SAR studies of 1,5-diarylpyrazole-based CCK1 receptor antagonists

A high throughput screening campaign revealed compound 1 as a potent antagonist of the human CCK(1) receptor. Here, we report the syntheses and SAR studies of 1,5-diarylpyrazole analogs with various structural modifications of the alkane side chain of the molecule. The difference in affinity between the two enantiomers for the CCK(1) receptor and the flexible nature of the linker led to the design of constrained analogs with increased potency.     

2-Substituted piperazine-derived imidazole carboxamides as potent and selective CCK1R agonists for the treatment of obesity

The discovery and structure–activity relationship of 1,2-diarylimidazole piperazine carboxamides bearing polar side chains as potent and selective cholecystokinin 1 receptor (CCK1R) agonists are described. Optimization of this series resulted in the discovery of isopropyl carboxamide 40, a CCK1R agonist with sub-nanomolar functional and binding activity as well as excellent potency in a mouse overnight food intake reduction assay.     

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.     

Anthranilic sulfonamide CCK1/CCK2 dual receptor antagonists II: Tuning of receptor selectivity and in vivo efficacy

In the previous article we demonstrated how certain CCK2R-selective anthranilic amides could be structurally modified to afford high-affinity, selective CCK1R activity. We now describe our efforts at modulating and optimizing the CCK1R and CCK2R affinities aimed at producing compounds with good pharmacokinetics properties and in vivo efficacy in rat models of gastric acid and pancreatic amylase secretion.     

CCK1 and CCK2 Receptors Are Expressed on Pancreatic Stellate Cells and Induce Collagen Production

The gastrointestinal hormone cholecystokinin (CCK) can induce acute pancreatitis in rodents through its action on acinar cells. Treatment with CCK, in combination with other agents, represents the most commonly used model to induce experimental chronic pancreatitis. Pancreatic stellate cells (PSC) are responsible for pancreatic fibrosis and therefore play a predominant role in the genesis of chronic pancreatitis. However, it is not known whether PSC express CCK receptors. Using real time PCR techniques, we demonstrate that CCK1 and CCK2 receptors are expressed on rat PSC. Interestingly both CCK and gastrin significantly induced type I collagen synthesis. Moreover, both inhibit proliferation. These effects are comparable with TGF-β-stimulated PSC. Furthermore, the natural agonists CCK and gastrin induce activation of pro-fibrogenic pathways Akt, ERK, and Src. Using specific CCK1 and CCK2 receptor (CCK2R) inhibitors, we found that Akt activation is mainly mediated by CCK2R. Akt activation by CCK and gastrin could be inhibited by the PI3K inhibitor wortmannin. Activation of ERK and the downstream target Elk-1 could be inhibited by the MEK inhibitor U0126. These data suggest that CCK and gastrin have direct activating effects on PSC, are able to induce collagen synthesis in these cells, and therefore appear to be important regulators of pancreatic fibrogenesis. Furthermore, similar to TGF-β, both CCK and gastrin inhibit proliferation in PSC.     

Discovery of pyrimidine carboxamides as potent and selective CCK1 receptor agonists

A series of six-membered heterocycle carboxamides were synthesized and evaluated as cholecystokinin 1 receptor (CCK1R) agonists. A pyrimidine core proved to be the best heterocycle, and SAR studies resulted in the discovery of analog 5, a potent and structurally diverse CCK1R agonist.     

Over-expression of CCK1 Receptor Reverse Morphine Dependence

Studies demonstrated that cholecystokinin (CCK) system involved in morphine dependence and withdrawal. Our previous study showed that endogenous CCK system were up-regulated after chronic morphine exposure. Additionally, CCK1 receptor significantly blocked the inhibitory effect of exogenous CCK-8 on morphine dependence, but CCK2 receptor appears to be necessary for low concentrations of endogenous CCK to potentiate morphine dependence. Therefore, CCK1R and CCK2R function differently in chronic morphine dependence, but the mechanism is still unclear. In this study, HEK-293 cells co-transfected with µ-opioid receptors (HEK293-hMOR) and CCK1R or CCK2R were established. Cells were treated with 10 µM morphine for 6, 12, 16, 24 h and 100 µM naloxone precipitation for 15 min. cAMP overshoot was appeared at 12 h and was increased time dependently after morphine exposure in HEK293-hMOR cells. The cAMP overshoot did not appear in CCK1R-overexpressing HEK293-hMOR cells, while still appeared in CCK2R-overexpressing HEK293-hMOR cells. Over-expression of CCK1R reversed CREB and ERK1/2 activation in HEK293-hMOR cells exposed to morphine. Our study identifies over-expression of CCK1R significantly blocked morphine dependence, which was related with phosphorylation of CREB, and ERK1/2 signaling activation. While over-expression of CCK2R promoted morphine dependence, which was related with phosphorylation of CREB but not ERK1/2 signaling activation.     

Anthranilic acid derivatives: a new class of non-peptide CCK1 receptor antagonists

Having successfully obtained new CCK(1) ligands holding appropriate groups on the anthranilic acid dimer used as molecular scaffold we were interested in increasing their micromolar affinity for the CCK(1) receptors by modifying the spatial relationship of the main pharmacophoric groups. Since, we have proposed simplified analogues reducing the anthranilic acid dimer to a monomer. In this stage of our research program we have prepared and tested on CCK receptors a series of N-substituted anthranilic acid derivatives keeping a Phe residue at the C-terminal site. The indole-2-carbonyl group imparts the best CCK(1) receptor binding affinity (compound 1: IC(50)=197.5 nM) while a sharp decrease in binding affinity is observed for the other indole containing derivatives. Moreover, in order to support the different binding behaviour observed for the synthesized compounds, a conformational investigation was carried out. Finally, on the basis of the main pharmacophoric groups of the obtained new lead compound (1) (coded VL-0395) a receptor binding hypothesis has been provided.     

In vivo and in vitro characterization of CCK8 bearing a histidine-based chelator labeled with 99mTc-tricarbonyl

The development of receptor targeting radiolabeled ligands has gained much interest in recent years for diagnostic and therapeutic applications in nuclear medicine. Cholecystokinin (CCK) receptors have been shown to be overexpressed in a subset of neuroendocrine and other tumors. We are evaluating binding and biodistribution properties of a CCK8 peptide derivative labeled with (99m)Tc(I)-tricarbonyl. The CCK8 peptide was modified at its N-terminus by adding to its N-terminus two lysine-histidine modules (KH), where histidine is coupled to the side chain of the lysine ((KH)(2)-CCK8). (99m)Tc(I)-tricarbonyl was generated with the IsoLinktrade mark kit. A431 cells stably transfected with a cDNA encoding for the human CCK2 receptor were utilized to determine binding affinity, internalization, and retention of the labeled peptide, in comparison with wild-type A431 cells. A nude mouse tumor model was obtained by generating A431-CCK2R and A431-control tumors in opposite flanks of the animals. High specific activity labeling with (99m)Tc was achieved. In A431-CCK2R cells, specific saturable binding was observed as well as evident internalization of the radiolabeled peptide after binding. Biodistribution experiments showed rapid, specific localization of (KH)(2)-CCK8 on A431-CCK2R xenografts compared with control tumors, although absolute uptake values were not markedly higher compared with background activity. Clearance of unbound radioactivity was both urinary and hepatobiliary. In imaging experiments, while targeting to CCK2R positive tumors could be appreciated, there was poor contrast between target and nontarget areas. (KH)(2)-CCK8 shows adequate in vitro and in vivo properties for CCK2R targeting although improvement of biodistribution warrant further development.     

Development of a CCK1R-membrane nanoparticle as a fish-out tool for bioactive peptides

The cholecystokinin receptor type 1 (CCK1R) is a G protein-coupled receptor (GPCR) that is involved in several biological processes including the regulation of the secretion of digestive enzymes. The peptide hormone cholecystokinin (CCK) binds to CCK1R, which is an important pharmacological target for several diseases, including obesity. Interestingly, nutritional dietary peptides also appear to activate CCK1R, and may play a role in CCK1R signaling in the gut. In this study, a novel technique to screen for CCK1R ligands based on affinity-selection is described. Functional expressed CCK1R is reconstituted into membrane nanoparticles called NABBs (nanoscale apo-lipoprotein bound bilayers). NABBs are native-like bilayer membrane systems for incorporation of GPCRs. CCK1R-NABBs were characterized using a fluorescently labeled CCK analog and can be used as a cutting-edge technology to screen for CCK1R ligands using affinity-selection mass spectrometry.     

Enterostatin reduces serum cholesterol levels by way of a CCK(1) receptor-dependent mechanism

Enterostatin (APGPR), an anorectic pentapeptide derived from the amino terminus of procolipase, significantly reduced serum cholesterol levels after oral administration at a dose of 100 mg/kg for 3 days in mice fed a high-cholesterol-cholic acid diet. The hypocholesterolemic effect of APGPR was inhibited by pretreatment with lorglumide, an antagonist for cholecystokinin 1 (CCK(1)) receptor, even though APGPR does not have any affinity for CCK(1) receptors. Similarly, the hypocholesterolemic activity of VPDPR, an APGPR analogue, was blocked by lorglumide. These results suggest that the hypocholesterolemic effects of APGPR and VPDPR are mediated by a CCK(1) receptor-dependent mechanism.     

Targeted disruption of the murine CCK1 receptor gene reduces intestinal lipid-induced feedback inhibition of gastric function

Cholecystokinin (CCK), acting at CCK1 receptors (CCK1Rs) on intestinal vagal afferent terminals, has been implicated in the control of gastrointestinal function and food intake. Using CCK1R(-/-) mice, we tested the hypothesis that lipid-induced activation of the vagal afferent pathway and intestinal feedback of gastric function is CCK1R dependent. In anesthetized CCK1R(+/+) ("wild type") mice, meal-stimulated gastric acid secretion was inhibited by intestinal lipid infusion; this was abolished in CCK1R(-/-) mice. Gastric emptying of whole egg, measured by nuclear scintigraphy in awake mice, was significantly faster in CCK1R(-/-) than CCK1R(+/+) mice. Gastric emptying of chow was significantly slowed in response to administration of CCK-8 (22 pmol) in CCK1R(+/+) but not CCK1R(-/-) mice. Activation of the vagal afferent pathway was measured by immunohistochemical localization of Fos protein in the nucleus of the solitary tract (NTS; a region where vagal afferents terminate). CCK-8 (22 pmol ip) increased neuronal Fos expression in the NTS of fasted CCK1R(+/+) mice; CCK-induced Fos expression was reduced by 97% in CCK1R(-/-) compared with CCK1R(+/+) mice. Intralipid (0.2 ml of 20% Intralipid and 0.04 g lipid), but not saline, gavage increased Fos expression in the NTS of fasted CCK1R(+/+) mice; lipid-induced Fos expression was decreased by 47% in CCK1R(-/-) compared with CCK1R(+/+)mice. We conclude that intestinal lipid activates the vagal afferent pathway, decreases gastric acid secretion, and delays gastric emptying via a CCK1R-dependent mechanism. Thus, despite a relatively normal phenotype, intestinal feedback in response to lipid is severely impaired in these mice.     

Discovery of new piperidine amide triazolobenzodiazepinones as intestinal-selective CCK1 receptor agonists

New cholecystokinin-1 receptor (CCK1R) agonist 'triggers' were identified using iterative library synthesis. Structural activity relationship studies led to the discovery of compound 10e, a potent CCK1R agonist that demonstrated robust weight loss in a diet-induced obese rat model with very low systemic exposure. Pharmacokinetic data suggest that efficacy is primarily driven through activation of CCK1R's located within the intestinal wall.     

Further evidence that the CCK2 receptor is coupled to two transduction pathways using site-directed mutagenesis

A heterogeneity of CCK2 receptors has been reported which could correspond to different states of coupling to G proteins and/or association with different second messenger systems. To investigate these hypotheses, the wild-type CCK2 receptor and three mutants F347A, D100N and K333M/K334T/R335L, expected to modify the coupling of the G protein with the third intracellular loop of the receptor, were transfected into Cos-7 cells and their binding and signalling properties were evaluated using the natural ligand CCK8. Activation of wild-type as well as F347A, D100N or K333M/K334T/R335L CCK2 receptors by this ligand led to a similar arachidonic acid release which was blocked by pertussis toxin and the phospholipase A2 inhibitor, mepacrine. Nevertheless, in contrast to the wild-type CCK2 receptor, addition of CCK8 to cells transfected with the F347A or K333M/K334T/R335L mutants did not result in the production of inositol phosphates while the maximum increase in this second messenger formation was reduced by 30% with the D100N mutant. Taken together, these results suggest that the CCK2 receptor is coupled to two G proteins and that Phe347 and the cluster of basic residues K333/K334/R335 probably play a key role in Gq protein stimulation leading to inositol phosphate production but not in activation of the G protein coupled to phospholipase A2. These data bring additional support at the molecular level to the existence of different affinity states of CCK2 receptors suggested from the results of binding assays and behavioural studies.     

Different affinity states of CCK(1) receptors on pancreatic acini and gastric smooth muscle in the rat.

It has recently been shown that--after chronic cholecystokinin (CCK) treatment--an adaptation of pancreatic secretory but not gastric motor function does occur. Recent studies indicate that the CCK(1)-receptor exists in two (i.e. high and low) affinity states, which could be distinguished by the CCK-analogue JMV-180. CCK occupancy of high and low affinity sites is thought to be related to the initiation of different intracellular events and consequent biological responses. Affinity states of CCK(1)-receptors on pancreas and gastrointestinal (GI) smooth muscle could be different and this can offer an explanation for the different effects of CCK on pancreatic and gastric growth. We therefore studied the affinity states of CCK(1)-receptors on isolated rat pancreatic acini and gastric smooth muscle preparations. When acini were incubated with increasing concentrations of CCK-8, a biphasic (i.e. stimulation followed by inhibition) effect on amylase release was observed. JMV-180 caused only stimulation of enzyme release and combined JMV-180 and CCK stimulation (at submaximal doses) resulted in an additive secretory response. CCK-8 induced contractions of pyloric, antral and fundic muscle in a concentration-dependent manner. The response was monophasic, reaching a plateau. JMV-180 had only a very weak effect on these preparations. On the contrary, it inhibited CCK-induced contractions in a competitive manner, the concentration-response curve to CCK being shifted to the right by the CCK analogue. Our data suggest that the affinity states of CCK(1)-receptors on rat pancreatic and gastric tissue are different. On pancreatic acini CCK(1)-receptors exist in both high- and low-affinity states whose occupation is followed by the sequence of intracellular events leading to growth. In contrast, occupation of low affinity receptors (the only ones present in the GI smooth muscle) does not lead to cell proliferation. This difference therefore explains the different adaptive response of the pancreas and the stomach to chronic CCK administration. Furthermore, different affinity states of CCK(1)-receptors may mediate different functions of the digestive tract.     

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.     

Conformational behavior of cyclic CCK-related peptides determined by 400-MHz 1H-NMR: relationships with affinity and selectivity for brain receptors

The conformational study of a homogenous series of cyclic analogues of CCK8, selective for central receptors, such as Boc-X-Tyr(SO3H)-Nle-D-Lys-Trp-Nle-Asp-Phe-NH2, where X = L-Glu, D-Glu, or gamma-D-Glu, was performed by 400-MHz 1H-nmr. The regular increase in affinity for central receptors when going from [L-Glu] to [gamma-D-Glu] is correlated to (a) an enhancement in internal flexibility of the cyclic moiety, (b) an external orientation of the tyrosine side chain, and (c) a restructuring of the C-terminal part of the peptide. All these results could permit a modeling of biologically active conformation of CCK8 for both receptors types to be performed.     

Somatic mutations in CCK2R alter receptor activity that promote oncogenic phenotypes

The roles of cholecystokinin 2 receptor (CCK2R) in numerous physiologic processes in the gastrointestinal tract and central nervous system are well documented. There has been some evidence that CCK2R alterations play a role in cancers, but the functional significance of these alterations for tumorigenesis is unknown. We have identified six mutations in CCK2R among a panel of 140 colorectal cancers and 44 gastric cancers. We show that these mutations increase receptor activity, activate multiple downstream signaling pathways, increase cell migration, and promote angiogenesis. Our findings suggest that somatic mutations in CCK2R may promote tumorigenesis through deregulated receptor activity and highlight the importance of evaluating CCK2R inhibitors to block both the normal and mutant forms of the receptor.     

The biologically crucial C terminus of cholecystokinin and the non-peptide agonist SR-146,131 share a common binding site in the human CCK1 receptor. Evidence for a crucial role of Met-121 in the activation process.

The cholecystokinin (CCK) receptor-1 (CCK1R) is a G protein-coupled receptor, which mediates important central and peripheral cholecystokinin actions. Our aim was to progress in mapping of the CCK1R binding site by identifying residues that interact with the methionine and phenylalanine residues of the C-terminal moiety of CCK because these are crucial for its binding and biological activity, and to determine whether CCK and the selective non-peptide agonist, SR-146,131, share a common binding site. Identification of putative amino acids of the CCK1R binding site was achieved by dynamics-based docking of the ligand CCK in a refined three-dimensional model of the CCK1R using, as constraints, previous results that identified contact points between residues of CCK and CCK1R (Kennedy, K., Gigoux, V., Escrieut, C., Maigret, B., Martinez, J., Moroder, L., Frehel, D., Gully, D., Vaysse, N., and Fourmy, D. (1997) J. Biol. Chem. 272, 2920-2926 and Gigoux, V., Escrieut, C., Fehrentz, J. A., Poirot, S., Maigret, B., Moroder, L., Gully, D., Martinez, J., Vaysse, N., and Fourmy, D. (1999) J. Biol. Chem. 274, 20457-20464). By this approach, a series of residues forming connected hydrophobic clusters were identified. Pharmacological and functional analysis of mutated receptors indicated that a network of hydrophobic residues including Cys-94, Met-121, Val-125, Phe-218, Ile-329, Phe-330, Trp-326, Ile-352, Leu-356, and Tyr-360, is involved in the binding site for CCK and in the activation process of the CCK1R. Within this hydrophobic network, the physico-chemical nature of residue 121 seems to be essential for CCK1R functioning. Finally, the biological properties of mutants together with dynamic docking of SR-146,131 in the CCK1R binding site demonstrated that SR-146,131 occupies a region of CCK1R binding site which interacts with the C-terminal amidated tripeptide of CCK, i.e. Met-Asp-Phe-NH(2). These new and important insights will serve to better understand the activation process of CCK1R and to design or optimize ligands.     

Gut bitter taste receptor signalling induces ABCB1 through a mechanism involving CCK

T2Rs (bitter taste-sensing type 2 receptors) are expressed in the oral cavity to prevent ingestion of dietary toxins through taste avoidance. They are also expressed in other cell types, including gut enteroendocrine cells, where their physiological role is enigmatic. Previously, we proposed that T2R-dependent CCK (cholecystokinin) secretion from enteroendocrine cells limits absorption of dietary toxins, but an active mechanism was lacking. In the present study we show that T2R signalling activates ABCB1 (ATP-binding cassette B1) in intestinal cells through a CCK signalling mechanism. PTC (phenylthiocarbamide), an agonist for the T2R38 bitter receptor, increased ABCB1 expression in both intestinal cells and mouse intestine. PTC induction of ABCB1 was decreased by either T2R38 siRNA (small interfering RNA) or treatment with YM022, a gastrin receptor antagonist. Thus gut ABCB1 is regulated through signalling by CCK/gastrin released in response to PTC stimulation of T2R38 on enteroendocrine cells. We also show that PTC increases the efflux activity of ABCB1, suggesting that T2R signalling limits the absorption of bitter tasting/toxic substances through modulation of gut efflux membrane transporters.