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J. Biol. Chem., Vol. 281, Issue 30, 21558-21565, July 28, 2006
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1
From the
Molecular Pathophysiology Section, NIDCD, National Institutes of Health, Bethesda, Maryland 20892, the Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, the ¶Computational Chemistry Core Laboratory, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, and the ||Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543
Received for publication, April 17, 2006 , and in revised form, May 19, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-amino butyric acid type B receptors. They have a characteristic Venus flytrap-like structure (3) in their N-terminal extracellular domain, which constitutes the orthosteric site for binding of endogenous agonists (Fig. 1A). A crystal structure of homodimeric mGluR1 Venus flytrap-like domain in agonist-bound form verified this (3). Exogenous allosteric modulators bind to sites in the seven transmembranespanning domains of the receptors topographically distinct from the orthosteric sites (Fig. 1A). Allosteric modulators offer advantages over classic orthosteric ligands as therapeutic agents, including the potential for greater GPCR subtype selectivity and safety.
A number of allosteric modulators have been identified targeting specifically the CaR, which plays a central role in the regulation of calcium homeostasis (4, 5). Positive allosteric modulators of the CaR, such as NPS R-568 (Fig. 1B), increase CaR activation, thereby decreasing secretion of parathyroid hormone, and thus may be useful in the treatment of primary and secondary hyperparathyroidism. Cinacalcet is the first-inclass GPCR allosteric modulator approved by the U. S. Food and Drug Administration recently for treatment of secondary hyperparathyroidism in patients with chronic kidney disease on dialysis and for treatment of hypercalcemia in patients with parathyroid cancer. Negative allosteric modulators of the CaR, such as NPS 2143 (Fig. 1B), decrease receptor activation, thereby stimulating endogenous parathyroid hormone secretion. This potentially offers a novel method for treatment of osteoporosis.
NPS R-568, cinacalcet, NPS 2143, and several other allosteric modulators of the CaR are structurally related phenylalkylamines with a positively charged central amino group (Fig. 1B). We and others recently reported that the residue Glu837 in transmembrane helix 7 of the CaR is crucial for action of those phenylalkylamines tested (6-8). Another mutation, I841A, has also been reported to abolish responsiveness to positive and negative allosteric modulators (8). It is speculated that allosteric binding sites of these phenylalkylamines partially overlap and that a critical salt bridge might form between the negatively charged Glu837 and the positively charged central amine in these compounds.
Recently, a novel negative allosteric modulator, Compound 1, was described (9). It is structurally distinct from those phenylalkylamines (Fig. 1B), and in a competition assay, it did not displace a radiolabeled analogue of NPS 2143 bound to the receptor, suggesting that its unique allosteric site is distinct from those of phenylalkylamines (9). In the present work, we synthesized and studied Compound 1 analogues and examined the effects of alanine substitutions at some key residues of the CaR, such as Glu767, Glu837, and Ile841, on allosteric modulation of the receptor by these compounds.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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strain). The sequence of mutant receptors was confirmed by automated DNA sequencing using a dRhodamine terminator cycle sequencing kit and ABI PRISM-373A DNA sequencer (Applied Biosystems, Foster City, CA). Transient Transfection of Wild Type and Mutant Receptors in HEK-293 Cells—Transfections were performed using 12 µg of plasmid DNA for each transfection in a 75-cm2 flask of HEK-293 cells. DNA was diluted in serum-free DMEM (BioFluids nc., Rockville, MD) and mixed with diluted Lipofectamine (Invitrogen), and then the mixture was incubated at room temperature for 30 min. The DNA-Lipofectamine complex was further diluted in 6 ml of serum-free DMEM and was to 80% confluent HEK-293 cells plated in 75-cm2 flasks. After 5 h of incubation, 15 ml of complete DMEM containing 10% fetal bovine serum (BioFluids Inc., Rockville, MD) was added. 24 h after transfection, transfected cells were split and cultured in complete DMEM.
Phosphoinositide (PI) Hydrolysis Assay—PI hydrolysis assay has been described previously (6). Briefly, 24 h after transfection, transfected cells from a confluent 75-cm2 flask were split and plated in two 12-well plates in complete DMEM medium containing 3.0 µCi/ml [3H]myoinositol (PerkinElmer Life Sciences) and cultured for another 24 h. Culture medium was replaced by 1x PI buffer (60 mM NaCl, 2.5 mM KCl, 2.8 mM glucose, 0.2 mM MgCl2, 10 mM LiCl in 12.5 mM PIPES buffer, pH 7.2) and incubated for 1 h at 37°C. After removal of PI buffer, cells were incubated for an additional 1 h with different concentrations of Ca2+ in 1x PI buffer with or without treatment of compounds. The reactions were terminated by the addition of 1 ml of acid:methanol (1:1,000 v/v) per well. Total inositol phosphates were purified by chromatography on Dowex 1-X8 columns, and radioactivity for each sample was counted with liquid scintillation counter. Graphs of concentration dependence for stimulation of PI hydrolysis by [Ca2+]o for each transfection were drawn by using GraphPad Prism version 2.0 software. Each value on a curve is the mean of duplicate determinations. Graphs shown in this study were representative ones from at least three independent experiments.
Synthetic Chemistry—Compound 1 was provided by Bristol-Myers Squibb Pharmaceutical Research Institute (9). For the synthesis of JKJ05, (S)-(-)-2-amino-3-phenyl-1-propanol (45 mg, 0.30 mmol) and potassium tert-butoxide (34 mg, 0.30 mmol) were added to a solution of the appropriately substituted 2-chloropyridine (88 mg, 0.20 mmol) (prepared in accordance with known literature methods (20)) in toluene (2.5 ml), and the resulting mixture was stirred for 48 h at 90 °C. The solution was allowed to cool to room temperature, and the solvent was removed under reduced pressure. Following the careful addition of H2O (10 ml), the resulting mixture was extracted with EtOAc (3 x 10 ml). The combined organic fractions were dried (MgSO4) and filtered, and solvent was removed under reduced pressure. The resulting crude product was purified by column chromatography (1:10 MeOH:EtOAc) to afford JKJ05 as a colorless solid; 100 mg, 90%. 1H NMR (CDCl3, 300 MHz) 
2.73 (dd, JHH = 13.5, 8.1 Hz, 1H), 2.93 (dd, JHH = 13.5, 5.7 Hz, 1H), 3.46-3.54 (m, 1H), 3.61 (q, JHH = 5.4 Hz, 2H), 3.819 (s, 3H), 3.824 (s, 6H), 3.87 (t, JHH = 5.4 Hz, 2H), 4.24 (dd, JHH = 10.8, 6.9 Hz, 1H), 4.42 (dd, JHH = 10.8, 4.2 Hz, 1H), 5.86 (t, JHH = 5.7 Hz, 1H), 6.74 (dd, JHH = 7.8, 1.2 Hz, 2H), 6.79 (d, JHH = 8.4 Hz, 1H), 6.85 (s, 2H), 6.96 (tt, JHH = 7.5, 1.2 Hz, 1H), 7.18-7.22 (m, 7H), 7.92 (d, JHH = 8.7 Hz, 1H); (time-of-flight mass spectrometry) m/z 558.2629 (M+H+) (calculated for C32H36N3O6+) 558.2604.
For the synthesis of JKJ05-Ac, pyridine (11.0 µl, 0.14 mmol), 4-dimethylaminopurine (0.5 mg, 0.0045 mmol), and Ac2O (13.2 µl, 0.14 mmol) were added to a solution of JKJ-05 (25 mg, 0.045 mmol) in CH2Cl2 (2 ml) and stirred for a 1-h period. Solvent was removed under reduced pressure, and the resulting crude product was purified by column chromatography (1:1 EtOAc: hexanes) to afford JKJ05-Ac as a colorless solid; 20 mg, 74%. 1H NMR (CDCl3, 300 MHz) 1.83 (s, 3H), 2.72-3.40 (m, 2H), 3.62 (q, JHH = 5.4 Hz, 2H), 3.807 (s, 3H), 3.814 (s, 6H), 3.88 (t, JHH = 5.4 Hz, 2H), 4.28-4.41 (m, 2H), 4.49-4.60 (m, 1H), 5.85 (t, JHH = 5.4 Hz, 1H), 6.05 (d, JHH = 8.1 Hz, 1H), 6.74 (dd, JHH = 8.7, 1.2 Hz, 2H), 6.81 (d, JHH = 8.7 Hz, 1H), 6.83 (s, 2H), 6.94 (tt, JHH = 7.5, 1.2 Hz, 1H), 7.18-7.29 (m, 7H), 7.96 (d, JHH = 8.7 Hz, 1H); (time-of-flight mass spectrometry) m/z 600.2700 (M+H+) (calculated for C34H38N3O7+) 600.2710.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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E837A Mutation in the CaR Converts JKJ 05 into a Positive Allosteric Modulator—We also tested the effects of JKJ05 on the E837A mutant CaR. Surprisingly, JKJ05 significantly increased sensitivity of the E837A mutant to Ca2+ with an EC50 of 2.5 µM (Fig. 3, B and C) rather than inhibiting response to Ca2+ by the receptor. E837A mutation abolished allosteric modulator action by phenylalkylamines tested, whereas this same mutation evidently does not eliminate binding of JKJ05 to the receptor, suggesting that JKJ05, similar to Compound 1, might not share a binding pocket with NPS 2143. On the other hand, different allosteric actions associated with Compound 1 and its analogue JKJ05 indicate distinct interactions between functional group(s) in JKJ05 and surrounding residues(s) in the CaR that did not occur with Compound 1.
Positive Modulation of E837A Mutant CaR by JKJ05 Critically Depends on the Primary Amino Group in the Compound—To verify whether positive modulation of the E837A mutant CaR by compound JKJ05 requires the positively charged primary amine in the compound, we synthesized JKJ05-Ac, an acetylated derivative of JKJ05, via treatment with of acetic anhydride, pyridine, and a catalytic amount of dimethylaminopurine (Fig. 1, B and C). We found that JKJ05-Ac remained a weak negative modulator of the wild type CaR, comparable with JKJ05 (Fig. 3A). However, the augmentation of E837A mutant receptor sensitivity to Ca2+ by compound JKJ05 was abolished by acetylation of this compound (Fig. 3B). Thus, JKJ05-Ac could be considered as a "silent" allosteric modulator of the E837A mutant CaR, defined as not altering the response of an orthosteric ligand but binding to an allosteric site of the GPCR.
Molecular Modeling of the CaR 7TM Domain and Docking of Compound 1 and JKJ05—In an effort to understand the mechanism of allosteric modulation exerted by Compound 1 and its analogues, we constructed a homology model of the CaR 7TM domain based on the crystal structure of bovine rhodopsin (10) and performed docking experiments with these compounds (see supplemental materials). No external bias was given on the precise location of the potential binding site. Instead, the docking region initially was loosely defined as the whole upper half of the helical bundle.
Compound 1 and related analogues could potentially bind to two adjacent pockets (Fig. 4), which here we designate as P1 (enclosed within TM3, TM4, TM5, TM6, and the second extracellular loop (EL2)) and P2 (enclosed within TM1, TM2, TM3, TM6, TM7, and EL2) (Fig. 4). It has been proposed that NPS 2143 and related phenylalkylamines bind to the P2 pocket, with their positively charged amino group engaged in an electrostatic interaction with the side chain of Glu837 (7.39) (6-8). As Compound 1 did not compete with a NPS 2143 analogue for binding to the receptor (9), we speculate that Compound 1 and its analogues JKJ05 and JKJ05-Ac bind to the P1 pocket. Our result that the E837A mutation in the P2 pocket blocked negative modulation by NPS 2143 but not by Compound 1 is consistent with our above modeling hypothesis.
Our model suggests that the striking positive modulation of the E837A mutant CaR by JKJ05 might result from the combined effects of three distinct factors (Fig. 5): 1) the alanine substitution of residue Glu837 (7.39), which altered the ground state interactions of this residue; 2) the formation of a salt bridge between the positively charged primary amino group of JKJ05 and the acidic residue Glu767 residing in EL2; and 3) the van der Waals interactions of the benzyl moiety adjacent to the amino group of JKJ05 with a critical cluster of hydrophobic amino acids located in the upper half of TM6 (Fig. 5). This TM6 region, Trp818 (6.48)-Tyr825 (6.55), is part of a "hot spot" of the CaR we found recently to be critical for receptor activation (7). Six out of the 8 residues in this region are potential sites for activating mutations (7). Among them, residue Phe821 (6.51), Ile822 (6.52), and Tyr825 (6.55) reside in the binding pocket for JKJ05 and analogues in our model (Fig. 5). Although alanine substitution of Trp818 (6.48) did not activate the CaR, aromatic residues at position 6.48 have been shown to play a key role in the activation of many other GPCRs, including rhodopsin (11, 12). Our model also suggests that besides these residues in TM6, adjacent residues in other TMs, such as Phe688 (3.36), might contribute to the formation of the hydrophobic cluster critical for the action of JKJ05 (Fig. 5). The fact that JKJ05 is able to left-shift the sensitivity of the receptor to Ca2+ only when Glu837 (7.39) is mutated to alanine suggests that the ground state interactions of this residue are sufficient to counteract the activating properties of JKJ05.
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Our in vitro assay shows that the function of this double mutant was impaired as seen in a decreased maximal response to extracellular calcium. However, consistent with our hypothesis, both JKJ05 and JKJ05-Ac, which interact with the critical hydrophobic cluster in a similar way, dramatically increased the calcium response of the receptor (Fig. 6). Conversely, Compound 1 interacted with the hydrophobic cluster in a different way (Fig. 5), due to its different chemical structure, leading to the slightly right-shifted calcium response of the receptor. These results support our model of the CaR 7TM and speculations on the mechanism of action of JKJ05.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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In addition to their therapeutic potential, allosteric modulators of the CaR offer unique insights into the mechanisms of receptor activation. How the signal of conformational changes in orthosteric sites upon ligand binding is transmitted to the 7TM, leading to receptor activation, is a major unanswered question. It is speculated that movements of the helices within 7TM and/or between two 7TMs in dimeric CaRs are ultimately responsible for receptor activation and G protein-coupling. Allosteric modulators bound to the allosteric sites in the 7TM domain of the CaR likely facilitate (positive modulators) or impede (negative modulators) these movements.
We published earlier that residue Glu837 in TM7 is crucial for positive allosteric modulation by NPS R-568 (6), and later, it was reported that this same residue is also crucial for allosteric modulation by other phenylalkylamines (8). It is speculated that a critical salt bridge is formed between the positively charged central group of these phenylalkylamines and the acidic side chain of residue Glu837. Here we report that Compound 1, a novel negative allosteric modulator of the CaR lacking a central amino group, retains activity against the E837A mutant. This, together with earlier competition assay data, suggests that Compound 1 binds to an allosteric site distinct from those of phenylalkylamines. We also found that JKJ05, an analogue of Compound 1, acts as a negative allosteric modulator on the wild type receptor, but surprisingly, as a positive modulator on the E837A mutant receptor. This positive modulation critically depends on the primary amine in JKJ05. These findings led us to speculate a novel mechanism of allosteric modulation by JKJ05 and its acetylation derivative JKJ05-Ac based on modeling of the CaR 7TM domain and docking of these compounds.
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Our model offers an interpretation of our findings, and at this stage, we cannot exclude other possible mechanisms. Further studies are necessary to understand why positive modulation of the CaR by JKJ05 and JKJ05-Ac critically depends on alteration of the ground state interactions of Glu767 and Glu837 by either alanine substitution or a salt bridge formation. Moreover, residue Ile841 seems to be crucial for action of all allosteric modulators of the CaR tested so far, including previously published phenylalkylamines and Compound 1 and its analogues. The exact role this residue plays in receptor activation remains to be elucidated.
We emphasize that a single missense mutation, E837A, converts a negative allosteric modulator (JKJ05) of the CaR into a positive modulator. To our knowledge, this is the first report of an allosteric modulator whose action, negative versus positive modulation, critically depends on a single residue in a GPCR. There have been reports that mutations in the orthosteric site of GPCRs converted antagonists to agonists (16, 17). We now show that such a phenomenon can also happen at the allosteric binding site of the CaR, suggesting the need for caution in therapeutic application of allosteric modulator-based agents and the need for identification of possible genetic variation in the allosteric site of therapeutically targeted GPCRs.
Genetic variants of many GPCRs have been reported (see Ref. 18 for a review), and the CaR genetic locus appears to harbor one of the largest numbers (
100) of variants with over 20 variations identified in the 7TM domain alone (19). Incorporation of studies of genetic variants into strategies for GPCR drug discovery and clinical drug testing has an important potential to improve efficacy and decrease toxicity of drugs (18). Given the risk of "paradoxical" effects if the receptor targeted carries an unsuspected missense mutation in the 7TM, further delineation of residues comprising allosteric binding sites, elucidation of allosteric modulation mechanism, and appreciation of potential sequence variation in the 7TM of the receptor among populations to be treated appear warranted.
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FOOTNOTES |
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The on-line version of this article (available at http://www.jbc.org) contains supplemental methods and supplemental references.
1 To whom correspondence should be addressed: National Institutes of Health, Bldg. 10, Rm. 8C-101, 9000 Rockville Pike, Bethesda, MD 20892. Tel.: 301-496-9212; Fax: 301-402-0374; E-mail: jianxinh{at}intra.niddk.nih.gov.
2 The abbreviations used are: GPCR, G protein-coupled receptor; CaR, extracellular Ca2+ receptor; hCaR, human CaR; mGluR, metabotropic glutamate receptor; PI, phosphoinositide; 7TM, seven transmembrane domain; WT, wild type; TM, transmembrane; EL, extracellular loop; DMEM, Dulbecco's modified Eagle's medium; PIPES, 1,4-piperazinediethanesulfonic acid.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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