Current Topics in Medicinal Chemistry - Volume 7, Issue 12, 2007

Cholecystokinin (CCK) and gastrin are among the first gastrointestinal hormones discovered. Since the first sequencing of these regulatory peptides in 1964 and 1968, a huge amount of basic and translational research work has been carried. Due to their wide tissue distribution in both the central nervous system and peripheral organs and to the number of biological processes which they regulate, CCK and gastrin receptors are undoubtedly important candidate targets in medicine. These receptors are still the subject of intensive research today. In this special issue of Current Topics in Medicinal Chemistry are gathered 10 articles related to cholecystokinin and gastrin receptors written by world-wide acknowledged experts in the field. In a first article, Jens Rehfeld and colleagues draw a picture of the available knowledge related to cellular synthesis, secretion and processing of cholecystokinin and gastrin peptides as well as to major physiological effects of the two peptides. In a second article Larry Miller and collegues describe the biochemical and cell biological mechanisms of cholecystokinin receptor regulation. These include receptor phosphorylation/dephosphorylation by specific enzymes, internalization/ intracellular trafficking and oligomerisation. In a third article, Florence Noble reports major data on the design of peptidic ligands specific for the CCK receptors and some pharmacological properties of these ligands, with a special focusing on the central nervous system. In the following two articles, Rosario Herranz and Barrett Kalindjian and colleagues review the last five literature years of medicinal chemistry of non peptide ligands of CCK1R and CCK2R. In a sixth article, Kyoko Miyasaka and colleagues describe available data on CCK1R polymorphism and its pathophysiological relevance in humans. In a seventh article, Marc Berna and Robert Jensen report in detail the established and possible roles of CCK1 and CCK2 receptors in gastrointestinal and metabolic diseases and discuss available results from human agonist/antagonist studies. In the following paper, Graham Baldwin reviews the most recent data in relation to CCK receptors and cancer and possible therapeutic implications, which has been a field of intensive investigations. In another article, Jean-Claude Reubi focuses his report on expression of CCK receptors in certain neuroendocrine tumours and on the promising use of CCK-related radiolabelled ligands to identify and cure these cancers. In the last article, Irina Tikhinova and colleagues, introduce and develop the concept of target-structure based design of ligands which could be applied to CCK receptors on the basis of data from CCK receptor binding site studies and further to other G-protein coupled receptors for which no satisfactory ligand is available. I wish to express my thanks to all contributors of this special issue which hopefully will be an helpful source of information for investigators in this field as well as in the wide field of G protein coupled receptors.

Cholecystokinin (CCK) and gastrin together constitute a family of homologous peptide hormones, which are both physiological ligands for the gastrin/CCK-B receptor, whereas the CCK-A receptor binds only sulfated CCKpeptides. CCK peptides are mainly produced in small intestinal endocrine I-cells and in cerebral neurons. CCK peptides regulate pancreatic enzyme secretion and growth, gallbladder contraction, intestinal motility, satiety and inhibit gastric acid secretion. Moreover, they are potent neurotransmitters in the brain and the periphery. CCK peptides are derived from proCCK and have the bioactive heptasequence -Tyr(SO4)-Met-Gly-Trp-Met-Asp-Phe-NH2 as their C-terminus. The dominant forms in plasma are CCK-58, CCK-33, CCK-22 and CCK-8, whereas CCK-8 is the major transmitterform. Due to scarcity of specific assays, knowledge about CCK in disease is still limited. Gastrin peptides are mainly synthetized in antroduodenal G-cells, from where they are released to blood to regulate gastric acid secretion and mucosal growth. Small amounts are synthetized further down the intestinal tract, in the foetal pancreas, in a few cerebral and peripheral neurons, in the pituitary gland and in spermatozoes. Gastrin peptides are derived from progastrin and all have the C-terminal bioactive hexasequence -Tyr (SO4)-Gly-Trp-Met-Asp-Phe-NH2. The major gastrin forms in tissue and plasma are gastrin- 34 and gastrin-17, but also gastrin-71, -14 and -6 have been identified. Gastrin peptides are secreted in excessive amounts from gastrinomas and are expressed at lower levels in bronchogenic, colorectal, gastric, ovarian and pancreatic cancers. A carcinogenetic significance of gastrin peptides remains, however, to be proven.

Regulation of the cholecystokinin receptor is accomplished by biochemical and cell biological mechanisms. The major mechanism for biochemical regulation involves phosphorylation of serine and threonine residues within the receptor's intracellular third loop and carboxyl-terminal tail. This form of rapid desensitization is achieved by protein kinase C, a kinase activated in the normal signaling cascade of this Gq-coupled receptor, and/or a member of the G protein-coupled receptor kinase family that recognizes the active conformation of the receptor. Conversely, a type 2A serine protein phosphatase has been shown to selectively act on this receptor in an agonist-regulated manner, resulting in receptor dephosphorylation and resensitization. Cell biological mechanisms for desensitization involve the net removal of receptors from exposure to circulating hormone via insulation within a specialized plasma membrane domain or internalization into the cell within endocytic compartments. Internalization has been shown to occur by both clathrindependent and clathrin-independent mechanisms, the latter believed to represent potocytosis in caveolae, that lead to recycling of receptors to the plasma membrane for resensitization or shuttling of receptors to lysosomes for degratory down-regulation. Interestingly, receptor phosphorylation has been shown to affect intracellular domain accessibility and receptor trafficking, although the specific proteins regulating this have not yet been identified. The cholecystokinin receptor can exist in the plasma membrane as oligomeric superstructures, namely as homomers with themselves or as heteromers with closely related class I G protein-coupled receptors. Agonist occupation results in oligomer dissociation, but the functional significance of this observation has yet to be defined.

Cholecystokinin (CCK) is a peptide originally discovered in the gastrointestinal tract but also found in high density in the mammalian brain. The C-terminal sulphated octapeptide fragment of cholecystokinin (CCK8) constitutes one of the major neuropeptides in the brain. CCK8, interacting with nanomolar affinities with two different receptors designated CCK1 and CCK2, has been shown to be involved in numerous physiological functions and is involved in the modulation/control of multiple central functions. In particular, CCK is involved in the neurobiology of anxiety, depression, psychosis, cognition, nociception and feeding behavior. The functional role of CCK has been facilitated thanks to the development of potent and selective CCK receptor antagonists and agonists. In this review, the strategies followed to design peptidic analog ligands will be reported, as the pharmacology of CCK receptors.

This review mainly covers last five year literature on CCK1R agonists and antagonists. These CCK1R ligands have been found following the two usual and complementary strategies for drug discovery: rational design based on structure activity relationships on the CCK-7 and CCK-4 peptide sequences of the endogenous ligands and random screening of diverse compounds, followed by hit optimization. The first group includes: chimeric bifunctional opioid/CCK peptides, designed as opioid agonists with balanced CCK1R/CCK2R antagonist activity for the treatment of neuropathic pain, antagonist and agonist dipeptoids, and 1,3-dioxoperhydropyrido[1,2-c]pyrimidine- and anthranilic acidbased antagonists. Among the ligands derived from random screening, a few new 1,4-benzodiazepine-, 1,5- benzodiazepine-, and five member ring heterocycle-based CCK1R ligands have been reported. Finally, taking into account the importance of receptor mapping studies for ligand optimization and future precise de novo receptor structurebased design of new selective and more effective ligands, the most significant conclusions of these studies have also been reviewed.

There has been an effort towards the design and preparation of non-peptide antagonists of the CCK2 receptor going back for over fifteen years. However, as no obvious unmet medical need for this type of molecule has emerged, the interest has somewhat declined. A number of comprehensive reviews have been written where much of the early work is described and so this article focuses on the information generated in the last five years. It is to be hoped that the area will regain some impetus following the recent disclosure of clinical trial data demonstrating the possible utility of a CCK2 antagonist in pancreatic cancer. When considering non-peptide agonists for the CCK2 receptor, traditionally, much less work has been reported in the area. However, recent suggestions of possible clinical utility in the treatment of diabetes, functionally different subtypes of the receptor and molecular models of receptor-ligand interactions should act as a spur for work towards potent small molecule ligands.

Since the isolation and sequencing of cholecystokinin (CCK), considerable advances have been made in understanding the roles played by this peptide as a hormone and as a neuropeptide. CCK-1(A) and 2(B) receptor (R) cDNAs have been cloned; shortly thereafter, the naturally occurring CCK-1R gene-deficient rat (the Otsuka Long-Evans Tokushima Fatty (OLETF) rat) was discovered. This strain develops adult-onset diabetes with obesity, and has a 6847 base-pair deletion of the CCK-1R gene in which the promoter lesion and the first two exons are missing. At the same time, the genomic structures of CCK-1R in rats, mice, and humans were clarified. The CCK-1R gene consists of five exons interrupted by four introns. It has been determined that there is species- and tissue-specific CCK receptor heterogeneity of expression; in particular, there is evidence that the human pancreas does not express CCK-1R, while the pancreas in rodents primarily expresses CCK-1R. Although CCK-1R polymorphisms with amino acid changes such as 21Gly to Arg, 71 Arg to Gly, and 364 Val to Ile were discovered in subjects with obesity and diabetes mellitus, these changes occur sporadically. We identified two sequence changes, a G to T change in nucleotide -128, and an A to G change in nucleotide -81, in the promoter region of the CCK-1R gene. This polymorphism is considered to be a single nucleotide polymorphism (SNP) related to weight control difficulties in obese subjects as well as to psychiatric disorders. The precise molecular mechanisms of this polymorphism remain to be clarified.

In this paper, the estabished and possible roles of CCK1 and CCK2 receptors in gastrointestinal (GI) and metabolic diseases are reviewed and available results from human agonist/antagonist studies are discussed. While there is evidence for the involvement of CCK1R in numerous diseases including pancreatic disorders, motility disorders, tumor growth, regulation of satiety and a number of CCK-deficient states, the role of CCK1R in these conditions is not clearly defined. There are encouraging data from several clinical studies of CCK1R antagonists in some of these conditions, but their role as therapeutic agents remains unclear. The role of CCK2R in physiological (atrophic gastritis, pernicious anemia) and pathological (Zollinger-Ellison syndrome) hypergastrinemic states, its effects on the gastric mucosa (ECL cell hyperplasia, carcinoids, parietal cell mass) and its role in acid-peptic disorders are clearly defined. Furthermore, recent studies point to a possible role for CCK2R in a number of GI malignancies. Current data from human studies of CCK2R antagonists are presented and their potential role in the treatment of these conditions reviewed. Furthermore, the role of CCK2 receptors as targets for medical imaging is discussed.

Over the past 20 years there has been considerable interest in the role of CCK receptors in gastrointestinal cancer. Initial excitement over reports of the detection by PCR of CCK-1 and CCK-2 receptor mRNA in a wide range of gastrointestinal tumours has been tempered by the realisation that the presence of receptor binding sites is much more restricted. The current consensus is that expression of CCK-1 and -2 receptor proteins is common only in tumours of neural or neuroendocrine origin. A striking example of this general rule has been provided by the detection of CCK-2 receptors by receptor autoradiography in more than 90% of medullary thyroid carcinomas. Despite the absence of CCK receptors from many gastrointestinal adenocarcinomas, evidence from animal models and from tumour cell lines in vitro suggests that the CCK-2 receptor may contribute to the development of esophageal and gastric adenocarcinomas, and further experimental work in these areas is clearly warranted. Promising therapeutic approaches include the development of radiolabelled gastrin/CCK derivatives for use in tumour imaging, and the use of appropriate selective antagonists for treatment of those tumour subtypes likely to express CCK receptors.

Gut hormone receptors can be over-expressed in several human cancers and represent the basis for receptortargeted tumor imaging and therapy. A promising receptor for such clinical applications is the cholecystokinin receptor. Cholecystokinin receptors are expressed in numerous neuroendocrine tumors, in particular medullary thyroid carcinomas and neuroendocrine gut tumors, as well as in stromal tumors. Moreover, several radiolabeled CCK or gastrin analogs have been developed allowing to detect these tumors and their metastases in patients using in vivo cholecystokinin receptor scintigraphy, proving the feasibility of targeting CCK receptors in human tumors in vivo.

Computer-aided drug design becomes an important part of G-protein coupled receptors (GPCR) drug discovery process that is applied for improving the efficiency of derivation and optimization of novel ligands. It represents the combination of methods that use structural information of a receptor binding site of known ligands to design new ligands. In this report, we give a brief description of ligand binding sites in cholecystokinin and gastrin receptors (CCK1R and CCK2R) which were delineated using experimental and computational methods, and then, we show how the validated ligand binding sites can be used to design and improve novel ligands. The translation of the knowledge of ligand-binding sites of different GPCRs to computer-aided design of novel ligands is summarized.

New FDA Approval of Cymbalta™ for Generalized Anxiety Disorder. Major depressive disorder is the 4th leading cause of disability worldwide and projected to rise significantly by 2020 [1]. In response, Eli Lilly and others developed Selective Serotonin Reuptake Inhibitors (SSRIs), such as Prozac™, as a first generation approach to combat this debilitating disorder [2]. However, serotonin reuptake monotherapy with SSRIs affords remission of symptoms for only one third of patients in clinical trials. This data drove researchers at Eli Lilly to develop a second generation of antidepressives that would be more clinically effective by targeting the reuptake of both serotonin (5-HT) and norepinephrine (NE). The result of their research was Cymbalta™, a potent, dual inhibitor of serotonin (5-HT, Ki = 4.6 nM) and norepinephrine (NE, Ki = 16 nM) that represented a new class of drugs referred to as a serotonin and norepinephrine reuptake inhibitors (SNRIs) [3,4]. Moreover, Cymbalta™ was a 10-fold more potent SSRI than Prozac™ (5-HT, Ki = 48 nM). Subsequent clinical trials with Cymbalta™ demonstrated that enhancing both serotonergic and noradrenergic neurotransmission results in improved antidepressant efficacy. Shortly thereafter, Cymbalta™ was approved by the FDA, and over 4.5 million patients have taken Cymbalta™ for the treatment of major depressive disorder or diabetic peripheral neuropathic pain [3-5]. In February of 2007, the FDA approved Cymbalta™ for the treatment of generalized anxiety disorder (GAD), a condition that affects over 6.5 million adults in America. GAD is a complex disorder characterized by exaggerated worry, chronic anxiety,irritability, sleep disturbances and fatigue which last for at least six months and cycle through periods of exacerbation and remission. In three randomized, double-blind, placebo-controlled GAD clinical studies, Cymbalta™ provided patients with a 46% improvement in anxiety symptoms compared to 32% with placebo, as evaluated by the Hamilton Anxiety Scale. In addition, patients taking Cymbalta™ experienced a 46% improvement in function compared to 26% with placebo, as measured by the Sheehan Disability Scale. In these trials, the dose range of Cymbalta™ was 60-120 mg/day, with the 120 mg dose provided the greatest efficacy [5]. REFERENCES [1] Murray, C.J.L.; Lopez, A.D., Eds. ‘The Global Burden of Disease: A Comprehensive Assessment of Mortality and Disability from Diseases, Injuries and Risk Factors in 1990 and Projected to 2020’ Cambridge, MA: Harvard University Press, 1996. [2] Wong, D.T.; Perry, K.W.; Bymaster, F.P. ‘Case History: The Discovery of Fluoxetine Hydrochloride ( Prozac )’ Nat. Rev. Drug Discov. 2005, 49, 764-774. [3] Bymaster, F.P.; Beedle, E.E.; Findlay, J.; Gallagher, P.T.; Krushinski, J.H.; Mitchell, S.; Robertson, D.W.; Thompson, D.C.; Wallace, L.; Wong, D.T. ‘Duloxetine (Cymbalta™), a Dual Inhibitor of Serotonin and Norepinephrine Reuptake’ Bioorg. Med. Chem. Lett. 2003, 13, 4477-4480. [4] Bymaster, F.P.; Lee, T.C.; Knadler, M.P.; Detke, M.J.; Iyengar, S. ‘The Dual Transporter Inhibitor Duloxetine: A Review of its Preclinical Pharmacology, Pharmacokinetic Profile, and Clinical Results in Depression’ Curr. Pharm. Design 2005, 11, 1475-1493. [5] For information on the FDA approval of Cymbalta™ for GAD see: www.lilly.com.