Anthranilic acid based CCK1 receptor antagonists: Blocking the receptor with the same ‘words’ of the endogenous ligand

The anthranilic acid diamides represent the more recent class of nonpeptide CCK₁ receptor antagonists. This class is characterized by the presence of anthranilic acid, used as a molecular scaffold, and two pharmacophores selected from the C-terminal tetrapeptide of CCK. The lead compound coded VL-0395, endowed with sub-micromolar affinity towards CCK₁ receptors, was characterized by the presence of Phe and 2-indole moiety at the C- and N-termini of anthranilic acid, respectively. Herein we describe the first step of the anthranilic acid C-terminal optimization using, instead of Phe, aminoacids belonging to the primary structure of CCK-8 and other not coded residues. Thus we demonstrate that the CCK₁ receptor affinity depends on the nature of the aminoacidic side chain as well as that the free carboxy group of the alpha-aminoacids is crucial for the binding. The R enantiomers of the most active compounds represent the eutomers of this class of antagonists confirming thus the stereo preference of the receptor. Moreover this SAR study demonstrates that the receptor binding pocket, that host the aminoacidic side chain, results much more tolerant respect to that accommodating the indole ring. As a result, an appropriate variation of the aminoacidic side chain could provide a better CCK₁ receptor affinity diorthosis.     

Use of the heterobifunctional cross-linker m-maleimidobenzoyl N-hydroxysuccinimide ester to affinity label cholecystokinin binding proteins on rat pancreatic plasma membranes

The binding of 125I-cholecystokinin-33 (125I-CCK-33) to its receptors on rat pancreatic membranes was decreased by modification of membrane protein sulfhydryl groups. Sulfhydryl modifying reagents also caused an accelerated release of bound 125I-CCK-33 from its receptor. Because of the presence of an essential sulfhydryl group(s) in CCK receptor binding we studied the application of the heterobifunctional (SH,NH2) cross-linker, m-maleimidobenzoyl N-hydroxysuccinimide ester (MBS), to affinity label 125I-CCK-33 binding proteins on rat pancreatic plasma membranes. Analysis of the cross-linked products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that this heterobifunctional cross-linker affinity labeled a major Mr = 80,000-95,000 protein previously identified as part of the CCK receptor on the basis of affinity labeling using homobifunctional and heterobifunctional photoreactive cross-linkers. Additional proteins of Mr greater than 200,000, and Mr = 130,000-140,000 were affinity labeled using MBS. The efficiency of the cross-linking reaction between 125I-CCK-33 and its membrane binding proteins with MBS was significantly greater than that obtained with NH2-directed homobifunctional reagents such as disuccinimidyl suberate. The efficiency of cross-linking could be dramatically improved by reduction of membrane proteins with low-molecular weight thiols prior to binding and cross-linking. The differential labeling patterns of the CCK binding proteins obtained with chemical cross-linkers of similar length but different chemical reactivity underscores the need for caution in predicting native receptor structure from affinity labeling data alone. Using the same pancreatic plasma membrane preparation and 125I-insulin, the Mr = 125,000 alpha-subunit of the insulin receptor was affinity labeled using MBS as cross-linker, demonstrating its utility in identifying other peptide hormone receptors.     

Is a nitrogen atom an important pharmacophoric element in sigma ligand binding?

A lingering question in sigma receptor ligand development is whether a nitrogen atom serves as an important pharmacophoric element in binding affinity. To address this question, we have synthesized several phenylalkylpiperidines and phenylalkylpiperazines and demonstrated that removal of the N atom from a typical phenylalkylpiperidine led to little or no binding to sigma receptors. In addition, where two N atoms occur in a compound, such as with phenylalkylpiperazines, the N atom on the longer alkyl chain appears to be more important. Thus, based on this study, the N atom is an important pharmacophoric element in the binding of phenylalkylpiperidines and phenylalkylpiperazines to sigma receptors.     

Synthesis and biological evaluation of cyclic and branched peptide analogues as ligands for cholecystokinin type 1 receptor

A library of cyclic CCK8 analogues, containing unnatural amino acids in the peptide sequence, is prepared using solid-phase synthesis. The structure of these cyclic peptides is based on a previously synthesised compound, cyclo-CCK8, selective for CCK(1) receptor. Structure-activity investigations are performed by evaluating the binding properties of the new analogues. In particular, the binding ability of the cyclic CCK8 analogues is tested by nuclear medicine studies on cell line transfected with CCK(1) receptor. Compounds named cyclo-A4-cyclo-A7 show binding constant in the range 6.0-8.0 microM, with an improved affinity over the previous described cyclo-CCK8, but almost comparable IC(50) values among new analogues towards CCK(1) were obtained.     

Characterization of cholecystokinin receptors on guinea pig gastric chief cell membranes

The binding of cholecystokinin (CCK) to its receptors on guinea pig gastric chief cell membranes were characterized by the use of 125I-CCK-octapeptide (CCK8). At 30 degrees C optimal binding was obtained at acidic pH in the presence of Mg2+, while Na+ reduced the binding. In contrast to reports on pancreatic and brain CCK receptors, scatchard analysis of CCK binding to chief cell membranes revealed two classes of binding sites. Whereas, in the presence of a non-hydrolyzable GTP analog, GTP gamma S, only a low affinity site of CCK binding was observed. Chief cell receptors recognized CCK analogs, with an order of potency of: CCK8 greater than gastrin-I greater than CCK4. Although all CCK receptor antagonists tested (dibutyryl cyclic GMP, L-364718 and CR1409) inhibited labeled CCK binding to chief cell membranes, the relative potencies of these antagonists in terms of inhibiting labeled CCK binding were different from those observed in either pancreatic membranes or brain membranes. The results indicate, therefore, that on gastric chief cell membranes there exist specific CCK receptors, which are coupled to G protein. Furthermore, chief cell CCK receptors may be distinct from pancreatic or brain type CCK receptors.     

Binding of a phenethyl ester analogue of cholecystokinin to the solubilized pancreatic cholecystokinin receptor: use in ligand-affinity chromatography.

Pancreatic acinar cells have both high and low affinity receptors for cholecystokinin (CCK), yet their membranes appear to possess only a single class of binding sites. Recently, gallbladder membrane CCK receptors were shown to undergo inter-conversion between two affinity states dependent on G protein coupling. Keys for that observation were the differential binding affinities of CCK and a phenethyl ester analogue of CCK (OPE), with the high affinity state binding CCK with higher affinity than OPE, and the low affinity state binding OPE with higher affinity than CCK. Here, we performed analogous experiments using these ligands and both pancreatic membranes and a solubilized preparation. Both preparations were found to have only single affinity states of this receptor. However, the state on membranes had a higher affinity for CCK than for OPE, and that on the solubilized preparation had a higher affinity for OPE than for CCK. This supports the hypothesis that the ternary complex of ligand-receptor-G protein found in membranes represents the high affinity state of this receptor, while the uncoupled form of this receptor after solubilization represents its low affinity state. The high affinity of OPE for the solubilized receptor can be utilized in a purification strategy to follow receptor-bearing fractions and to provide an efficient and specific affinity-binding step.     

Design and synthesis of tricyclic tetrahydroquinolines as a new series of nonsteroidal selective androgen receptor modulators (SARMs)

Some tricyclic tetrahydroquinolines (THQs) were found to have the potential of a new series of nonsteroidal selective androgen receptor modulators (SARMs). Compound 5b was first designed and synthesized under our hypothesis based on a four-point pharmacophoric requirement of the 3-carbonyl, 18-methyl, 17-hydroxyl, and 13-quaternary carbon groups of dihydrotestosterone (DHT). It was revealed that this compound exhibits not only a strong androgen receptor (AR) agonistic activity (EC₅₀ = 9.2 nM) but also the highest selectivity in binding affinity to AR among the steroid hormone receptors. Furthermore, this compound showed a weak virilizing effect with retention of the desired anabolic effect as compared with DHT in vivo.     

Conformationally constrained analogs of BAY 59-3074 as novel cannabinoid receptor ligands

To obtain information on the pharmacophoric requirements of the CB1/CB2 partial agonist BAY 59-3074 we have synthesized a series of new conformationally constrained dibenzofuran (4a-d) and dibenzopyran analogs (5). All constrained analogs exhibited reduced binding affinity at both cannabinoid receptor subtypes, suggesting that planar conformations of these ligands are less favored by both receptors. We also found that 4c, 4d, and 5 exhibited 3- to 12-fold selectivity for hCB2 over rCB1 receptors and may serve as new chemotypes for the development of CB2-selective cannabinergics.     

Synthesis and biological characterisation of [3H]BBL454, a new CCK2 selective radiolabelled agonist displaying original pharmacological properties

[(3)H]BBL454, a new CCK(2) selective tritiated agonist was prepared via the reductive tritiation of a 5-aminopentyn-1-yl moiety introduced on the N-terminal end of a pentapeptide derivative of cholecystokinin. The binding properties of this labelled compound were determined on CHO cells transfected with the rat CCK(2) receptor. [(3)H]BBL454 is able to discriminate two affinity states of the CCK(2) receptor a supplementary indication of its validity for further exploring the heterogeneity of this receptor.     

Solubilization and characterization of CCK receptors from mouse pancreas

To study the characteristics of the CCK receptor, plasma membranes were prepared from mouse pancreatic acini, and CCK receptors solubilized with 1% digitonin. To measure hormone binding, the solubilized receptors were incubated with 125I-CCK at 4 degrees C and the hormone-receptor complex was precipitated with 10% polyethylene glycol. Specific 125I-CCK binding by the solubilized CCK receptor was compared to that by the plasma membrane-bound CCK receptor. Both the solubilized and the membrane-bound receptor displayed optimal binding at an acidic pH (between 6.0 and 7.0) and showed a similar sensitivity to monovalent and divalent cations. The solubilized receptors preserved their relative specificity for CCK molecules: CCK-8 greater than CCK-33 greater than desulfated CCK-8 greater than CCK-4. However, the soluble CCK receptor had a lower binding affinity than plasma membrane-bound receptor. Solubilized receptors preserved their relative specificity for inhibitors of CCK binding and action: dibutyryl cyclic GMP greater than N-CBZ-tryptophan greater than proglumide. Solubilized receptors had affinities for these antagonists that were similar to receptors on intact plasma membranes. These data indicate, therefore, that the specific binding properties of the CCK receptor are inherent to the solubilized glycoprotein molecules.     

Purification of the pancreatic cholecystokinin receptor

We have previously shown that the pancreatic cholecystokinin (CCK) receptor can be solubilized in 1% digitonin. In this study, digitonin-solubilized CCK receptors from rat pancreas were purified using sequential affinity chromatography on ricin-II agarose and on AffiGel-CCK. Electrophoresis of the radioiodinated purified receptors on SDS-polyacrylamide gels followed by autoradiography revealed two proteins: a major band of Mr = 80,000-90,000, and a minor band of Mr = 55,000. Through the purification procedure, the receptors preserved their agonist specificity (CCK-8 less than CCK-33 less than desulfated CCK-8 less than CCK-4) and binding affinity. Scatchard transformations of binding data for the purified receptor preparation were best fit by linear plots compatible with a single class of binding sites with Kd = 9.4 nM. The estimated purification was about 80,000 fold and consistent with the expected Bmax for a pure Mr = 80,000 protein binding one CCK molecule. This two-step purification procedure opens the possibility for molecular studies of the CCK receptor.     

Analysis of structure-activity relationships with the N-(3-acyloxy-2-benzylpropyl)-N'-[4-(methylsulfonylamino)benzyl]thiourea template for vanilloid receptor 1 antagonism

In a continuing effort to elucidate the structure-activity relationships of the lead antagonist N-[2-(3,4-dimethylbenzyl)-3-pivaloyloxypropyl]-N'-[4-(methylsulfonylamino)benzyl]thiourea (1), the distances between the proposed four pharmacophores in 1 have been varied by insertion or deletion of one carbon to optimize their fit to the receptor. In addition, the acyloxy group of the C region was replaced with amide and N-hydroxy amide to identify the pharmacophoric importance of the ester group in the C2 region. The results indicated that the pharmacophoric arrangement of 1 was optimal for receptor binding affinity and antagonism, and the ester of the C2 region was significant for receptor binding. Among the derivatives, compound 19 showed distinct behavior with a 2-fold improvement in antagonism but a 13-fold reduction in binding affinity compared to 1. The partial separation of pharmacophoric requirements of these two assays has been noted before and compound 19 is thus selective for the calcium entry-linked receptor population. The conformational analysis of 1 generated three distinct conformers having different types of hydrophobic interactions, which will be utilized for exploring the active conformation of the VR1 ligand.     

The somatostatin receptor on isolated pancreatic acinar cell plasma membranes. Identification of subunit structure and direct regulation by cholecystokinin

Somatostatin binding to its receptors on rat pancreatic acinar membranes was characterized with [125I-Tyr1]somatostatin. Binding at 24 degrees C was rapid reaching a maximum after 60 min and was reversible upon the addition of 1 microM unlabeled ligand. Scatchard analysis revealed a single class of binding sites, with a Kd of 0.32 +/- 0.03 nM and a binding capacity of 600 +/- 54 fmol/mg of protein. Specificity for the somatostatin was demonstrated with the inhibition of labeled hormone binding by somatostatin analogs in proportion to their biological activities. When [125I-Tyr1]somatostatin was cross-linked to its receptors with the photoreactive cross-linker n-hydroxysuccinimidyl-4-azidobenzoate, the hormone was associated with Mr = 90,000 protein. Similar mobilities of the radioactive band were observed in the presence and absence of dithiothreitol. In contrast to other unrelated peptides, cholecystokinin (CCK) and its analogs directly reduced [125I-Tyr1] somatostatin binding to isolated membranes. The effect of CCK was one-half-maximal at 3 nM and maximal at 100 nM. In the presence of 3 nM CCK8, the binding capacity for somatostatin was decreased to 237 +/- 39 fmol/mg of protein without a significant change in affinity. Dibutyryl cyclic GMP, a CCK receptor antagonist, blocked this action of CCK8 indicating that the CCK receptor mediated the decrease in [125-Tyr1]somatostatin binding. In contrast cerebral cortex membranes, which also possess a somatostatin receptor, were not regulated by CCK. These results indicate, therefore, that 1) purified pancreatic acinar plasma membranes contain specific receptors for somatostatin, 2) the receptor has an apparent Mr of about 90,000, and 3) the binding of somatostatin to its receptor on pancreatic plasma membranes is regulated by CCK analogs acting via the CCK receptor.     

Importance of sulfation of gastrin or cholecystokinin (CCK) on affinity for gastrin and CCK receptors

We investigated the importance of sulfation of gastrin or cholecystokinin (CCK) on influencing their affinity for gastrin or CCK receptors by comparing the abilities of sulfated gastrin-17 (gastrin-17-II), desulfated gastrin-17 (gastrin-17-I), CCK-8 and desulfated CCK-8 [des(SO3)CCK-8] to interact with CCK or gastrin receptors on guinea pig pancreatic acini. For inhibiting binding of 125I-gastrin to gastrin receptors, gastrin-17-II (Kd 0.08 nM) greater than CCK-8 (Kd 0.4 nM) greater than gastrin-17-I (Kd 1.5 nM) greater than des(SO3)CCK-8 (Kd 28 nM). For inhibiting binding of 125I-Bolton Hunter-labeled CCK-8 to CCK receptors the relative potencies were: CCK-8 much greater than des(SO3)CCK-8 = gastrin-17-II greater than gastrin-17-I. Each peptide interacted with both high and low affinity CCK binding sites. The relative abilities of each peptide to interact with high affinity CCK receptors showed a close correlation with their abilities to cause half-maximal stimulation of enzyme secretion. These results demonstrate that, in contrast to older studies, sulfation of both CCK and gastrin increase their affinities for both gastrin and CCK receptors. Moreover, the gastrin receptor is relatively insensitive to the position of the sulfate moiety, whereas the CCK receptor is extremely sensitive to both the presence and exact position of the sulfate moiety.     

Refinement of the structure of the ligand-occupied cholecystokinin receptor using a photolabile amino-terminal probe

Affinity labeling is a powerful tool to establish spatial approximations between photolabile residues within a ligand and its receptor. Here, we have utilized a cholecystokinin (CCK) analogue with a photolabile benzoylphenylalanine (Bpa) sited in position 24, adjacent to the pharmacophoric domain of this hormone (positions 27-33). This probe was a fully efficacious agonist that bound to the CCK receptor saturably and with high affinity (K(i) = 8.9 +/- 1.1 nm). It covalently labeled the CCK receptor either within the amino terminus (between Asn(10) and Lys(37)) or within the third extracellular loop (Glu(345)), as demonstrated by proteolytic peptide mapping, deglycosylation, micropurification, and Edman degradation sequencing. Truncation of the receptor to eliminate residues 1-30 had no detrimental effect on CCK binding, stimulated signaling, or affinity labeling through a residue within the pharmacophore (Bpa(29)) but resulted in elimination of the covalent attachment of the Bpa(24) probe to the receptor. Thus, the distal amino terminus of the CCK receptor resides above the docked ligand, compressing the portion of the peptide extending beyond its pharmacophore toward the receptor core. Exposure of wild type and truncated receptor constructs to extracellular trypsin damaged the truncated construct but not the wild type receptor, suggesting that this domain also may play a protective role. Use of these additional insights into molecular approximations provided key constraints for molecular modeling of the peptide-receptor complex, supporting the counterclockwise organization of the transmembrane helical domains.     

Effects of cholecystokinin-58 on type 1 cholecystokinin receptor function and regulation

Cholecystokinin, like many peptide hormones, is present as multiple molecular forms. CCK-58 has been identified as the dominant form in the circulation, whereas most of the studies of CCK-receptor interactions have been performed with CCK-8. Despite both sharing the pharmacophoric region of CCK, representing its carboxy terminal heptapeptide amide, studies in vivo have demonstrated biological diversity of action of the two peptides, with CCK-58, but not CCK-8, stimulating pancreatic fluid secretion and lengthening the interval between meals. Here, we have directly studied the ability of these two CCK peptides to bind to the type 1 CCK receptor and to stimulate it to elicit an intracellular calcium response. The calcium response relative to receptor occupation was identical for CCK-58 and CCK-8, with the longer peptide binding with approximately fivefold lower affinity. We also examined the ability of the two peptides to elicit receptor internalization using morphological techniques and to disrupt the constitutive oligomerization of the CCK receptor using receptor bioluminescence resonance energy transfer. Here, both full agonist peptides had similar effects on these regulatory processes. These data suggest that both molecular forms of CCK act at the CCK1 receptor quite similarly and elicit similar regulatory processes for that receptor, suggesting that the differences in biological activity observed in vivo most likely reflect differences in the clearance and/or metabolism of these long and short forms of CCK peptides.     

Probing receptor binding activity of interleukin-8 dimer using a disulfide trap

Interleukin-8 (IL-8), a member of the chemokine superfamily, exists as both monomers and dimers, and mediates its function by binding to neutrophil CXCR1 and CXCR2 receptors that belong to the G protein-coupled receptor class. It is now well established that the monomer functions as a high-affinity ligand, but the binding affinity of the dimer remains controversial. The approximately 1000-fold difference between monomer-dimer equilibrium constant (microM) and receptor binding constant (nM) of IL-8 does not allow receptor-binding affinity measurements of the native IL-8 dimer. In this study, we overcame this roadblock by creating a "trapped" nondissociating dimer that contains a disulfide bond across the dimer interface at the 2-fold symmetry point. The NMR studies show that the structure of this trapped dimer is indistinguishable from the native dimer. The trapped dimer, compared to a trapped monomer, bound CXCR1 with approximately 70-fold and CXCR2 with approximately 20-fold lower affinities. Receptor binding involves two interactions, between the IL-8 N-loop and receptor N-domain residues, and between IL-8 N-terminal and receptor extracellular loop residues. In contrast to a trapped monomer that bound an isolated CXCR1 N-domain peptide with microM affinity, the trapped dimer failed to show any binding, indicating that dimerization predominantly perturbs the binding of only the N-loop residues. These results demonstrate that only the monomer is a high-affinity ligand for both receptors, and also provide a structural basis for the lower binding affinity of the dimer.     

Insights into the binding and activation sites of the receptors for cholecystokinin and gastrin

CCK receptors represent potential targets in a number of diseases. Knowledge of CCK receptor binding sites is a prerequisite for the understanding of the molecular basis for their ligand recognition, partial agonism, ligand-induced trafficking of signalling. In the current paper, we report studies from our laboratory and others which have provided new data on the molecular basis of the pharmacology and functioning of CCK1 and CCK2 receptors. It has been shown that: 1) homologous regions of the two receptors are involved in the binding site of CCK, however, positioning of CCK slightly differs in agreement with distinct pharmacophores of CCK toward the two receptors and receptor sequence variations; 2) Binding sites of most of non-peptide agonists/ antagonist are buried in the pocket formed by transmembrane helices and overlap that of CCK; Aromatic amino acids within and near the binding site, especially in helix VI, are involved in receptor activation; 4) Like for other members of family A of G-protein coupled receptors, residues of the binding sites as well as of conserved motifs such as E/DRY, NPXXY are crucial for receptor activation.     

Receptors for cholecystokinin and gastrin peptides display specific binding properties and are structurally different in guinea-pig and dog pancreas

In the light of the strong potency of gastrin-related peptides on pancreatic exocrine secretion in dog, we analyzed the binding properties of peptides related to cholecystokinin (CCK) and gastrin on dog pancreatic acini compared to guinea-pig acini. Moreover, we determined apparent molecular masses of photoaffinity labelled CCK/gastrin receptors in the two models. Using the CCK radioligand, receptor selectivity towards CCK/gastrin agonists and antagonists was found to be lower in dog acini than in guinea-pig acini. Performing the binding with CCK and gastrin radioligands in combination with N2,O2'-dibutyryl-guanosine 3',5'-monophosphate, revealed that in dog acini there exist two different sub-classes of CCK/gastrin receptors having high and low selectivity, the latter ones being able to bind gastrin with high affinity (Kd = 2.1 nM). SDS-PAGE analysis of covalently cross-linked receptors using several photosensitive CCK and gastrin probes of different peptide chain lengths demonstrated that in guinea-pig, CCK peptides bound to a 84-kDa component whereas in dog pancreas, CCK and gastrin peptides bound to three distinct molecular species (Mr approximately equal to 78,000, 45,000, 28,000). Performing cross-linking in the presence of 1 microM CCK indicated that a 45-kDa protein is the putative CCK/gastrin receptor in dog pancreas. Our results support the concept of heterogeneity of CCK/gastrin receptors.     

Haloperidol: towards further understanding of the structural contributions of its pharmacophoric elements at D2-like receptors

An attempt to understand the pharmacophore-relevant position of the alcoholic moiety in haloperidol and the contributions of other pharmacophoric elements led to the re-synthesis of its tropane analogue (compound 2). An analysis of the binding data suggests that haloperidol binds to the DA receptors with the OH group in the axial position and the OH group, while not essential for binding, enhances binding especially at the D2 receptor. It also became clear that shortening the butyrophenone chain not only reduces binding affinity at the DA receptors but eliminates subtype selectivity.