (v/v) or 5% (v/v) as appropriate for a given ligand. These DMSO concentrations did not shift pK_i and pEC₅₀ values of histamine.

[³H]Mepyramine Saturation Binding Assay. Membranes expressing a given H₁R isoform and RGS4 were thawed, suspended in binding buffer (12.5 mM MgCl₂, 1 mM EDTA, and 75 mM Tris-HCl, pH 7.4), and sedimented by a 10-min centrifugation at 4°C and 13,000g. The membrane pellet was resuspended in binding buffer. Each reaction mixture contained 50 to 75 µg of membrane protein. All saturation binding assays were performed in a concentration range from 0.2 to 20 nM [³H]mepyramine. Nonspecific binding was determined in the presence of 10 µM diphenhydramine. Incubations were conducted for 90 min at room temperature and shaking at 250 rpm. Bound [³H]mepyramine was separated from free [³H]mepyramine by filtration through GF/C filters (Whatman, Maidstone, UK) followed by three steps of washing with 2 ml of binding buffer (4°C). The radioactivity remaining on filters was determined with liquid scintillation counting, after an equilibration phase of at least 4 h with scintillation cocktail.

[³H]Mepyramine Competition Binding Assay. Preparation of membrane was performed as described for the [³H]mepyramine saturation binding assay. All competition binding experiments were carried out in presence of 5 nM [³H]mepyramine and unlabeled ligands at final concentrations from 1 nM to 100 µM as appropriate to obtain saturated competition curves. Incubations were conducted for 90 min at room temperature and shaking at 250 rpm. Bound [³H]mepyramine was determined as described for the [³H]mepyramine saturation binding assay.

Steady-State GTPase Assay. Membranes expressing a given H₁R isoform and RGS4 were thawed, suspended in 10 mM Tris-HCl, pH 7.4, and sedimented by a 10-min centrifugation at 4°C and 13,000g. The membrane pellet was resuspended in 10 mM Tris-HCl, pH 7.4. Each assay tube contained Sf9 cell membranes (10–20 µgof protein/tube), 1.0 mM MgCl₂, 0.1 mM EDTA, 0.1 mM ATP, 100 nM GTP, 0.1 mM adenylyl imidodiphosphate, 5 mM creatine phosphate, 40 µg of creatine kinase, 0.2% (w/v) bovine serum albumin in 50 mM Tris-HCl, pH 7.4, and several ligands in a concentration range from 1 nM to 1 mM as appropriate to obtain saturated concentration/response curves. Reaction mixtures (80 µl) were incubated for 3 min at 25°C. Thereafter, 20 µlof[-³²P]GTP (0.1 µCi/tube) were added to each tube. Reactions were conducted for 20 min at 25°C and terminated by the addition of 900 µl of slurry consisting of 5% (w/v) activated charcoal and 50 mM NaH₂PO₄, pH 2.0. Charcoal-quenched reaction mixtures were centrifuged for 15 min at room temperature at 15,000g. Six hundred microliters of the supernatant fluid of reaction mixtures was removed, and ³²P_i was determined by liquid scintillation counting.

The steady-state GTPase assay was performed in the agonist and antagonist mode. In the agonist mode, the percentage of increase in GTP hydrolysis relative to histamine was determined with increasing concentrations of each ligand from 0.1 nM to 100 µM. In the antagonist mode, the percentage of GTP hydrolysis was determined as described in agonist experiments, but assay tubes additionally contained 1 µM histamine.

Data Analysis. All data were analyzed with the software Prism 4.02 (GraphPad Software Inc., San Diego, CA). K_B and pK_B values were calculated according to Cheng and Prusoff (1973). All data are the means ± S.E.M. of at least three independent experiments. To compare two pairs of data, the significance of the deviation of zero p was calculated using the t test.

Construction of an Active gpH₁R Model with Dimeric Histaprodifen in the Binding Pocket. A homology model of the inactive gpH₁R was constructed as described previously (Strasser and Wittmann, 2007). In addition, essential internal water molecules were placed into the receptor model. Afterward, the whole model was embedded into an environment consisting of 104 1-palmitoyl-2-oleoyl-phosphatidylcholine molecules, 12,675 intracellular and extracellular water molecules, and 8 sodium and 25 chloride ions to achieve electroneutrality. This template was used to generate a model of the active gpH₁R based on the distance restraints given by Niv et al. (2006). Therefore, we performed restrained MD simulations with the software package GROMACS 3.2 (van der Spoel et al., 2004). This procedure will be described in detail elsewhere. In brief, a positively charged dimeric histaprodifen 17 was manually docked into the proposed binding pocket of the active gpH₁R, and one sodium ion was deleted to conserve electroneutrality. The whole simulation box was energetically minimized. Thereafter, MD simulations were performed. The whole equilibration phase was divided in 12 cycles with different simulation times and position constraints as described in the following. The constraints for the backbone atoms (bb), side chain atoms (sc), and ligand atoms (lig) are given in kiloJoules per milliliter per squared nanometer. Cycle 1: 250 ps, bb 5000, sc 5000, lig 5000; cycle 2: 100 ps, bb 4000, sc 4000, lig 4000; cycle 3: 100 ps, bb 4000, sc 3000, lig 3000; cycle 4: 100 ps, bb 3000, sc 3000, lig 3000; cycle 5: 100 ps, bb 2000, sc 1000, lig 1000; cycle 6: 100 ps, bb 1000, sc 800, lig 800; cycle 7: 100 ps, bb 800, sc 600, lig 600; cycle 8: 100 ps, bb 600, sc 400, lig 400; cycle 9: bb 400, sc 200, lig 200; cycle 10: 100 ps, bb 200, sc 100, lig 100; cycle 11: 100 ps, bb 100, sc 0, lig 0; cycle 12: 100 ps, bb 0, sc 0, lig 0. A 1-ns productive phase without position constraints followed. Simulations were carried out at 310 K and 1 atmosphere. For MD simulations Berendsen, temperature, and pressure coupling was used. For all calculations we used the software package GROMACS 3.2 (van der Spoel et al., 2004), with the ffG53A6 force field (Oostenbrink et al., 2004). The force field parameters for dimeric histaprodifen 17 were adopted from the ffG53A6 force field.

	Results

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Immunological Detection of the H₁R Constructs. Based on the amino acid sequences of H₁R isoforms, we calculated the molecular masses (without N-glycosylation) to be 55,784 Da for hH₁R, 55,957 Da for bH₁R, 55,693 Da for rH₁R, and 55,620 Da for gpH₁R. All H₁R species isoforms were immunologically detected with the M1 antibody (Fig. 4). bH₁R and rH₁R exhibited strong bands at 60 kDa. The increase by 4 kDa relative to the theoretical mass is probably due to N-glycosylation. hH₁R showed a strong band at 85 kDa, probably because of a higher degree of N-glycosylation. In addition, weak bands were visible in a range from 25 to 30 kDa, and at 50 kDa. gpH₁R showed very different behavior in migration, i.e., a strong band was detected at 25 kDa, intermediate bands were visible at 30 and 36 kDa, and faint bands were detected at 50 and 100 kDa, respectively. The results with hH₁R and gpH₁R are in accordance with previous studies from our laboratory (Seifert et al., 2003).

View larger version (84K): [in this window] [in a new window]   Fig. 4. Immunological detection of hH1R, bH1R, rH1R, and gpH1RinSf9 cell membranes. Sf9 cell membranes expressing one of the H1Rs and RGS4 were analyzed in an immunoblot as described under Materials and Methods. For immunological detection, the M1 antibody was used. Each line represents one of the four analyzed H1R species isoforms. Numbers at the left indicate the apparent molecular masses of the proteins in kilodaltons.

Some of the differences in electrophoretic mobility between the four H₁R species isoforms may be explained with different N-glycosylation states. At the N terminus, the four species isoforms exhibit different numbers of N-glycosylation sites: hH₁R (two), bH₁R (three), rH₁R (two), and gpH₁R (one). In E2 (last amino acid), there is an additional potential N-glycosylation site in bH₁R and rH₁R because the homology model shows that this amino acid should be positioned at the surface of the receptor (Fig. 2).

Analysis of H₁R Species Isoforms in the [³H]Mepyramine Saturation Binding Assay. The K_D and B_max values determined in the [³H]mepyramine saturation binding assay are given in Table 2. The K_D values at hH₁R, bH₁R and rH₁R were approximately 2-fold higher (p < 0.05) than at gpH₁R. The B_max value for bH₁R was approximately 2-fold higher (p < 0.05) than for hH₁R, rH₁R and gpH₁R. Representative saturation binding isotherms for the four H₁R species isoforms are shown in Fig. 5. The K_D values determined in the Sf9 cell system are in good accordance with the K_D values reported in the literature for native tissues, cultured cell lines and recombinant mammalian expression systems (Chang et al., 1979; Aceves et al., 1985; Nakahata et al., 1986; Yamashita et al., 1991; Fujimoto et al., 1993; Gillard et al., 2002; Bruysters et al., 2005). The nonspecific binding of [³H]mepyramine in Sf9 cell membranes expressing H₁R species isoforms ranged between 10 and 30% of total [³H]mepyramine binding, providing an excellent signal-to-noise ratio for competition experiments even for the H₁R species isoforms exhibiting lower expression levels, i.e., gpH₁R.

View larger version (16K): [in this window] [in a new window]   Fig. 5. [3H]Mepyramine saturation binding isotherms at hH1R, bH1R, rH1R, and gpH1R. The saturation binding experiments were performed using Sf9 cell membranes expressing hH1R, bH1R, rH1R, or gpH1R and RGS4 as described under Materials and Methods. Data were analyzed by nonlinear regression and were best fitted to hyperbolic one-site saturation isotherms. The square () shows the data for the total binding, the upward-facing triangle () shows the nonspecific, and the downward-facing triangle () shows the specific binding curves. A, hH1R. B, bH1R. C, rH1R. D, gpH1R. Data shown are the means of a representative binding experiment performed in triplicate for each membrane. For clarity, SD values are not shown. Those values generally varied by less than 10% of the means.

Analysis of Histaprodifens at H₁R Species Isoforms in the [³H]Mepyramine Competition Binding Assay. The results of the competition binding assays are summarized in Table 3. The pK_i values of histamine 1 at the four H₁R species isoforms did not significantly differ from each other. The pK_i values of histaprodifen 2 were significantly higher than the pK_i values of histamine 1 at all H₁R species isoforms (p = 0.0006 for hH₁R, p = 0.03 for bH₁R, p = 0.0011 for rH₁R, and p = 0.0267 for gpH₁R). The introduction of chlorine 3 or fluorine 4 in one of the phenyl moieties of histaprodifen did not result in significant differences in the binding affinity compared with histaprodifen 2 at all four species isoforms. N-Methylhistaprodifen 5 had similar pK_i values as histaprodifen 2 at all four H₁R species isoforms. Compounds 6 and 7 differ from each other in the substitution pattern of the imidazolyl moiety. The pK_i values of these compounds were in a range from 6.33 to 7.11 at all four H₁R species isoforms. The pK_i value of suprahistaprodifen 7 at gpH₁R was significantly higher than at hH₁R(p = 0.0012). Otherwise, there were no significant differences between these two compounds at H₁R species isoforms.

The additional methyl group in suprahistaprodifen in (R)- and (S)-configuration (compounds 8R and 8S) resulted in significant differences between species isoforms and the (R)- and (S)-configuration as well, as illustrated in Fig. 6. There was no significant difference in the pK_i values between 7 and 8R at hH₁R and gpH₁R, but the pK_i values of 8S were significant lower (p = 0.0026 for hH₁R and p = 0.0006 for gpH₁R) at both species isoforms compared with the (R)-configuration of 8. The decrease amounted to 0.8 log units at gpH₁R and 0.7 log units at hH₁R. In contrast, at bH₁R and rH₁R, the pK_i values of 8R and 8S were significantly lower than of 7, but there was no significant difference between 8R and 8S. Thus, the largest difference in affinity between the (R)- and (S)-configuration was found at gpH₁R, followed by hH₁R. The additional phenyl moiety in 9 did not result in significant differences in pK_i values relative to suprahistaprodifen 7 at all four H₁R species isoforms. Again, the highest pK_i value was found at gpH₁R, which was significantly higher than at hH₁R(p = 0.0082) and bH₁R(p = 0.0057). The pK_i values between the related phenyl-substituted 10R and 10S did not significantly differ from each other at H₁R species isoforms. At gpH₁R, the pK_i value of 9 was significantly higher than for 10R or 10S (p = 0.004 for 10R and p = 0.0072 for 10S). The pK_i values of 11 and 12 were similar at each H₁R species isoform. Compounds 13 to 16, possessing a different substitution pattern of the terminal imidazolyl moiety compared with suprahistaprodifen 7 in combination with a varying length of the CH₂-spacer, showed decreased affinity compared with histaprodifen 2 at all four H₁R species isoforms. Except for 13 at bH₁R, the decrease of approximately 0.4–1 log units was significant (p < 0.01). For the largest histaprodifen known so far, dimeric histaprodifen 17, we observed significant increases in pK_i values compared with histaprodifen 2 for bH₁R, rH₁R and gpH₁R. The pK_i values for histamine 1, suprahistaprodifen 7, and dimeric histaprodifen 17 at hH₁R and gpH₁R are in good accordance with data published for mammalian expression systems (Gillard et al., 2002; Bruysters et al., 2005). Figure 7 provides an overview of the binding affinities of several histaprodifen derivatives compared with histaprodifen 2 itself. Affinities are given as pK_i values. There are significant species-differences between hH₁R and gpH₁R, whereas bH₁R and rH₁R exhibit an intermediate behavior. For some compounds, bH₁R and rH₁R are more similar to gpH₁R, and for other compounds, bH₁R and rH₁R are more similar to hH₁R.

View larger version (9K): [in this window] [in a new window]   Fig. 6. Competition binding isotherms for 8R and 8S at hH1R, bH1R, rH1R, and gpH1R. The competition binding experiments were performed using Sf9 cell membranes expressing hH1R, bH1R, rH1R, or gpH1R and RGS4 in presence of 5 nM [3H]mepyramine ([3H]MEP) as described under Materials and Methods. Data were analyzed by nonlinear regression and were best fit to one-site (monophasic) competition curves. The square () shows the data for the (R)-configured derivative 8R; the triangle () shows the data for the (S)-configured derivative 8S.A, hH1R. B, bH1R. C, rH1R. D, gpH1R.

View larger version (22K): [in this window] [in a new window]   Fig. 7. pKi values of selected histaprodifen derivatives relative to the pKi value of histaprodifen at hH1R, bH1R, rH1R, and gpH1R in the competition binding assay. The pKi values are calculated relative to histaprodifen 2, with the following equation: pKi = pKi (cpd.)–pKi (2). The original binding data for this analysis are shown in Table 3.

Constitutive Activity and Maximum Stimulation with Histamine of hH₁R, bH₁R, rH₁R, and gpH₁Rinthe Steady-State GTPase Assay. The basal activity (Fig. 8) of hH₁R, rH₁R, and gpH₁R in the steady-state GTPase assay ranged from 1.1 to 1.4 pmol/(mg min) without significant difference between these three species isoforms. However, the basal activity with bH₁R [1.8 pmol/(mg min)] was significantly higher (p < 0.05) than with hH₁R, rH₁R, and gpH₁R. In addition, the maximum stimulation with 100 µM histamine 1 relative to the basal activity (HA) was significantly higher (p < 0.0001) at bH₁R than at hH₁R, rH₁R and gpH₁R (Table 4). The ratio HA/B_max was not significantly different between the four species isoforms, indicating that the large stimulatory effect of histamine at bH₁R was a result of the high expression level of bH₁R in Sf9 cell membranes. The inverse agonist mepyramine 18 (Fitzsimons et al., 2004) exhibited only small inhibitory effects on basal GTP hydrolysis, indicative for low constitutive activity of H₁R species isoforms. The pEC₅₀ values of 18 ranged from 7.66 to 8.96 (Table 4). Only the pEC₅₀ values of bH₁R and rH₁R were significantly different from gpH₁R(p < 0.0025).

View larger version (15K): [in this window] [in a new window]   Fig. 8. Constitutive activity and maximal stimulation with histamine at hH1R, bH1R, rH1R, and gpH1R in the steady-state GTPase assay. Sf9 cell membrane coexpressing hH1R, bH1R, rH1R, or gpH1R and RGS4 were used for the GTPase assay as described under Materials and Methods. Basal, GTPase activity without any ligand; HA, maximal stimulation with 100 µM histamine; and MEP, GTPase activity in presence of 10 µM mepyramine. Data are means ± S.E.M. at least three independent experiments performed in duplicates or triplicates each. Membranes were used from independent membrane preparations.

Analysis of Histaprodifens at H₁R Species Isoforms in the Steady-State GTPase Assay. The results of the GTPase assays are summarized in Tables 5 (agonist mode) and 6 (antagonist mode). There were no significant differences in the pEC₅₀ values of the endogenous ligand histamine 1 among H₁R species isoforms. Histaprodifen 2 was a partial agonist at all four species isoforms. At gpH₁R and rH₁R, the pEC₅₀ of 2 increased approximately 0.8 log units relative to histamine 1. In contrast, there was no significant difference in the pEC₅₀ values of histaprodifen 2 relative to histamine 1 at hH₁R and bH₁R. The pEC₅₀ values of 2 between gpH₁R and hH₁R(p = 0.0005) or bH₁R(p = 0.0302) were significantly different. However, there was no significant difference in the pEC₅₀ values of 2 between gpH₁R and rH₁R, which were higher than at hH₁R and bH₁R. Thus, the additional diphenylpropyl moiety in histaprodifen 2 compared with histamine increased the pEC₅₀ at gpH₁R and rH₁R but had no effect on hH₁R and bH₁R. The additional Cl 3 and F 4 substituent in the diphenylpropyl moiety of 3 had no significant influence on the pEC₅₀ relative to histaprodifen 2. The pEC₅₀ of 4 at gpH₁R versus hH₁R(p = 0.0102) and bH₁R(p = 0.0121) and at rH₁R versus hH₁R(p = 0.0203) and bH₁R (p = 0.0111) were significantly different. The pEC₅₀ of 2 compared with the pEC₅₀ of 5 at hH₁R, bH₁R, and rH₁R were not significantly different. In contrast, the pEC₅₀ of 5 relative to 2 was significantly different (p = 0.0006) at gpH₁R. These data show that the pEC₅₀ values of the small histaprodifens 2 to 5 were similar between gpH₁R and rH₁R and higher than at hH₁R and bH₁R. Compared with histamine 1, pEC₅₀ values increased at gpH₁R and rH₁R, but not at hH₁R and bH₁R, except for 5 at bH₁R.

The additional imidazolyl moieties in the histaprodifen derivatives 6, 7, and 13 differ in the position of substitution. Compounds 6, 7, and 13 exhibited only small differences in pEC₅₀ values at H₁R species isoforms compared with histaprodifen 2. Again, higher pEC₅₀ values were found at gpH₁R and rH₁R for 6, 7, and 13. The substitution position of the imidazolyl moiety had only a small influence on the pEC₅₀ values at a given species isoform. Compounds 11, 12, and 9 have additional moieties at the R₃ position compared with suprahistaprodifen 7. The pEC₅₀ values of 11 were only significantly different between gpH₁R and bH₁R (p = 0.0061). The additional ethyl group in 11 slightly decreased pEC₅₀ values at rH₁R, but it had a small increasing influence at gpH₁R and hH₁R with respect to suprahistaprodifen 7. There was no significant difference between the four H₁R species isoforms with respect to the pEC₅₀ values of the thienylmethyl-substituted suprahistaprodifen 12. Again, the additional substituent slightly decreased the pEC₅₀ values relative to suprahistaprodifen 7. The pEC₅₀ value of 9 at gpH₁R was significantly higher (p < 0.05) than at hH₁R, bH₁R, and rH₁R.

The chiral histaprodifens 8R and 8S showed partial agonism at gpH₁R. The (R)-enantiomer 8R was significantly more potent than the (S)-enantiomer 8S (p = 0.0068). Only the (R)-enantiomer 8R showed partial agonism at hH₁R, bH₁R, and rH₁R. The potencies and the efficacies were significantly lower than at gpH₁R(p < 0.05). The (S)-enantiomer 8S showed antagonism at hH₁R, bH₁R, and rH₁R, with similar pK_B values (Table 6). Thus, the additional methyl group, introducing a center of chirality constitutes a unique agonism/antagonism-switch between the species [(S)-configuration 8S] and within the species [(R)/(S)-configuration 8R, 8S]. Figure 9 illustrates this switch for gpH₁R and hH₁R.

View larger version (16K): [in this window] [in a new window]   Fig. 9. Concentration-response curves for 8R and 8S at hH1R and gpH1R in the steady-state GTPase assay. The experiments were performed using Sf9 cell membranes coexpressing hH1R, bH1R, rH1R, or gpH1R and RGS4 as described under Materials and Methods. The square () shows the data for the (R)-configured derivative 8R, the upward-facing triangle () shows the data for the (S)-configured derivative 8S, the downward-facing triangle () shows the data for the (S)-configured derivative 8S performed with a final concentration of 1 µM histamine 1 (HA) in each assay tube. A, data for hH1R + RGS4. B, data for gpH1R + RGS4.

In compounds 13 to 16, the length of the CH₂-spacer was varied, resulting in differences in pEC₅₀ values of approximately 0.7 log units at each species isoform between the compounds of this series. Compound 15, with a (CH₂)₄-spacer, showed the lowest potency at all four species isoforms. The highest potency in this series was evident for 13 or 14. For dimeric histaprodifen 17, species differences were detected for all pairs of H₁R species isoforms (p < 0.05), except for the pairs gpH₁R/rH₁R and bH₁R/rH₁R. The potency increased in the order hH₁R < bH₁R < rH₁R < gpH₁R.

At gpH₁R, all histaprodifens were partial agonists. The compounds showed E_max values from 0.23 to 0.92. Only 10S showed an E_max value <0.5. E_max values at gpH₁R and rH₁R were higher than at bH₁R and hH₁R in most cases. In addition, the E_max values between hH₁R and bH₁R, on one hand, and between rH₁R and gpH₁R, on the other hand, were similar.

Binding Mode of Dimeric Histaprodifen in gpH₁R. MD simulations with docked dimeric histaprodifen 17 in gpH₁R showed that the ligand interacts with a binding pocket straight through the entire receptor (Fig. 10A, only hydrogen bonds and electrostatic interactions between ligand and receptor are shown). The following amino acids participate in the binding of 17. Leu-39 (TM1, 1.35), Leu-43 (TM1, 1.39), Leu-97 (TM2, 1.65), Trp-112 (TM3, 3.28), Asp-116 (TM3, 3.32), Tyr-117 (TM3, 3.33), Ser-120 (TM3, 3.36), Ile-124 (TM3, 3.40), Trp-167 (TM4, 4.56), backbone carbonyl of Lys-187 (E2), Glu-190 (E2), Trp-429 (TM6, 6.48), Tyr-432 (TM6, 6.51), Trp-456 (TM7, 7.40), and Tyr-459 (TM7, 7.43). The quaternary amine moiety in the center of dimeric histaprodifen is electrostatically trapped between the negatively charged Asp-116 (TM3, 3.32) and Glu-190 (E2). One of the histamine moieties forms a stable hydrogen bond interaction with Ser-120 (TM3, 3.36) and Tyr-432 (TM6, 6.51). The second histamine moiety forms a stable hydrogen bond to the backbone carbonyl of Lys-187 (E2). Both diphenylpropyl moieties are embedded in hydrophobic pockets formed by amino acids Trp-429 (TM6, 6.48), Ile-124 (TM3, 3.40) and Trp-167 (TM4, 4.56) as well as Leu-39 (TM1, 1.35), Leu-43 (TM1, 1.39), Leu-97 (TM2, 1.65), Trp-112 (TM3, 3.28), and Trp-456 (TM7, 7.40), respectively.

View larger version (41K): [in this window] [in a new window]   Fig. 10. Binding mode of dimeric histaprodifen 17 at gpH1R. Stable conformations of dimeric histaprodifen 17 (A), suprahistaprodifen 7 in orientations I (B) and orientation II (C), and the chiral histaprodifens 8R and 8S in orientations I and orientation II (D) in the binding pocket of gpH1R are shown. For clarity, 8R with the corresponding Ile-455 (TM7, 7.39) side chain conformation is rendered in red, and 8S with the corresponding Ile-455 (TM7, 7.39) side chain conformation is rendered in blue. The blue circle highlights the movement of the Ile-455 side chain. MD simulations and docking were performed as described under Materials and Methods. Hydrogen bonds and electrostatic interaction between ligand and amino acids are marked as red dashed lines. To increase clarity, only the most important parts of the transmembrane helices and the E2 loop around the binding pocket are shown.

	Discussion

Top Abstract Materials and Methods Results Discussion Conclusions References

 
Pharmacological Differences between hH₁R, bH₁R, rH₁R, and gpH₁R Concerning the Interaction with Chiral Histaprodifens. The results of the steady-state GTPase assay with the novel chiral histaprodifens 8R, 8S, 10R, and 10S showed unexpected and unique pharmacological differences between gpH₁R versus hH₁R, bH₁R, and rH₁R. At gpH₁R, all four chiral histaprodifens were partial agonists. However, only 8R was a partial agonist at hH₁R, bH₁R, and rH₁R, whereas 8S, 10R, and 10S were antagonists at those H₁R species isoforms. For GPCR activation, a conformational change in Trp^6.48/Phe^6.52, referred to as aromatic switch, is important (Crocker et al., 2006). Trp^6.48 (Trp-429 in gpH₁R) is important for direct interaction of histaprodifens with H₁R species isoforms (Fig. 2). Thus, it is possible that the additional methyl group in 8S, 10R, and 10S occupies a part of the binding pocket in hH₁R, bH₁R and rH₁R like a molecular wedge and prevents reorientation of the aromatic amino acids Phe^6.52 and Trp^6.48, with the consequence that the receptor cannot be activated.

Chiral histaprodifens 8R, 8S, 10R, and 10S were also analyzed at the guinea pig ileum (Striegl, 2006). All four compounds acted as partial agonists, and the (R)-enantiomers 8R (pEC₅₀ = 7.67) and 10R (pEC₅₀ = 7.36) were approximately 1 log unit more potent than the corresponding (S)-enantiomers (8S) (pEC₅₀ = 6.81) and 10S (pEC₅₀ = 6.44). These results are in good agreement with the results obtained in the recombinant system (Table 5; Fig. 9). Similar results were obtained for some ergoline derivatives at H₁R. Specifically, 8S-lisuride acted as potent partial agonist at the guinea pig ileum, whereas the 8R-lisuride showed only weak antagonism (Pertz et al., 2006). An analogous agonism/antagonism-switch as for the enantiomeric pairs 8R/8S and 10R/10S was observed for 1-allyl-8S-lisuride in the GTPase assay. This compound is a partial agonist at gpH₁R, but an antagonist at hH₁R (Pertz et al., 2006). Species-dependent stereoselectivity of receptor ligands is well documented (Soudjin et al., 2003) and extends beyond GPCRs. As an example, for chiral C-cyclopropylalkylamides, the (+)-enantiomer preferentially activates the human pregnane X receptor, whereas the (–)-enantiomer preferentially activates the mouse receptor (Mu et al., 2005).

H₁R Binding Pocket for Histaprodifens. The MD simulations showed that dimeric histaprodifen 17 is docked into a binding pocket straight through the entire gpH₁R (Fig. 10A). Because of the symmetrical properties of dimeric histaprodifen 17, all histaprodifen derivatives with a dimeric histamine moiety (7-12) could bind in two different orientations. Suprahistaprodifen 7 and the chiral histaprodifens 8R and 8S are predicted to interact with Asp-116 (TM3, 3.32), Ser-120 (TM3, 3.36), Lys-187 (E2), Glu-190 (E2), and Tyr-432 (TM6, 6.51) in both orientations. The diphenylpropyl moiety is predicted to interact with Ile-124 (TM3, 3.40), Trp-167 (TM4, 4.56), and Trp-429 (TM6, 6.48) in orientation I (Fig. 10B) and with Leu-39 (TM1, 1.35), Leu-43 (TM1, 1.39), Leu-97 (TM2, 1.65), Trp-112 (TM3, 3.28), and Trp-456 (TM7, 7.40) in orientation II (Fig. 10C).

The chiral histaprodifen 8R fits well into the binding pocket in both orientations. However, 8S only fits well in orientation I due to a steric clash between the additional methyl group and Ile-455 (TM7, 7.39) (Fig. 10D). At gpH₁R the affinities of suprahistaprodifen 7 and the chiral histaprodifen 8R are similar, whereas a decrease in affinity is observed for 8S. Based on these results two hypotheses can be proposed. First, suprahistaprodifen 7 and the chiral histaprodifens 8R and 8S may only bind in orientation II into the binding pocket. As a consequence of the sterical clash, the affinity of 8S is decreased. Second, suprahistaprodifen 7 and the chiral histaprodifen 8R may bind in orientations I and II, but 8S may only bind in orientation I. Thus, the experimentally observed K_i value for 7 and 8R may result from summation of the K_i values for orientations I and II, whereas the K_i value for 8S may only be derived from orientation I. With our present experimental data, we cannot distinguish which of the two hypotheses is correct. By analogy to our data, Dezi et al. (2007) discuss two possible orientations for some butyrophenone derivatives at the serotonin 5-hydroxytryptamine_2A receptor.

Molecular Basis for Pharmacological Differences between hH₁R, bH₁R, rH₁R, and gpH₁R. The alignment of the amino acid sequences is given in Fig. 2. Therein, the amino acids that interact directly with dimeric histaprodifen 17 in gpH₁R are highlighted. The same amino acids are found in hH₁R, bH₁R and rH₁R, too. The only difference occurs in the E2-loop at position 187 (gpH₁R), with a lysine in gpH₁R but an aspartate in hH₁R, bH₁R, and rH₁R. However, our simulations showed that dimeric histaprodifen 17 forms a stable hydrogen bond only to the backbone of Lys-187 in gpH₁R, but not to the side chain, which is pointing away from the binding pocket. Thus, the differences in this position should not be responsible for the observed species differences in pharmacology.

The alignment shows a difference between gpH₁R versus hH₁R, bH₁R and rH₁R in TM2 in position 2.61. In hH₁R, bH₁R, and rH₁R, there is an asparagine, which is changed to a serine in the gpH₁R. Bruysters et al. (2005) reported that this mutation is responsible for species differences between gpH₁R and hH₁R concerning suprahistaprodifen 7 and dimeric histaprodifen 17. Our studies showed an analogous species difference in pK_i values of suprahistaprodifen 7 and dimeric histaprodifen 17 between hH₁R and gpH₁R. If the amino acid exchange in position 2.61 alone had been exclusively responsible for the observed species differences between hH₁R and gpH₁R, the pK_i values of dimeric histaprodifen 17 at bH₁R and rH₁R would have been expected to be identical to that at hH₁R. However, we noticed a trend that the pK_i values and pEC₅₀ values of suprahistaprodifen 7 and dimeric histaprodifen 17 exhibited the order gpH₁R > rH₁R > bH₁R > hH₁R. Thus, it can be concluded that the amino acid exchange in position 2.61 is responsible for part of the species differences between hH₁R and gpH₁R, but other differences in amino acid sequences also influence pEC₅₀ and pK_i values.

In the competition binding assay, we observed that, in general, histaprodifens exhibited higher pK_i values at gpH₁R and lower pK_i values at hH₁R and bH₁R. The pK_i values at rH₁R were intermediate between those at gpH₁R and hH₁R/bH₁R. In addition, in the steady-state GTPase assay, histaprodifens were, in general, more potent and efficacious at gpH₁R, followed by rH₁R, bH₁R, and hH₁R. However, as pointed out above, there are no species differences in the amino acid side chains that directly interact with dimeric histaprodifen in the binding pocket of gpH₁R. Therefore, it can be concluded, that amino acids that are different between the four species isoforms and not in direct contact with the binding pocket, have an indirect influence onto the binding pocket and to the binding mode of ligands.

	Conclusions

Top Abstract Materials and Methods Results Discussion Conclusions References

 
Our studies document substantial pharmacological differences between H₁R species isoforms. With respect to their pharmacological profile, bH₁R and rH₁R can be classified intermediate between hH₁R and gpH₁R, bH₁R being more similar to hH₁R and rH₁R being more similar to gpH₁R. This trend is approximately reflected in Table 1, where the level of similarity of the amino acid sequences between hH₁R, bH₁R, rH₁R, and gpH₁R is given. Our studies support the usefulness of GPCR species isoforms as tools to analyze the binding mode of ligands. However, our data also clearly show that in some cases, it is difficult to explain species differences in pharmacology with a single defined difference in amino acid sequence. Finally, the chiral histaprodifens are novel unique ligands to dissect subtle differences in the activation mechanism of the H₁R in various species.

	Acknowledgements

 
We thank Dr. H. Fukui for providing cDNAs for bH₁R and rH₁R; Dr. S. Elz (Department of Pharmaceutical and Medicinal Chemistry I, University of Regensburg, Germany) for helpful discussions and support; K. Wohlfart, A. Seefeld, A.-C. Groß, and C. Huber for performing the GTPase and binding assays; and G. Wilberg for competent help with the cell culture and immunoblot analysis. We also thank the reviewers for constructive critique.

	Footnotes

 
This work was supported by the Research Training Program (Graduierten-kolleg) GRK760 "Medicinal Chemistry: Molecular Recognition–Ligand-Receptor Interactions" of the Deutsche Forschungsgemeinschaft.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.129601.

ABBREVIATIONS: H₁R, histamine H₁ receptor; GPCR, G protein-coupled receptor; TM transmembrane domain; gp, guinea pig; h, human; b, bovine; r, rat; RGS, regulator of G protein signaling; PCR, polymerase chain reaction; S, signal peptide; F, FLAG; DMSO, dimethyl sulfoxide; MD, molecular dynamics; bb, backbone; sc, side chain; lig, ligand; E, extracellular loop; HA, histamine; cpd., compound; I, intracellular loop, MEP, mepyramine; ps, picosecond.

Address correspondence to: Dr. Roland Seifert, Department of Pharmacology and Toxicology, University of Regensburg, Universitätsstraße 31, D-93053 Regensburg, Germany. E-mail: roland.seifert{at}chemie.uni-regensburg.de

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