From the Department of Medicine and Center for Gastroenterology Research on Absorptive and Secretory Processes, Tupper Research Institute, New England Medical Center, Tufts University School of Medicine, Boston, Massachusetts 02111
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ABSTRACT |
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The development of non-peptide agonists for
peptide hormone receptors would markedly expand the treatment options
for a large number of diseases. However, difficulty in identifying
non-peptide molecules which possess intrinsic activity has been a major
obstacle in achieving this goal. At present, most of the known
non-peptide ligands for peptide hormone receptors appear in standard
functional assays to be antagonists. Here, we report that a
constitutively active mutant of the human cholecystokinin-B/gastrin
receptor, Leu325 Glu, offers the potential to
detect even trace agonist activity of ligands which, at the wild type
receptor isoform, appear to lack efficacy. The enhanced functional
sensitivity of the mutant receptor enabled us to detect intrinsic
activity of L-365,260, an established non-peptide antagonist for the
cholecystokinin-B/gastrin receptor. Extending from this observation, we
were able to demonstrate that minor structural modifications could
convert L-365,260 into either: (i) an agonist or (ii) an inverse
agonist (attenuates ligand-independent signaling). The ability to
confer functional activity to small non-peptide ligands suggests that
the properties of endogenous peptide hormones can be mimicked, and even
extended, by considerably less complex molecules.
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INTRODUCTION |
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Current understanding of G-protein-coupled receptor activation has in large part been based on the study of biogenic amine receptors (1). The corresponding endogenous ligands, together with synthetic derivatives of these small molecules, cover a spectrum of functional activities ranging from full agonists to antagonists. With the discovery of constitutively active receptor mutants, this range has been further extended to include inverse agonists, distinguished by their ability to attenuate ligand-independent signaling (2).
Another major group of G-protein-coupled receptors is activated by endogenous peptide molecules. Compared with biogenic amines, these peptide agonists are significantly larger and structurally more complex. Since endogenous peptides exert important hormone and neurotransmitter functions, there is considerable interest in whether their function can be mimicked by non-peptide drugs (3). This possibility is suggested by the opioid receptor system. Naturally occurring opioid receptor non-peptide agonists (e.g. morphine) as well as synthetic derivatives have been utilized since the early 19th century (4). Over the last 10 years, numerous non-peptide compounds have been identified which recognize specific peptide hormone receptor subtypes with high affinity. Unlike the corresponding endogenous peptide agonists, the vast majority of these new ligands appear to lack intrinsic activity and have been pharmacologically classified as antagonists (5).
The difficulty in generating non-peptide agonists is exemplified by the extensive efforts which have focused on the identification of ligands for the human cholecystokinin-B/gastrin receptor (CCK-BR).1 This receptor has been implicated in modulating memory, anxiety, and pain perception, as well as in regulating gastrointestinal mucosal growth and secretion (6-8). With the exception of some peptide-derived compounds (9, 10), all of the synthetic CCK-BR non-peptide ligands which have been discovered to date are reported to be antagonists and thus appear unable to satisfy the structural requirements for receptor activation.
The prototype of such non-peptide, selective CCK-BR antagonists is L-365,260, a benzodiazepine-based ligand which was discovered in 1989 (11). Widely tested both in vivo and in vitro, this compound has become a cornerstone in the characterization and pharmacological classification of CCK receptors. We now report that L-365,260 has unexpected residual intrinsic activity, which we were able to detect using a constitutively active CCK-BR mutant. Extending from this finding, we demonstrate that slight structural modifications of L-365,260 convert this ligand into either: (i) a non-peptide agonist for the wild type CCK-BR, or (ii) an inverse agonist. These findings illustrate that existing non-peptide ligands may have considerably higher potential to activate peptide hormone receptors than was previously appreciated, and suggest that constitutively active receptor mutants provide promising tools to detect and optimize such functional properties.
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EXPERIMENTAL PROCEDURES |
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Materials-- Cell culture media and fetal calf serum were obtained from Life Technologies, Inc. (Gaithersburg, MD) and from Intergen (Purchase, NY), respectively. 125I-CCK-8 (2,200 Ci/mmol) and myo-[3H]inositol (45-80 Ci/mmol) were purchased from NEN Life Science Products (Boston, MA). Unlabeled gastrin heptadecapeptide (G-17, unsulfated form), CCK-8 (sulfated form), CCK-8US (unsulfated form), and CCK-4 were obtained from Peninsula Laboratories (Belmont, CA). L-365,260, L-740,093 (R and S forms), YM022, and L-364,718 were generously provided by Wyeth Research Ltd. (Taplow, United Kingdom). The "peptoid" compounds PD-135,158 and PD-136,450 were a gift from Parke-Davis Research Center (Cambridge, UK).
Measurement of Inositol Phosphate Accumulation-- COS-7 cells (1 × 106) were plated onto 10-cm culture dishes (Costar, Cambridge, MA) and grown overnight in Dulbecco's modified Eagle's medium, 10% fetal calf serum at 37 °C. The cells were transfected with 5 µg of wild type or mutant CCK-BR cDNA (12), subcloned into the expression vector pcDNAI (Invitrogen). The following day, cells were split into 6-well plates (3 × 105/well) (Nunc). The cells were then prelabeled overnight with 3 µCi/ml myo-[3H]inositol in serum-free Dulbecco's modified Eagle's medium. To assess ligand-induced inositol phosphate production, the medium was replaced with phosphate-buffered saline containing 10 mM LiCl, and cells were incubated with the respective ligands for 30 min at 37 °C. Each ligand was tested at a concentration which was at least 25-fold higher than the corresponding dissociation constant (Ki value) determined in radioligand binding experiments (see below, Table I). According to the simple Langmuir isotherm (fractional receptor occupancy = ligand concentration/[Ligand concentration + Ki]) the ligand concentrations which were utilized result in >95% receptor occupancy (13), and will thus induce near maximal receptor stimulation and inositol phosphate production. After incubation, cells were lysed and extracted with methanol/chloroform; the upper phase was analyzed for inositol phosphates by strong anion exchange chromatography (14). Inositol phosphate production was expressed as a percentage of the total cellular tritium content which was incorporated during an overnight exposure to myo-[3H]inositol. Concentration-response curves were calculated using the GraphPad Prizm software (GraphPad, San Diego, CA).
Binding Experiments--
Twenty-four hours after transfection,
COS-7 cells were seeded into 24-well dishes (1 × 104/well) (Costar). After an additional 24 h,
competition binding experiments were performed in Hank's balanced salt
solution supplemented with 25 mM HEPES, pH 7.3, 0.2%
bovine serum albumin, and 0.15 mM phenylmethylsulfonyl
fluoride, using 20 pM 125I-CCK-8 as the
radioligand. After an 80-min incubation in the absence or presence of
unlabeled ligands, cell monolayers were washed three times with Hank's
balanced salt solution and then hydrolyzed in 1 N NaOH for
-counting (15). All binding affinities were calculated by nonlinear
curve fitting using the LIGAND computer program (16). Receptor
densities in transfected COS-7 cells were calculated from
125I-CCK-8 binding experiments with increasing
concentrations of unlabeled CCK-8 as the homologous competitor.
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RESULTS AND DISCUSSION |
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Molecular characterization of the third intracellular loop of the
human CCK-BR led to the identification of a point mutation (Leu325 Glu) which results in constitutive receptor
activity (17). When transiently expressed in COS-7 cells, the
Leu325
Glu mutant triggers agonist independent
production of inositol phosphates, to levels exceeding cells expressing
the wild type receptor at similar densities (Fig.
1A). Despite this difference in ligand-independent signaling, the wild type and the mutant receptors
increase inositol phosphate production to comparable levels when
stimulated with saturating concentrations of either CCK-8 or G-17, two
of the principal endogenous agonists for the CCK-BR (6, 7, 18). In
addition, both CCK-8 and G-17 have similar potencies and binding
affinities when respective values at the wild type and the mutant
receptor isoforms are compared (Fig. 1B, Table
I).
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Contrary to expectation, we found that the non-peptide ligand,
L-365,260, has significant partial agonist activity when stimulating the mutant (versus the wild type) receptor (Fig.
2A). L-365,260, previously
considered a prototype CCK-BR antagonist (5, 11), is able to activate
the Leu325 Glu receptor mutant with approximately half
the efficacy of the full agonist, G-17. On closer examination, it
became apparent that L-365,260 also induces a small yet significant
(p < 0.01) increase of inositol phosphate production
in cells expressing the wild type CCK-BR (Fig. 2A, left
panel).
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These findings prompted us to investigate the functional properties of
compounds which are structurally related to L-365,260, including YM022
(19) and (R)-L-740,093, as well as its stereoisomer, (S)-L-740,093 (20). All of these molecules share a
1,4-benzodiazepine backbone (Fig. 2B), and are reported to
function as antagonists. However, when assessed with the constitutively
active receptor mutant (Fig. 2A), it became readily apparent
that these compounds cover a broad range of intrinsic activities. The
amplification of second messenger signaling observed with the
Leu325 Glu receptor allowed us to demonstrate that
minor structural changes of L-365,260 result in considerable functional
alterations. Replacing the C5-phenyl moiety of the core benzodiazepine
structure with an azabicyclo[3.2.2]nonane substituent, combined with
changing the C3 stereochemistry, converts L-365,260 into
(S)-L-740,093 (20), the most efficacious agonist of the
tested ligands. Reflecting its relatively strong signaling potential,
(S)-L-740,093 also functions as a partial agonist at the
wild type CCK-BR (Fig. 2A). Further confirming the agonist
activity of (S)-L-740,093, stimulation of the wild type
CCK-BR with this compound triggers a
concentration-dependent increase in second messenger
signaling (Fig. 3). Therefore,
(S)-L-740,093 provides the first example of a "true"
non-peptide agonist for the human CCK-BR.
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It is noteworthy that the mirror image (R-enantiomer) of the non-peptide agonist (S)-L-740,093 has the opposite functional properties. (R)-L-740,093 reduces basal signaling of the constitutively active receptor close to that of the wild type isoform (Fig. 2A) and thereby satisfies the criterion of an inverse agonist. Non-peptide inverse agonists have recently been identified for two other types of peptide receptors, i.e. the thyrotropin-releasing hormone and the AT1A angiotensin II receptors (21, 22). Like (R)-L-740,093, each of these non-peptide inverse agonists appears to have no intrinsic activity at the respective wild type receptors and was therefore originally classified as an antagonist. Together, these examples illustrate that inverse agonism is likely a hidden property of many ligands which are currently considered "antagonists." To enable more definitive classification of these compounds, further functional characterization using constitutively active receptors will be required. The discovery of non-peptide inverse agonists provides a compelling rationale for developing a new class of drugs, targeted at constitutively active peptide hormone receptors which result in human disease (23, 24). For example, the pathogenesis of thyroid adenomas has been linked to constitutively active thyroid stimulating hormone receptors (25); drugs which "silence" these overactive proteins could potentially be utilized to inhibit tumor growth. Similarly, inverse agonists could delay the onset of precocious puberty in patients with constitutively active luteinizing hormone receptors (26).
The concentration-dependent activity of (S,R)-L-740,093 at the wild type and mutant CCK-BRs supports the pharmacological classification of these compounds as agonist and inverse agonist, respectively (Figs. 3 and 4A). It is intriguing that altering the steric conformation of these compounds can interconvert their function. An observation which parallels our findings with the L-740,093 enantiomers was recently reported for synthetic derivatives of the carboxyl-terminal cholecystokinin tetrapeptide fragment, CCK-4. Two of these peptoid ligands, PD-149,164 and PD-151,932, which are distinguished only by their steric conformation, were found to act as agonist and antagonist, respectively, at the CCK-A receptor subtype (27). It remains to be established whether stereoisomers of ligands for other peptide hormone receptors will also have opposite functional properties.
Unlike the L-740,093 enantiomers, YM022 has minimal effect on the basal activity of either the wild type or the mutant CCK-B/gastrin receptors (Fig. 2A). This lack of intrinsic activity, and the ability of YM022 to block CCK-8 induced inositol phosphate formation (17), are consistent with the expected properties of an antagonist. To further validate the functional classification of the CCK-BR non-peptide ligands, the interaction of YM022 with S- and R-740,093 was studied. (S)-L-740,093 induced inositol phosphate production was inhibited by YM022 in a concentration-dependent manner (Fig. 4B, top), further supporting that (S)-L-740,093 functions as a non-peptide agonist. In addition, YM022 was able to attenuate the inhibitory effect of (R)-L-740,093 on the constitutively active CCK-BR mutant (Fig. 4B, bottom). This observation is in agreement with the principle that the activity of inverse agonists should also be sensitive to inhibition by antagonists (2).
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To determine whether the enhanced signaling observed with the
Leu325 Glu mutant applied to a structurally different
class of molecules, we compared activation of the wild type and the
mutant CCK-BRs by two peptoid ligands, PD-135,158 and PD-136,450. Both
of these compounds have in previous in vivo studies been
shown to be partial CCK-BR agonists (9, 10). Using recombinant human
CCK-BRs expressed in COS-7 cells we confirmed the agonist properties of PD-135,158 and PD-136,450 in vitro, and in addition
demonstrated that these peptoids are almost full agonists at the
constitutively active mutant (Fig.
5).
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Despite significant structural differences between tested compounds,
respective efficacy values at the wild type CCK-BR are systematically
amplified at the Leu325 Glu mutant. The correlation of
ligand efficacies at both receptor isoforms is illustrated by the
dashed line in Fig. 5. This systematic amplification
contrasts with several reports of mutant receptors where observed
efficacy changes were clearly ligand-specific. For the
-adrenergic,
-opoid, and CCK-B receptors, mutations have been identified which
only result in the amplification of ligand efficacies for selected
compounds. In contrast, the efficacies of other, structurally different
ligands remain unchanged or are even reduced when examined with these
mutant receptors (28-30). It is of note that all of the mutations
which differentially affect the function of individual ligands
correspond to amino acid substitutions within the transmembrane domains
of the receptor, close to the extracellular surface, whereas the
Leu325
Glu mutation of the CCK-BR is found in the third
intracellular loop. Whether mutations induce a ligand-specific or a
generalized amplification of ligand efficacies may depend on the
location of these mutations within the receptor molecule. We have
previously demonstrated that CCK-BR transmembrane domain mutations
differentially modulate the interaction of individual compounds with
the putative transmembrane domain "ligand binding pocket" (31). In
contrast, we hypothesize that intracellular mutations can globally
affect receptor isomerization by altering the equilibrium between
"inactive" and "active" states (2).
Our observations can in part be explained by an extended ternary
complex model of receptor activation which has been proposed based on
study of a constitutively active 2-adrenergic receptor (32). Consistent with this model, we noted that the efficacies of
partial CCK-BR agonists were systematically enhanced when comparing respective values at the wild type receptor and the Leu325
Glu mutant (Fig. 5). The extended ternary complex model of receptor
activation further postulates that with constitutive receptor activity,
the binding affinities of ligands should increase commensurate with
their signaling efficacies, i.e. the largest affinity shifts
are expected for full agonists. This prediction is supported by the
observation that agonist affinities at constitutively active biogenic
amine receptors were increased by up to 100-fold when compared with
respective wild type values (33-37). When comparing the binding of
different ligands to the wild type CCK-BR and to the Leu325
Glu mutant, we observed only minor (
2-fold) affinity increases (Table I) despite the marked alterations in efficacy (Fig. 5). Although
it is difficult to draw any firm conclusions based on the extremely
small affinity changes at the CCK-BR, the observed shifts roughly
correlate with the efficacies of tested ligands as would be expected
based on previous studies with biogenic amine receptors.
Enhanced signaling potencies of agonists have been proposed as another general property of constitutively active (versus wild type) receptors (38). It has been demonstrated by detailed pharmacological characterization of a constitutively active muscarinic receptor that the degree of potency increase for individual ligands correlates with respective agonist efficacies (34). Consistent with the expectation of increased agonist potencies, our data reveal that (S)-L-740,093 is slightly more potent at the constitutively active CCK-BR (EC50 of inositol phosphate formation = 0.7 nM; see Fig. 4A) than at the wild type receptor (EC50 = 2.5 nM; see Fig. 3). However, we were unable to detect any potency shifts for the peptide ligands CCK-8 and gastrin (see Fig. 1B), despite the fact that both of these full agonists have considerably higher efficacy than the partial agonist, (S)-L-740,093. The latter findings suggest limitations of the extended ternary model of receptor activation in predicting how constitutive receptor activity affects agonist potency for a given ligand. Consistent with our observations, it has been previously noted for other constitutively active mutants that the potencies of peptide agonists show little or no changes versus corresponding values at the respective wild type receptor isoforms (22, 39-41). It is possible that the extended ternary complex model of receptor activation is most applicable to small ligands (e.g. (R)-L-740,093 and biogenic amines), whereas larger ligands (e.g. peptides) may interact with the receptor in a less predictable fashion.
The apparent lack of generalizable rules regarding potency shifts at constitutively active receptors can be best explained by the "cubic ternary complex" model of receptor activation (42). This recently proposed model refines earlier theories by considering additional receptor- and ligand-specific variables and multiple receptor states which may be involved in agonist-mediated receptor activation. The model acknowledges that several of these factors may be altered by receptor mutations which confer constitutive activity, and implies that these changes will not necessarily result in potency increases.
In addition to revealing both consistencies with and limitations of existing models of receptor activation, our observations extend current knowledge regarding the versatility of benzodiazepine-based molecules as potent and selective ligands for a wide range of different receptors (43). Based on the precedent provided, it appears likely that such molecules are preferred structures not only for the development of specific antagonists, but may also provide promising templates for novel receptor agonists. Consistent with this generalization, it has been recently reported that certain benzodiazepine derivatives can act as mixed CCK-A receptor agonists/CCK-BR antagonists (44). However, although structural similarities exist between these 1,5-benzodiazepine-derived CCK-A receptor agonists and 1,4-benzodiazepine-based CCK-BR ligands (tested in the present study), the two groups of compounds are clearly distinguished by the configuration of their respective benzodiazepine cores and by the different composition of attached substituents. As a common theme, the respective substituents appear to play a key role in determining the level of ligand intrinsic activity both at the CCK-A and the CCK-B/gastrin receptors.
At present, there are only a few other examples of non-peptide agonists
which can mimic the function of endogenous peptide hormones (45, 46).
Constitutively active receptors, as exemplified by the
Leu325 Glu mutant, hold promise as sensitive probes for
the systematic screening of non-peptide ligands for intrinsic activity
since these receptors lead to a systematic efficacy increase,
regardless of ligand structure (see above). As an important advantage
in the search for agonist potential, the proposed strategy may be applicable independent of the chemical structure of non-peptide ligands, and could therefore be utilized to re-assess a large variety
of already known non-peptide ligands which have been classified as
antagonists for different peptide hormone receptors. Once identified, lead agonist mimetics can then be stucturally modified as necessary, to
optimize receptor specificity, oral bioavailability, and
pharmacokinetic properties. This approach should accelerate the
identification and development of new drugs, applicable for the
treatment of a broad spectrum of diseases.
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ACKNOWLEDGEMENTS |
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We thank Wyeth-Lederle for providing benzodiazepine-derived ligands, and Parke-Davis for peptoid compounds. We also thank M. Bläker, A. Kane, I. J. Kopin, S. Lee, A. Leiter, and F. Schmitz for careful reading of the manuscript, and C. Chen, B. Desai (Microbiology Core of the GRASP Digestive Disease Center, P30-DK34928) for technical assistance.
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FOOTNOTES |
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* This work was supported by NIDDK, National Institutes of Health, Grant DK46767 and The Medical Foundation, Boston.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 617-636-5875;
Fax: 617-636-4207; E-mail: akopin{at}msn.com.
1 The abbreviations used are: CCK-BR, cholecystokinin-B/gastrin receptor; G-17, gastrin heptadecapeptide (unsulfated form); CCK-8, cholecystokinin octapeptide (sulfated form); CCK-8US, cholecystokinin octapeptide (unsulfated form).
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REFERENCES |
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