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Corticotropin-Releasing Factor and Urocortin Receptors
C H A P T E R
6 Corticotropin-Releasing Factor and Urocortin Receptors D.E. Grigoriadis Neurocrine Biosciences Inc., San Diego, CA, United States
O U T L I N E Introduction57
Small Molecule Nonpeptide CRF1 Receptor Antagonists
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Corticotropin-Releasing Factor and Urocortin Receptor Family Receptor Family Subtypes Endogenous Ligands
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Clinical Experience and Relevance Central Nervous System Indications Endocrine Indications
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Pharmacology of CRF1 and CRF2 Receptors Interaction of Peptide and Nonpeptide Ligands The Argument for Receptor Kinetics
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Summary and Conclusions
63
References64
Abstract
INTRODUCTION
The key mediator of the innate physiological stress response has long been hypothesized to be the hypothalamic peptide corticotropin-releasing factor (CRF). This 41 amino acid peptide modulates the endocrine, autonomic, behavioral, and immune responses to stress through actions at specific receptors both in the central nervous system as well as the periphery. These seven transmembrane proteins belong to the superfamily of G protein–coupled receptors and play a role in central nervous system, cardiovascular, gastrointestinal, and pituitary-adrenal function. Decades of research have implied a direct relationship between stress-related disorders and CRF activity in the brain. As a result, this system has been the focus of many drug discovery programs aimed at identifying selective and specific orally active molecules that can block the pathophysiological increase in activity of this peptide. This review will summarize this system, the evidence supporting the utility of molecules that block the CRF system in human disease and the current state of the drug discovery programs that have yet to produce a viable therapeutic.
More than 2000 years ago, Hippocrates referred to the concept of homeostasis and equilibrium in health and disease as a dysregulation of this equilibrium. He further observed that there were individual differences in the severity of disease symptoms and that some individuals were better able to cope with their disease or illness than others. More than a half century ago, Hans Selye proposed that stress is the nonspecific (autonomic) response of a human body to any demand made upon it, real or perceived and that concept was broadened by Geoffrey Harris who first defined the role of the hypothalamus in the regulation of the pituitary– adrenocortical axis extending the notion that an organism’s ability to maintain homeostasis in the face of external stressors was coordinated by a specific portion
Stress: Neuroendocrinology and Neurobiology http://dx.doi.org/10.1016/B978-0-12-802175-0.00006-1
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© 2017 Elsevier Inc. All rights reserved.
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of the CNS operating in an automatic fashion. During the mid-1950s, Guillemin and Rosenberg, and Saffran and Schally independently recognized the presence of a specific factor in extracts of the hypothalamus that could stimulate the release of corticotropin [adrenocorticotropic hormone (ACTH)] from anterior pituitary cells in vitro. This extract was termed corticotropinreleasing factor (CRF). It was not until 1981, following the development of radioimmunoassays for ACTH and quantitative in vitro methods for assaying hypophysiotropic hormones along with the utility of ion exchange and high performance liquid chromatographic techniques, that enabled a team of Salk Institute scientists led by Wylie Vale to successfully isolate and purify the peptide CRF from sheep hypothalamic extracts.37 Thus, from the first basic principles in the understanding of an organism’s homeostatic response to stress that began centuries ago, this neuropeptide has been studied for its role in mediating the hypothalamic–pituitary–adrenal (HPA) axis and regulating the response to physical, emotional, and environmental stress. The discovery and structural elucidation of CRF ushered in a unique and focused hypothesis-driven KEY POINTS • C orticotropin-releasing factor (CRF) and the peptide family of urocortins activate two subtypes of Class-B G protein–coupled receptors (CRF1 and CRF2 receptors) and regulate the endocrine, autonomic, and behavioral response to stress. • Nonpeptide antagonists for the CRF1 receptor have been discovered that act allosterically and demonstrate efficacy in a wide range of animal models for stress behaviors. • The allosteric nature of the nonpeptide interaction requires a long residence time (slow dissociation) of the small molecule on the receptor for optimal efficacy in vivo. • Multiple small molecules have been examined in Phase 2 human studies for major depressive disorder, social anxiety, generalized anxiety, posttraumatic stress disorder, irritable bowel syndrome, and alcohol dependence. • Despite the tremendous body of evidence in animal models from different species providing an indication in human psychiatric disease, none of the CRF1 receptor antagonists to date have progressed beyond Phase 2 compelling the field to rethink the clinical application of these potent and selective compounds.
era of scientific study. While the initial understanding of its direct role at the pituitary pointed to the regulation of the endocrine stress response, it was the distribution of the CRF peptide in extrahypothalamic regions of the brain that intensified study into its role as a bona fide neurotransmitter and neuromodulator in the CNS. Multiple reports have detailed the anatomical distribution of both CRF-containing cell bodies and fibers in the CNS and more importantly, in regions of the brain shown to clearly regulate autonomic function and mediate the behavioral responses to stress. For example, in the cortex, high densities of CRF containing neurons that are localized primarily to prefrontal, cingulate, and insular areas, appear to regulate behavioral actions of the peptide. Many studies in rodents have demonstrated that exogenous administration of CRF into the CNS, has demonstrated direct interactions between the CRF system and virtually all monoaminergic neurotransmitter systems including the norepinephrine system in the locus coeruleus, the serotonergic systems in the dorsal and median raphe nuclei, and the dopaminergic system in the ventral tegmental area indicating that this system can impact multiple facets of the stress response of an organism. This critical convergent relationship between the hormone CRF and the monoaminergic systems in the brain further implicates this particular neuropeptide in mediating the central mechanisms through which various stressors can alter behavior. The cloning of multiple CRF receptor subtypes and the identification of a specific binding protein for the peptide (known as CRF-BP) have also enhanced the ability to dissect the behavioral consequences of activation or inhibition of this system. In addition to the receptors, the identification of endogenous-related family members of the CRF peptide (termed the urocortins) has expanded the notion that this system is under the control of multiple regulatory factors and plays a much more complex role in the CNS than previously thought. This chapter will focus primarily on the molecular biological, pharmacological, and functional characteristics of the CRF system and describe the recent challenges in the discovery and late-stage development of small molecule nonpeptide inhibitors of this system as potential therapeutics.
CORTICOTROPIN-RELEASING FACTOR AND UROCORTIN RECEPTOR FAMILY As a member of the superfamily of Class-B G-protein coupled receptors, the receptors for CRF and the urocortins have been shown to contain seven putative transmembrane domains and function through the
I. NEUROENDOCRINE CONTROL OF THE STRESS RESPONSE
Corticotropin-Releasing Factor and Urocortin Receptor Family
coupling of a stimulatory guanine-nucleotide–binding protein. These receptor subtypes all fall within the now well-described and still-growing, family of “gut-brain” neuropeptide receptors, which includes receptors for calcitonin, vasoactive intestinal peptide, parathyroid hormone, secretin, pituitary adenylate cyclase–activating peptide, glucagon, and growth hormone–releasing factor. While these receptors all share considerable sequence homology, they have been shown to activate a variety of intracellular signaling pathways. Cloning of the CRF receptor subtypes has enabled the pharmacological and biochemical characterization of these proteins and allowed the discovery of potential therapeutics through a variety of chemical screening strategies.
Receptor Family Subtypes The discovery of the CRF peptide led to the first radiolabeled form of the peptide that was used to probe the localization, distribution, and functional mechanisms of receptors in a variety of tissues from the brain and periphery in many different species. These studies served to define the physiology of the system and enabled a detailed investigation using molecular, biochemical, and behavioral tools to try and understand the role of this system in the normal and pathological state. In addition to the discovery of CRF, Vale and colleagues were the first to clone the CRF1 receptor in 1993 from a human Cushing anterior pituitary corticotropic adenoma using expression cloning.10 Several other groups later also identified the CRF1 form of the receptor from a variety of animal species. Interestingly, all species of CRF1 receptor mRNAs thus far identified encode proteins of 415 amino acids that are 98% identical to one another. In fact, the family of peptides has been well characterized in species ranging from insects to high-order vertebrates indicating the evolutionary importance of this system.24 The human CRF1 receptor gene contains at least two introns and is found in a number of alternative splice forms none of which to date have been found to have any physiological significance. Characteristic of most G protein–coupled receptors there are potential N-linked glycosylation sites on the large N-terminal extracellular domain and these molecularly identified sites confirm the glycosylation profiles determined previously using chemical affinity cross-linking studies. Indeed, the predicted molecular weight of the CRF1 receptor derived from deglycosylation studies was virtually identical to that obtained from the cloned amino acid sequence. Furthermore, mRNA distribution of the CRF1 receptor in the CNS of both rat and human tissues correlates extremely well with the previously identified biding sites for radiolabeled CRF using autoradiographic techniques in frozen
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brain sections. Alternative splice variants of the CRF1 receptor have been described localized to discrete tissues. For example, a unique splice variant of the CRF1 receptor has been reported in human pregnant myometrium and while the pharmacology appears to be similar to the original subtype, the different signaling characteristics suggest that this isoform may be expressed under specific physiological conditions.14 Shortly following the cloning and characterization of the CRF1 receptor, a second subtype of this family was identified. This receptor (termed the CRF2 receptor subtype) exists as three individual splice variants termed CRF2(a) and CRF2(b) and CRF2(c). These receptor isoforms differ only in the extracellular region where each has a unique N-terminal sequence generating 411, 431, and 397 amino acid proteins for the CRF2(a), CRF2(b), and CRF2(c) receptors, respectively. Thus far, the CRF2(c) receptor has been identified exclusively in human brain and its function still remains to be determined. Comparing the CRF1 and CRF2 receptor subtypes, there are very large regions of amino acid identity, particularly between transmembrane domains five and six. This similarity indicates that biochemical action is conserved and these receptors act through G protein coupling and signal transduction. Detailed distribution and receptor autoradiographic studies have localized CRF-binding sites and CRF receptor mRNA, respectively, in slide-mounted sections of many tissues from a variety of species identifying these proteins in anatomically and physiologically relevant areas. Of primary interest, the CRF1 receptor is highly expressed in the pituitary gland, specifically in corticotrophs where it regulates the release of ACTH as a key mediator of the stress response. This discrete localization has facilitated the utility of selective receptor antagonists in disorders related to increased HPA activity. This will be detailed later in this chapter. While the CRF2 receptor has been documented extensively with respect to its pharmacology, its role in behavior and physiological function has been defined largely through its localization and the effects of the endogenous agonists. Specific chemical tools that block this receptor subtype systemically are few and restricted to peptide analogs (illustrated in the next section). In addition, the distribution of the CRF2 receptor is isoform-dependent. Specifically, the precise localization the splice variants, CRF2(a) and CRF2(b), indicates discrete anatomical distribution. The predominant form in the CNS is the CRF2(a) receptor where localization studies and those using peptide agonists have implicated a role for this subtype in anxiety and feeding behaviors. The CRF2(b) splice variant is localized almost exclusively in the CNS to nonneuronal elements, such as the choroid plexus of the ventricular system and cerebral arterioles. In the periphery, however, the CRF2(b) receptor is highly expressed in both cardiac
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and skeletal muscle with lower levels evident in both lung and intestine. Its role peripherally has been described in cardiovascular function and gastrointestinal motility. In fact, for a short period of time, the CRF2(b) receptor was the target of intense study for the treatment of acute decompensated heart failure (ADHF) owing to the very potent vasodilatory effects of the endogenous peptide urocortin 2. Studies performed in rats, sheep, and some initial studies in humans showed early promise as a potential treatment for ADHF.1,9 The CRF2(c) isoform has yet to be identified in the rodent, however, in the human brain, this isoform was shown to be highly expressed in the septum, amygdala, hippocampus, and frontal cortex.
Endogenous Ligands Since the elucidation of the amino acid sequence of CRF, the peptide was found to bear strong similarities to two nonmammalian peptides, sauvagine from the frog and urotensin I from the teleost fish. These mammalian and nonmammalian peptides all potently release ACTH from cultured rat pituitary cells directly through the CRF1 receptor in the anterior pituitary. One striking difference between these peptides was that only the nonmammalian forms retained a high affinity for the CRF2 receptor. Thus it became highly unlikely that CRF itself was the endogenous ligand for the CRF2 receptor in mammals. Using antibodies for fish urotensin I, the first mammalian peptide with high affinity for the CRF2 receptor was discovered and termed urocortin 1. This unique peptide discovered first in the rat was observed to retain its high affinity for the CRF1 receptor and the CRF-binding protein as well. Furthermore, the peptide was found to be localized in areas corresponding to the distribution of the CRF2 receptor itself. The human homolog of urocortin 1 was subsequently cloned from a human brain library and defined as the endogenous ligand for the CRF2 receptor. Since then, two other mammalian peptides have been identified from the mouse and human termed urocortin 2 (UCN2, also referred to as stresscopin-related peptide) and urocortin 3 (UCN3, also referred to as stresscopin). Urocortin two and urocortin 3, unlike urocortin 1, have much higher selectivity for the CRF2 receptor subtype and have little or no affinity for the CRF-binding protein. A great deal of literature now exists on the discrete and independent physiologic function of the urocortins and their activity on the CRF2 receptor subtypes (for a comprehensive review see Ref. 34). However, the current paucity of selective small molecule antagonists that could be administered chronically, hinders our ability to fully understand the role of the CRF2 receptor subtype in normal or pathophysiology. Nonetheless, major advances have been made in elucidating the function of these ligands and the CRF2 receptor in
anxiety, cardiovascular disease, inflammatory disease, and gastrointestinal and feeding disorders. Interestingly, there are no known endogenous physiological antagonists for any of the CRF receptor subtypes known to date. However, various truncations and modifications of the agonist peptides have resulted in potent peptide antagonists. A large body of work has been dedicated to identifying specific and selective peptide antagonists of these receptor subtypes.8,35 These ligands were designed to extend duration of action in vivo and demonstrated high affinity and activity for both the CRF1 and CRF2 receptor subtypes. Two of these synthetic highaffinity peptides, Antisauvagine-30, an N-terminally truncated form of sauvagine, and the cyclized Astressin-2B have been shown to be selective for the CRF2 receptor subtype and used to dissect out the specific role of this receptor in a variety of in vitro and in vivo studies. The pharmacological profiles of these peptide agonists and antagonists greatly enhanced the understanding of the physiology of the CRF system and the important role this neurohormone plays in mediating a variety of physiological and pathophysiological responses.
PHARMACOLOGY OF CRF1 AND CRF2 RECEPTORS The pharmacology of the class B GPCRs CRF1 and CRF2 have been described, characterized, and studied since the discovery of the peptide itself. Given the importance of the system and the desire to link human pathology to interventions that would have some therapeutic benefit, basic biochemical studies became focused on the distinct interactions of both peptide and nonpeptide ligands. As more and more ligand tools were developed, there was a clear understanding of the relationships between the peptide and small molecule interactions and the functional relationship between the two.
Interaction of Peptide and Nonpeptide Ligands The peptide interactions of the CRF family of ligands with the receptors has been extensively reviewed and reported to be the same for both receptor subtypes. Studies using the CRF1 receptor have posited that the C-terminal region of CRF adopts an α-helical conformation, which binds to the extracellular domain of the receptor. This initial low-affinity interaction then allows the binding of the N-terminal portion of CRF to a portion of the transmembrane domain that ultimately translocates the signal to function within the cell. An important concept of this mechanism is that this tethering of the N-terminal portion of CRF greatly increases its local concentration at the receptor. This two-domain binding of the peptide implies that the peptide CRF may remain tethered to the
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PHARMACOLOGY OF CRF1 AND CRF2 RECEPTORS
N-terminus of the receptor when the receptor is in its inactive state.3,16,29,32 This has direct implications when examining the abilities of non-peptide small molecule ligands to block the function of these receptors for therapeutic benefit. It is interesting to note that truncated forms of CRF such as D-Phe CRF(12-41) or the synthetic peptides Astressin or Astressin-2 bind with very high affinity to the N-terminus of their receptors but since they lack the ability to interact with the transmembrane domain, act as complete antagonists. Small molecule tools have enabled the understanding of the mechanisms of receptor blockade for this family of receptors. It is important to note from the outset that while there have been thousands of CRF1-specific small molecule receptor antagonists reported in the academic and patent literature, the tremendous effort that was put forth in identifying selective CRF2 receptor antagonists has yet to provide molecules that do not interact with the CRF1 receptor. This is an interesting scientific problem given the high degree of similarity of the two receptor subtypes especially in the transmembrane domain regions. The discovery and characterization of these small molecules will be described below. As will be illustrated in the next sections, these molecules were found to bind well within the transmembrane domain region of the CRF1 receptor with virtually no overlap in the binding sites of the peptides, thus defining these interactions as allosteric to each other. Therefore, any inhibition of CRF1 receptor function by nonpeptide small molecules that are allosteric to the peptide-binding domain must by definition be a result of a physical conformational change in the receptor protein rather than a simple blockade of peptide agonist binding. Furthermore, as postulated above, agonist peptides can be bound to the N-terminus of the receptor protein despite the existence of a small molecule occupying its binding
site in the transmembrane region. This has clear clinical and therapeutic implications. If the peptide is essentially tethered to the N-terminus, then it is positioned to signal through the receptor as soon as the small molecule dissociates from its binding site. Given these dynamics, the longer the small molecule can occupy the receptor and maintain its inactive conformation, the longer the therapeutic benefit. It became clear that in addition to the high affinity and selectivity these small molecules needed as potential therapeutics, they would also have to maintain the property of a long kinetic receptor off-rate to maximize their therapeutic efficacy.
The Argument for Receptor Kinetics Accounting for the receptor kinetics in the discovery of potential therapeutics is not a novel concept. This has been described in elegant detail for compounds blocking the angiotensin receptor in hypertension.20 Applying this concept to molecules that block the function of the CRF1 receptor allosterically, adds another dimension to the drug discovery and lead optimization process. Correlating the apparent affinity of the molecules with in vitro and in vivo efficacy became the critical criterion for progressing compounds into advanced stage development. As described above, the receptor occupancy and the pharmacokinetic and pharmacodynamic properties of these molecules all had to be optimized simultaneously. A great deal of work has been published in this area examining the relationships, both physical and experimental, of small molecule interaction that have led to their testing in large clinical trials.12,33,39 An illustration of this effort is shown in Table 6.1 using molecules that have in fact progressed into Phase 2 human clinical studies. Extracted from previously published reports,
TABLE 6.1 Comparison of the Properties of Select CRF1 Receptor Antagonists That Have Been in Human Clinical Trials for Anxiety or Depression Compound
Clinical Trials Identifier
[125I]-Sauvagine Binding Affinity (Ki nM)
Dissociation Time (t1/2 min)
Kinetic Affinity (Ki nM)
In Vivo ACTH (% Veh AUC @ 120 min)
CP-316,311
NCT00143091
1.9
4.1
12
76
Emicerfont/GW-876008
NCT00397722
20
10
39
87
Pexacerfont/BMS-562086
NCT00135421 NCT00481325
7.4
14
19
96
ONO-2333Ms
NCT00514865
1.2
17
15
78
Verucerfont/GSK-561679
NCT00733980 NCT01018992
1.3
70
3.9
43
NBI 30775/R121919
Ref. 40
2.6
130
0.36
42
Comparison of clinical compounds in their ability to inhibit [125I]-sauvagine, their dissociation times from the receptor and calculated kinetic affinity, and their ability to inhibit ACTH release in adrenalectomized rats. Clinical trials highlighted are those directly testing efficacy of these molecules for anxiety or depression only and listed on www.ClinicalTrials.gov with the exception of NBI-30775 (R121919), which was tested in an open label study in Major Depression. Multiple other exploratory clinical trials exist for each of these compounds. Data adapted from Fleck BA, Hoare SR, Pick RR, Bradbury MJ, Grigoriadis DE. Binding kinetics redefine the antagonist pharmacology of the corticotropin-releasing factor type 1 receptor. J Pharmacol Exp Ther. 2012;341(2):518–531. doi:10.1124/jpet.111.188714.
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Table 6.1 summarizes the most advanced chemical compounds compared using their binding affinity, dissociation time, the affinity determined via kinetic analysis, and the in vivo efficacy for inhibition of ACTH release. It is clear that all molecules had high affinity for the receptor as defined by [125I]-sauvagine binding. What is also clear is that if the compounds are rank ordered by their receptor off rates or dissociation times, this correlated very well with their ability to block the ACTH release from adrenalectomized rats. Coupled with the notion described above that compounds bind allosterically, and the fact that adrenalectomy causes an uncontrolled release of both CRF from the hypothalamus and ACTH from the pituitary, it is not surprising that compounds would have to have very long residence times on the receptor to effectively block the response of CRF in vivo. The data accumulated in Table 6.1 provides evidence for a link between ligand residence time, defined by receptor off-rate, and offset kinetics and insurmountable/ noncompetitive antagonism at the CRF1 receptor.
small molecules compared to the binding domains of the peptides.22 Regardless of the initial chemical starting points identified through large chemical library screening, when these molecules were ultimately modified into compounds that could be tested in the clinic, they all seem to bind to exactly the same pocket deep within the transmembrane domain of this receptor. In fact, this has now been clearly demonstrated in studies that solved the crystal structure of the CRF1 receptor bound to a typical CRF1 receptor antagonist.17 One inescapable conclusion from all these studies is that so far, this receptor has been shown to have a defined binding pocket for small molecules that can modulate its activity but more importantly, it will not tolerate structures that deviate from this well-defined pocket. As will be hypothesized below, if the current lack of progress of clinical candidates is due to the physical limitations of this particular binding site, then a complete revision of the mechanisms through which we can block this system will have to be understood and exploited.
SMALL MOLECULE NONPEPTIDE CRF1 RECEPTOR ANTAGONISTS
CLINICAL EXPERIENCE AND RELEVANCE
The discovery of small molecule receptor antagonists did not emerge from any advances in the peptide arena. Although peptide antagonists had been synthesized, their poor pharmacokinetics (oral bioavailability, rapid clearance, and minimal brain penetration) and general lack of CRF1 receptor selectivity did not yield any information useful in the development of nonpeptide blockers. Utilizing large screening libraries, many small molecule antagonists were developed and profiled for their pharmacokinetic and pharmacodynamics properties. The first disclosure of a small molecule CRF1 receptor antagonist was in 1991 and since then, numerous low-molecular weight ligands have been developed that potently bind and antagonize the CRF1 receptor (for detailed reviews see13,15,19). What is important to note is that the chemical starting points for optimization of these varied structures were as diverse as the chemical libraries amassed following years of small molecule research in the pharmaceutical industry. These structurally distinct, specific and selective CRF1 receptor antagonists were discovered through extensive screening methodologies utilizing these diverse chemical libraries and painstakingly optimized for drug-like characteristics.21 As eluded to above, these molecules were found to bind well within the transmembrane domain region of the CRF1 receptor with virtually no overlap in the binding sites of the peptides. First described in 1997, point mutation studies of the CRF1 receptor identified transmembrane domains three and five as critical points of interaction of the
The first publication of a CRF1 receptor antagonist used in the human population was a small open-label study in a group of major depression patients.40 This preliminary study energized the field and instigated a tremendous effort in the design and development of subsequent molecules. Multiple compounds were discovered and evaluated clinically for efficacy ranging from major depression, social anxiety generalized anxiety, posttraumatic stress disorder, and irritable bowel syndrome as well as studies in alcohol abuse. In these well-controlled placebo and many times comparator trials, the efficacy of a CRF1 antagonist has not been demonstrated. The clinical indications that have been examined have all been based on the following premise that CRF is the primary regulator of the stress response, and since these diseases have been reported to have a stress component in their etiology or manifestation, a CRF1 receptor blocker would be a viable therapeutic.
Central Nervous System Indications In the CNS, the stress axis has been implicated in playing a major role in depression and anxiety-related disorders and has been extensively reviewed.6,7,23 Many patients with major depression have been shown to have a perturbed HPA axis and are hypercortisolemic defined by an abnormal dexamethasone suppression test.5,18 Since it was demonstrated that CRF is the primary regulator of the stress response through the HPA
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Summary and Conclusions
axis in animals, it was a logical extension that hypersecretion or hyperactivity of CRF in brain might underlie the symptomatology seen in major depression or anxiety. Major clinical observations collectively provided overwhelming evidence that implicated the CRF system in the pathologic development of CNS diseases. For example, the first indication was the observation that the concentration of CRF was found to be significantly higher in the cerebrospinal fluid (CSF) of drug-free individuals with major depression28 and subsequently, it was found that the levels of CRF in the CSF were elevated while the CRF receptors were downregulated in the brains of suicide victims.2,27 Furthermore, this elevation could be reversed following successful treatment with electroconvulsive therapy.26 Since then, a myriad of studies have provided evidence for the role of CRF in depression and anxiety-related disorders.4,11,30 Beyond all of the animal studies where CRF1 antagonists have demonstrated efficacy in anxiety and depression behaviors, the now routinely available genetic analyses for disease states have offered a new approach in the selection of candidate populations with which to test the system.25,31,38 All of these data, evidence generated over the past three decades, created the overwhelming motivation to test this hypothesis in the clinic in a number of diseases of the CNS. To date however, no CRF1 receptor antagonist has progressed in development beyond Phase 2 proof-of-concept trials. A reevaluation of the hypothesis and specific disease-state or population is therefore warranted.
Endocrine Indications From a potential therapeutic perspective, while decades of study have focused on CNS and peripheral disorders like irritable bowel syndrome, the potential therapeutic indications at the level of the pituitary have been largely ignored. This is not entirely surprising or unexpected given the large potential patient populations with psychiatric disease, stress disorders, and immune and gastrointestinal disorders. A very small but unmet medical need does however exist for individuals whose HPA axis is chronically activated either through overexpression of hypothalamic CRF or genetic disorders that disrupt the axis. One example of such a disorder is congenital adrenal hyperplasia. This disease was first described in the 1950’s and is a complex genetic disorder in which the adrenals produce little or no cortisol. The most predominant form of this disease is a mutation in the 21-hydroxylase enzyme in the adrenal, which is directly responsible for the conversion of steroid precursors ultimately to glucocorticoids. This leads to a significant reduction in the negative feedback loop of cortisol at the level of both the hypothalamus and the pituitary, in effect causing substantial increases in both CRF and
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ACTH respectively. The elevated ACTH stimulates the adrenal steroidogenic pathways and without the ability to convert steroid precursors to cortisol, upstream precursors, such as 17α-hydroxyprogesterone, accumulate and generate high levels of androgens that are the root pathology of this disease. Current and most common treatment therapies are direct administration of glucocorticoids and are intended to not only replace the missing cortisol but also decrease the ACTH drive of androgen excess. In order to achieve this level of control, supraphysiological doses of glucocorticoids are required to effectively suppress adrenal androgens leading to issues associated with high steroid use. CRF receptor antagonists, which act at the level of the pituitary and can directly block CRF from stimulating ACTH release, may help in the overall treatment and well-being of these patients. By inhibiting the release of ACTH directly, the pressure on the adrenal to produce androgens should be lessened and administered cortisol may be reduced to physiologic replacement levels. A very recent clinical pharmacology study has been performed and shown some promising results.36
SUMMARY AND CONCLUSIONS Corticotropin-releasing factor has long been described as the primary regulator of the HPA axis and a target for the discovery of molecules that may have therapeutic utility in stress-related disorders. Despite heroic efforts in evaluating multiple CRF1 receptor antagonists in diseases such as major depressive disorder, generalized anxiety, social anxiety, posttraumatic stress disorder, irritable bowel disease, and stress in alcoholism (see www.Cinicaltrials.gov for details on numerous studies), none of the molecules examined have shown any clinical efficacy. While there may be fundamental issues with selection of the appropriate patient population, the specific design of the clinical trials, or the disease hypotheses themselves, it is evident that the tools we have currently at our disposal cannot demonstrate a therapeutic benefit in psychiatric disease. One common feature of these clinical trials (where actually measured) was that there was an effect on HPA activity. In this patient pool, there has been evidence of inhibition of ACTH release and this at least supports the utility of these agents as potential therapeutics in endocrine disorders where the primary requirement is modulation of ACTH release from the pituitary. The last three decades of intense study of this system should be examined with a critical view and the hypotheses modified to account for what the field has learned from these failures. CRF remains an important biochemical system both in the CNS and in the periphery and deserves a next-generation investigation.
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References 1. Adao R, Santos-Ribeiro D, Rademaker MT, Leite-Moreira AF, Bras-Silva C. Urocortin 2 in cardiovascular health and disease. Drug Discov Today. 2015;20(7):906–914. http://dx.doi.org/10.1016/ j.drudis.2015.02.012. 2. Arato M, Banki CM, Bissette G, Nemeroff CB. Elevated CSF CRF in suicide victims. Biol Psychiatry. 1989;25(3):355–359. 3. Assil IQ, Qi LJ, Arai M, Shomali M, Abou-Samra AB. Juxtamembrane region of the amino terminus of the corticotropin releasing factor receptor type 1 is important for ligand interaction. Biochemistry. 2001;40(5):1187–1195. 4. Aubry J-M. CRF system and mood disorders. J Chem Neuroanat. 2013;54:20–24. http://dx.doi.org/10.1016/j.jchemneu.2013.09.003. 5. Aubry JM, Gervasoni N, Osiek C, et al. The DEX/CRH neuroendocrine test and the prediction of depressive relapse in remitted depressed outpatients. J Psychiatr Res. 2006;41(3–4):290–294. 6. Bali A, Singh N, Jaggi AS. Neuropeptides as therapeutic targets to combat stress-associated behavioral and neuroendocrinological effects. CNS Neurol Disord Drug Targets. 2014;13(2):347–368. http://dx.doi.org/10.2174/1871527313666140314163920. 7. Binder EB, Nemeroff CB. The CRF system, stress, depression and anxiety-insights from human genetic studies. Mol Psychiatry. 2010;15(6):574–588. http://dx.doi.org/10.1038/mp.2009.141. 8. Broadbear JH, Winger G, Rivier JE, Rice KC, Woods JH. Corticotropin-releasing hormone antagonists, astressin B and antalarmin: differing profiles of activity in rhesus monkeys. Neuropsychopharmacology. 2004;29(6):1112–1121. 9. Chan WY, Frampton CM, Crozier IG, Troughton RW, Richards AM. Urocortin-2 infusion in acute decompensated heart failure: findings from the UNICORN study (urocortin-2 in the treatment of acute heart failure as an adjunct over conventional therapy). JACC Heart Fail. 2013;1(5):433–441. http://dx.doi.org/10.1016/j.jchf.2013.07.003. 10. Chen R, Lewis KA, Perrin MH, Vale WW. Expression cloning of a human corticotropin-releasing-factor receptor. Proc Natl Acad Sci USA. 1993;90(19):8967–8971. 11. Devi M, Sharma R. A review on animal models of depression. Int J Pharm Sci Res. 2013;4(10):3731–3736. http://dx.doi.org/10.13040/ IJPSR.0975-8232.4(10).3731-36. 12. Fleck BA, Hoare SR, Pick RR, Bradbury MJ, Grigoriadis DE. Binding kinetics redefine the antagonist pharmacology of the corticotropin-releasing factor type 1 receptor. J Pharmacol Exp Ther. 2012;341(2):518–531. http://dx.doi.org/10.1124/jpet.111.188714. 13. Gilligan PJ, Li YW. Corticotropin-releasing factor antagonists: recent advances and exciting prospects for the treatment of human diseases. Curr Opin Drug Discov Devel. 2004;7(4):487–497. 14. Grammatopoulos DK, Dai Y, Randeva HS, et al. A novel spliced variant of the type 1 corticotropin-releasing hormone receptor with a deletion in the seventh transmembrane domain present in the human pregnant term myometrium and fetal membranes. Mol Endocrinol. 1999;13(12):2189–2202. 15. Griebel G, Holsboer F. Neuropeptide receptor ligands as drugs for psychiatric diseases: the end of the beginning? Nat Rev Drug Discov. 2012;11(6):462–478. http://dx.doi.org/10.1038/nrd3702. 16. Hoare SR, Sullivan SK, Schwarz DA, et al. Ligand affinity for amino-terminal and juxtamembrane domains of the corticotropin releasing factor type I receptor: regulation by G-protein and nonpeptide antagonists. Biochemistry. 2004;43(13):3996–4011. http://dx.doi.org/10.1021/bi036110a. 17. Hollenstein K, Kean J, Bortolato A, et al. Structure of class B GPCR corticotropin-releasing factor receptor 1. Nature. 2013;499(7459): 438–443. http://dx.doi.org/10.1038/nature12357. 18. Ising M, Kunzel HE, Binder EB, Nickel T, Modell S, Holsboer F. The combined dexamethasone/CRH test as a potential surrogate marker in depression. Prog Neuropsychopharmacol Biol Psychiatry. 2005;29(6):1085–1093.
19. Kehne J, De LS. Non-peptidic CRF1 receptor antagonists for the treatment of anxiety, depression and stress disorders. Curr Drug Targets CNS Neurol Disord. 2002;1:467–493 (Copyright © 2013 U.S. National Library of Medicine.). 20. Lacourciere Y, Asmar R. A comparison of the efficacy and duration of action of candesartan cilexetil and losartan as assessed by clinic and ambulatory blood pressure after a missed dose, in truly hypertensive patients: a placebo-controlled, forced titration study. Candesartan/Losartan study investigators. Am J Hypertens. 1999;12(12 Pt 1–2):1181–1187. 21. Lanier M, Williams JP. Small molecule corticotropin-releasing factor antagonists. Expert Opin Ther Pat. 2002;12(11):1619–1630. 22. Liaw CW, Grigoriadis DE, Lorang MT, De Souza EB, Maki RA. Localization of agonist- and antagonist-binding domains of human corticotropin-releasing factor receptors. Mol Endocrinol. 1997;11(13):2048–2053. 23. Lin E-JD. Neuropeptides as therapeutic targets in anxiety disorders. Curr Pharm Des. 2012;18(35):5709–5727. 24. Lovejoy DA, Balment RJ. Evolution and physiology of the corticotropin-releasing factor (CRF) family of neuropeptides in vertebrates. Gen Comp Endocrinol. 1999;115(1):1–22. http://dx.doi.org/ 10.1006/gcen.1999.7298. 25. Menke A, Domschke K, Czamara D, et al. Genome-wide association study of antidepressant treatment-emergent suicidal ideation. Neuropsychopharmacology. 2012;37(3):797–807. http://dx.doi.org/ 10.1038/npp.2011.257. 26. Nemeroff CB, Bissette G, Akil H, Fink M. Neuropeptide concentrations in the cerebrospinal fluid of depressed patients treated with electroconvulsive therapy: corticotropin-releasing factor, β-endorphin and somatostatin. Br J Psychiatry. 1991;158:59–63. 27. Nemeroff CB, Owens MJ, Bissett G, Andorn AC, Stanley M. Reduced corticotropin-releasing factor receptor binding sites in the frontal cortex of suicide victims. Arch Gen Psychiatry. 1988;45:577–579. 28. Nemeroff CB, Widerlov E, Bissett G, et al. Elevated concentration of CSF corticotropin-releasing factor-like immunoreactivity in depressed patients. Science. 1984;226:1342–1344. 29. Nielsen SM, Nielsen LZ, Hjorth SA, Perrin MH, Vale WW. Constitutive activation of tethered-peptide/corticotropin-releasing factor receptor chimeras. Proc Natl Acad Sci USA. 2000;97(18): 10277–10281. 30. Overstreet DH. Modeling depression in animal models. Methods Mol Biol. 2012;829:125–144. http://dx.doi.org/10.1007/978-1-61779 -458-2_7 (Psychiatric Disorders). 31. Paez-Pereda M, Hausch F, Holsboer F. Corticotropin releasing factor receptor antagonists for major depressive disorder. Expert Opin Invest Drugs. 2011;20(4):519–535. http://dx.doi.org/ 10.1517/13543784.2011.565330. 32. Perrin MH, Sutton S, Bain DL, Berggren WT, Vale WW. The first extracellular domain of corticotropin releasing factor-R1 contains major binding determinants for urocortin and astressin. Endocrinology. 1998;139(2):566–570. 33. Ramsey SJ, Attkins NJ, Fish R, van der Graaf PH. Quantitative pharmacological analysis of antagonist binding kinetics at CRF1 receptors in vitro and in vivo. Br J Pharmacol. 2011;164(3):992–1007. http://dx.doi.org/10.1111/j.1476-5381.2011.01390.x. 34. Richards M, Rademaker M. Urocortins; 2013. 35. Rivier J, Gulyas J, Kirby D, et al. Potent and long-acting corticotropin releasing factor (CRF) receptor 2 selective peptide competitive antagonists. J Med Chem. 2002;45(21):4737–4747. 36. Turcu AF, Spencer-Segal JL, Farber RH, et al. Single-Dose study of a corticotropin-releasing factor receptor-1 antagonist in women with 21-hydroxylase deficiency. J Clin Endocrinol Metab. 2016;101(3):1174–1180. http://dx.doi.org/10.1210/jc.2015-3574. 37. Vale W, Spiess J, Rivier C, Rivier J. Characterization of a 41-residue ovine hypothalamic peptide that stimulates secretion of corticotropin and β-endorphin. Science. 1981;213:1394–1397.
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References
38. White S, Acierno R, Ruggiero KJ, et al. Association of CRHR1 variants and posttraumatic stress symptoms in hurricane exposed adults. J Anxiety Disord. 2013;27(7):678–683. http://dx.doi.org/10.1016/j.janxdis.2013.08.003. 39. Zhou L, Dockens RC, Liu-Kreyche P, Grossman SJ, Iyer RA. In vitro and in vivo metabolism and pharmacokinetics of BMS562086, a potent and orally bioavailable corticotropin-releasing factor-1 receptor antagonist. Drug Metab Dispos. 2012;40(6): 1093–1103. http://dx.doi.org/10.1124/dmd.111.043596.
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40. Zobel AW, Nickel T, Kunzel HE, et al. Effects of the high-affinity corticotropin-releasing hormone receptor 1 antagonist R121919 in major depression: the first 20 patients treated. J Psychiatr Res. 2000;34(3):171–181.
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6 Corticotropin-Releasing Factor and Urocortin Receptors D.E. Grigoriadis Neurocrine Biosciences Inc., San Diego, CA, United States
O U T L I N E Introduction57
Small Molecule Nonpeptide CRF1 Receptor Antagonists
62
Corticotropin-Releasing Factor and Urocortin Receptor Family Receptor Family Subtypes Endogenous Ligands
58 59 60
Clinical Experience and Relevance Central Nervous System Indications Endocrine Indications
62 62 63
Pharmacology of CRF1 and CRF2 Receptors Interaction of Peptide and Nonpeptide Ligands The Argument for Receptor Kinetics
60 60 61
Summary and Conclusions
63
References64
Abstract
INTRODUCTION
The key mediator of the innate physiological stress response has long been hypothesized to be the hypothalamic peptide corticotropin-releasing factor (CRF). This 41 amino acid peptide modulates the endocrine, autonomic, behavioral, and immune responses to stress through actions at specific receptors both in the central nervous system as well as the periphery. These seven transmembrane proteins belong to the superfamily of G protein–coupled receptors and play a role in central nervous system, cardiovascular, gastrointestinal, and pituitary-adrenal function. Decades of research have implied a direct relationship between stress-related disorders and CRF activity in the brain. As a result, this system has been the focus of many drug discovery programs aimed at identifying selective and specific orally active molecules that can block the pathophysiological increase in activity of this peptide. This review will summarize this system, the evidence supporting the utility of molecules that block the CRF system in human disease and the current state of the drug discovery programs that have yet to produce a viable therapeutic.
More than 2000 years ago, Hippocrates referred to the concept of homeostasis and equilibrium in health and disease as a dysregulation of this equilibrium. He further observed that there were individual differences in the severity of disease symptoms and that some individuals were better able to cope with their disease or illness than others. More than a half century ago, Hans Selye proposed that stress is the nonspecific (autonomic) response of a human body to any demand made upon it, real or perceived and that concept was broadened by Geoffrey Harris who first defined the role of the hypothalamus in the regulation of the pituitary– adrenocortical axis extending the notion that an organism’s ability to maintain homeostasis in the face of external stressors was coordinated by a specific portion
Stress: Neuroendocrinology and Neurobiology http://dx.doi.org/10.1016/B978-0-12-802175-0.00006-1
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© 2017 Elsevier Inc. All rights reserved.
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of the CNS operating in an automatic fashion. During the mid-1950s, Guillemin and Rosenberg, and Saffran and Schally independently recognized the presence of a specific factor in extracts of the hypothalamus that could stimulate the release of corticotropin [adrenocorticotropic hormone (ACTH)] from anterior pituitary cells in vitro. This extract was termed corticotropinreleasing factor (CRF). It was not until 1981, following the development of radioimmunoassays for ACTH and quantitative in vitro methods for assaying hypophysiotropic hormones along with the utility of ion exchange and high performance liquid chromatographic techniques, that enabled a team of Salk Institute scientists led by Wylie Vale to successfully isolate and purify the peptide CRF from sheep hypothalamic extracts.37 Thus, from the first basic principles in the understanding of an organism’s homeostatic response to stress that began centuries ago, this neuropeptide has been studied for its role in mediating the hypothalamic–pituitary–adrenal (HPA) axis and regulating the response to physical, emotional, and environmental stress. The discovery and structural elucidation of CRF ushered in a unique and focused hypothesis-driven KEY POINTS • C orticotropin-releasing factor (CRF) and the peptide family of urocortins activate two subtypes of Class-B G protein–coupled receptors (CRF1 and CRF2 receptors) and regulate the endocrine, autonomic, and behavioral response to stress. • Nonpeptide antagonists for the CRF1 receptor have been discovered that act allosterically and demonstrate efficacy in a wide range of animal models for stress behaviors. • The allosteric nature of the nonpeptide interaction requires a long residence time (slow dissociation) of the small molecule on the receptor for optimal efficacy in vivo. • Multiple small molecules have been examined in Phase 2 human studies for major depressive disorder, social anxiety, generalized anxiety, posttraumatic stress disorder, irritable bowel syndrome, and alcohol dependence. • Despite the tremendous body of evidence in animal models from different species providing an indication in human psychiatric disease, none of the CRF1 receptor antagonists to date have progressed beyond Phase 2 compelling the field to rethink the clinical application of these potent and selective compounds.
era of scientific study. While the initial understanding of its direct role at the pituitary pointed to the regulation of the endocrine stress response, it was the distribution of the CRF peptide in extrahypothalamic regions of the brain that intensified study into its role as a bona fide neurotransmitter and neuromodulator in the CNS. Multiple reports have detailed the anatomical distribution of both CRF-containing cell bodies and fibers in the CNS and more importantly, in regions of the brain shown to clearly regulate autonomic function and mediate the behavioral responses to stress. For example, in the cortex, high densities of CRF containing neurons that are localized primarily to prefrontal, cingulate, and insular areas, appear to regulate behavioral actions of the peptide. Many studies in rodents have demonstrated that exogenous administration of CRF into the CNS, has demonstrated direct interactions between the CRF system and virtually all monoaminergic neurotransmitter systems including the norepinephrine system in the locus coeruleus, the serotonergic systems in the dorsal and median raphe nuclei, and the dopaminergic system in the ventral tegmental area indicating that this system can impact multiple facets of the stress response of an organism. This critical convergent relationship between the hormone CRF and the monoaminergic systems in the brain further implicates this particular neuropeptide in mediating the central mechanisms through which various stressors can alter behavior. The cloning of multiple CRF receptor subtypes and the identification of a specific binding protein for the peptide (known as CRF-BP) have also enhanced the ability to dissect the behavioral consequences of activation or inhibition of this system. In addition to the receptors, the identification of endogenous-related family members of the CRF peptide (termed the urocortins) has expanded the notion that this system is under the control of multiple regulatory factors and plays a much more complex role in the CNS than previously thought. This chapter will focus primarily on the molecular biological, pharmacological, and functional characteristics of the CRF system and describe the recent challenges in the discovery and late-stage development of small molecule nonpeptide inhibitors of this system as potential therapeutics.
CORTICOTROPIN-RELEASING FACTOR AND UROCORTIN RECEPTOR FAMILY As a member of the superfamily of Class-B G-protein coupled receptors, the receptors for CRF and the urocortins have been shown to contain seven putative transmembrane domains and function through the
I. NEUROENDOCRINE CONTROL OF THE STRESS RESPONSE
Corticotropin-Releasing Factor and Urocortin Receptor Family
coupling of a stimulatory guanine-nucleotide–binding protein. These receptor subtypes all fall within the now well-described and still-growing, family of “gut-brain” neuropeptide receptors, which includes receptors for calcitonin, vasoactive intestinal peptide, parathyroid hormone, secretin, pituitary adenylate cyclase–activating peptide, glucagon, and growth hormone–releasing factor. While these receptors all share considerable sequence homology, they have been shown to activate a variety of intracellular signaling pathways. Cloning of the CRF receptor subtypes has enabled the pharmacological and biochemical characterization of these proteins and allowed the discovery of potential therapeutics through a variety of chemical screening strategies.
Receptor Family Subtypes The discovery of the CRF peptide led to the first radiolabeled form of the peptide that was used to probe the localization, distribution, and functional mechanisms of receptors in a variety of tissues from the brain and periphery in many different species. These studies served to define the physiology of the system and enabled a detailed investigation using molecular, biochemical, and behavioral tools to try and understand the role of this system in the normal and pathological state. In addition to the discovery of CRF, Vale and colleagues were the first to clone the CRF1 receptor in 1993 from a human Cushing anterior pituitary corticotropic adenoma using expression cloning.10 Several other groups later also identified the CRF1 form of the receptor from a variety of animal species. Interestingly, all species of CRF1 receptor mRNAs thus far identified encode proteins of 415 amino acids that are 98% identical to one another. In fact, the family of peptides has been well characterized in species ranging from insects to high-order vertebrates indicating the evolutionary importance of this system.24 The human CRF1 receptor gene contains at least two introns and is found in a number of alternative splice forms none of which to date have been found to have any physiological significance. Characteristic of most G protein–coupled receptors there are potential N-linked glycosylation sites on the large N-terminal extracellular domain and these molecularly identified sites confirm the glycosylation profiles determined previously using chemical affinity cross-linking studies. Indeed, the predicted molecular weight of the CRF1 receptor derived from deglycosylation studies was virtually identical to that obtained from the cloned amino acid sequence. Furthermore, mRNA distribution of the CRF1 receptor in the CNS of both rat and human tissues correlates extremely well with the previously identified biding sites for radiolabeled CRF using autoradiographic techniques in frozen
59
brain sections. Alternative splice variants of the CRF1 receptor have been described localized to discrete tissues. For example, a unique splice variant of the CRF1 receptor has been reported in human pregnant myometrium and while the pharmacology appears to be similar to the original subtype, the different signaling characteristics suggest that this isoform may be expressed under specific physiological conditions.14 Shortly following the cloning and characterization of the CRF1 receptor, a second subtype of this family was identified. This receptor (termed the CRF2 receptor subtype) exists as three individual splice variants termed CRF2(a) and CRF2(b) and CRF2(c). These receptor isoforms differ only in the extracellular region where each has a unique N-terminal sequence generating 411, 431, and 397 amino acid proteins for the CRF2(a), CRF2(b), and CRF2(c) receptors, respectively. Thus far, the CRF2(c) receptor has been identified exclusively in human brain and its function still remains to be determined. Comparing the CRF1 and CRF2 receptor subtypes, there are very large regions of amino acid identity, particularly between transmembrane domains five and six. This similarity indicates that biochemical action is conserved and these receptors act through G protein coupling and signal transduction. Detailed distribution and receptor autoradiographic studies have localized CRF-binding sites and CRF receptor mRNA, respectively, in slide-mounted sections of many tissues from a variety of species identifying these proteins in anatomically and physiologically relevant areas. Of primary interest, the CRF1 receptor is highly expressed in the pituitary gland, specifically in corticotrophs where it regulates the release of ACTH as a key mediator of the stress response. This discrete localization has facilitated the utility of selective receptor antagonists in disorders related to increased HPA activity. This will be detailed later in this chapter. While the CRF2 receptor has been documented extensively with respect to its pharmacology, its role in behavior and physiological function has been defined largely through its localization and the effects of the endogenous agonists. Specific chemical tools that block this receptor subtype systemically are few and restricted to peptide analogs (illustrated in the next section). In addition, the distribution of the CRF2 receptor is isoform-dependent. Specifically, the precise localization the splice variants, CRF2(a) and CRF2(b), indicates discrete anatomical distribution. The predominant form in the CNS is the CRF2(a) receptor where localization studies and those using peptide agonists have implicated a role for this subtype in anxiety and feeding behaviors. The CRF2(b) splice variant is localized almost exclusively in the CNS to nonneuronal elements, such as the choroid plexus of the ventricular system and cerebral arterioles. In the periphery, however, the CRF2(b) receptor is highly expressed in both cardiac
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and skeletal muscle with lower levels evident in both lung and intestine. Its role peripherally has been described in cardiovascular function and gastrointestinal motility. In fact, for a short period of time, the CRF2(b) receptor was the target of intense study for the treatment of acute decompensated heart failure (ADHF) owing to the very potent vasodilatory effects of the endogenous peptide urocortin 2. Studies performed in rats, sheep, and some initial studies in humans showed early promise as a potential treatment for ADHF.1,9 The CRF2(c) isoform has yet to be identified in the rodent, however, in the human brain, this isoform was shown to be highly expressed in the septum, amygdala, hippocampus, and frontal cortex.
Endogenous Ligands Since the elucidation of the amino acid sequence of CRF, the peptide was found to bear strong similarities to two nonmammalian peptides, sauvagine from the frog and urotensin I from the teleost fish. These mammalian and nonmammalian peptides all potently release ACTH from cultured rat pituitary cells directly through the CRF1 receptor in the anterior pituitary. One striking difference between these peptides was that only the nonmammalian forms retained a high affinity for the CRF2 receptor. Thus it became highly unlikely that CRF itself was the endogenous ligand for the CRF2 receptor in mammals. Using antibodies for fish urotensin I, the first mammalian peptide with high affinity for the CRF2 receptor was discovered and termed urocortin 1. This unique peptide discovered first in the rat was observed to retain its high affinity for the CRF1 receptor and the CRF-binding protein as well. Furthermore, the peptide was found to be localized in areas corresponding to the distribution of the CRF2 receptor itself. The human homolog of urocortin 1 was subsequently cloned from a human brain library and defined as the endogenous ligand for the CRF2 receptor. Since then, two other mammalian peptides have been identified from the mouse and human termed urocortin 2 (UCN2, also referred to as stresscopin-related peptide) and urocortin 3 (UCN3, also referred to as stresscopin). Urocortin two and urocortin 3, unlike urocortin 1, have much higher selectivity for the CRF2 receptor subtype and have little or no affinity for the CRF-binding protein. A great deal of literature now exists on the discrete and independent physiologic function of the urocortins and their activity on the CRF2 receptor subtypes (for a comprehensive review see Ref. 34). However, the current paucity of selective small molecule antagonists that could be administered chronically, hinders our ability to fully understand the role of the CRF2 receptor subtype in normal or pathophysiology. Nonetheless, major advances have been made in elucidating the function of these ligands and the CRF2 receptor in
anxiety, cardiovascular disease, inflammatory disease, and gastrointestinal and feeding disorders. Interestingly, there are no known endogenous physiological antagonists for any of the CRF receptor subtypes known to date. However, various truncations and modifications of the agonist peptides have resulted in potent peptide antagonists. A large body of work has been dedicated to identifying specific and selective peptide antagonists of these receptor subtypes.8,35 These ligands were designed to extend duration of action in vivo and demonstrated high affinity and activity for both the CRF1 and CRF2 receptor subtypes. Two of these synthetic highaffinity peptides, Antisauvagine-30, an N-terminally truncated form of sauvagine, and the cyclized Astressin-2B have been shown to be selective for the CRF2 receptor subtype and used to dissect out the specific role of this receptor in a variety of in vitro and in vivo studies. The pharmacological profiles of these peptide agonists and antagonists greatly enhanced the understanding of the physiology of the CRF system and the important role this neurohormone plays in mediating a variety of physiological and pathophysiological responses.
PHARMACOLOGY OF CRF1 AND CRF2 RECEPTORS The pharmacology of the class B GPCRs CRF1 and CRF2 have been described, characterized, and studied since the discovery of the peptide itself. Given the importance of the system and the desire to link human pathology to interventions that would have some therapeutic benefit, basic biochemical studies became focused on the distinct interactions of both peptide and nonpeptide ligands. As more and more ligand tools were developed, there was a clear understanding of the relationships between the peptide and small molecule interactions and the functional relationship between the two.
Interaction of Peptide and Nonpeptide Ligands The peptide interactions of the CRF family of ligands with the receptors has been extensively reviewed and reported to be the same for both receptor subtypes. Studies using the CRF1 receptor have posited that the C-terminal region of CRF adopts an α-helical conformation, which binds to the extracellular domain of the receptor. This initial low-affinity interaction then allows the binding of the N-terminal portion of CRF to a portion of the transmembrane domain that ultimately translocates the signal to function within the cell. An important concept of this mechanism is that this tethering of the N-terminal portion of CRF greatly increases its local concentration at the receptor. This two-domain binding of the peptide implies that the peptide CRF may remain tethered to the
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PHARMACOLOGY OF CRF1 AND CRF2 RECEPTORS
N-terminus of the receptor when the receptor is in its inactive state.3,16,29,32 This has direct implications when examining the abilities of non-peptide small molecule ligands to block the function of these receptors for therapeutic benefit. It is interesting to note that truncated forms of CRF such as D-Phe CRF(12-41) or the synthetic peptides Astressin or Astressin-2 bind with very high affinity to the N-terminus of their receptors but since they lack the ability to interact with the transmembrane domain, act as complete antagonists. Small molecule tools have enabled the understanding of the mechanisms of receptor blockade for this family of receptors. It is important to note from the outset that while there have been thousands of CRF1-specific small molecule receptor antagonists reported in the academic and patent literature, the tremendous effort that was put forth in identifying selective CRF2 receptor antagonists has yet to provide molecules that do not interact with the CRF1 receptor. This is an interesting scientific problem given the high degree of similarity of the two receptor subtypes especially in the transmembrane domain regions. The discovery and characterization of these small molecules will be described below. As will be illustrated in the next sections, these molecules were found to bind well within the transmembrane domain region of the CRF1 receptor with virtually no overlap in the binding sites of the peptides, thus defining these interactions as allosteric to each other. Therefore, any inhibition of CRF1 receptor function by nonpeptide small molecules that are allosteric to the peptide-binding domain must by definition be a result of a physical conformational change in the receptor protein rather than a simple blockade of peptide agonist binding. Furthermore, as postulated above, agonist peptides can be bound to the N-terminus of the receptor protein despite the existence of a small molecule occupying its binding
site in the transmembrane region. This has clear clinical and therapeutic implications. If the peptide is essentially tethered to the N-terminus, then it is positioned to signal through the receptor as soon as the small molecule dissociates from its binding site. Given these dynamics, the longer the small molecule can occupy the receptor and maintain its inactive conformation, the longer the therapeutic benefit. It became clear that in addition to the high affinity and selectivity these small molecules needed as potential therapeutics, they would also have to maintain the property of a long kinetic receptor off-rate to maximize their therapeutic efficacy.
The Argument for Receptor Kinetics Accounting for the receptor kinetics in the discovery of potential therapeutics is not a novel concept. This has been described in elegant detail for compounds blocking the angiotensin receptor in hypertension.20 Applying this concept to molecules that block the function of the CRF1 receptor allosterically, adds another dimension to the drug discovery and lead optimization process. Correlating the apparent affinity of the molecules with in vitro and in vivo efficacy became the critical criterion for progressing compounds into advanced stage development. As described above, the receptor occupancy and the pharmacokinetic and pharmacodynamic properties of these molecules all had to be optimized simultaneously. A great deal of work has been published in this area examining the relationships, both physical and experimental, of small molecule interaction that have led to their testing in large clinical trials.12,33,39 An illustration of this effort is shown in Table 6.1 using molecules that have in fact progressed into Phase 2 human clinical studies. Extracted from previously published reports,
TABLE 6.1 Comparison of the Properties of Select CRF1 Receptor Antagonists That Have Been in Human Clinical Trials for Anxiety or Depression Compound
Clinical Trials Identifier
[125I]-Sauvagine Binding Affinity (Ki nM)
Dissociation Time (t1/2 min)
Kinetic Affinity (Ki nM)
In Vivo ACTH (% Veh AUC @ 120 min)
CP-316,311
NCT00143091
1.9
4.1
12
76
Emicerfont/GW-876008
NCT00397722
20
10
39
87
Pexacerfont/BMS-562086
NCT00135421 NCT00481325
7.4
14
19
96
ONO-2333Ms
NCT00514865
1.2
17
15
78
Verucerfont/GSK-561679
NCT00733980 NCT01018992
1.3
70
3.9
43
NBI 30775/R121919
Ref. 40
2.6
130
0.36
42
Comparison of clinical compounds in their ability to inhibit [125I]-sauvagine, their dissociation times from the receptor and calculated kinetic affinity, and their ability to inhibit ACTH release in adrenalectomized rats. Clinical trials highlighted are those directly testing efficacy of these molecules for anxiety or depression only and listed on www.ClinicalTrials.gov with the exception of NBI-30775 (R121919), which was tested in an open label study in Major Depression. Multiple other exploratory clinical trials exist for each of these compounds. Data adapted from Fleck BA, Hoare SR, Pick RR, Bradbury MJ, Grigoriadis DE. Binding kinetics redefine the antagonist pharmacology of the corticotropin-releasing factor type 1 receptor. J Pharmacol Exp Ther. 2012;341(2):518–531. doi:10.1124/jpet.111.188714.
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6. CORTICOTROPIN-RELEASING FACTOR AND UROCORTIN RECEPTORS
Table 6.1 summarizes the most advanced chemical compounds compared using their binding affinity, dissociation time, the affinity determined via kinetic analysis, and the in vivo efficacy for inhibition of ACTH release. It is clear that all molecules had high affinity for the receptor as defined by [125I]-sauvagine binding. What is also clear is that if the compounds are rank ordered by their receptor off rates or dissociation times, this correlated very well with their ability to block the ACTH release from adrenalectomized rats. Coupled with the notion described above that compounds bind allosterically, and the fact that adrenalectomy causes an uncontrolled release of both CRF from the hypothalamus and ACTH from the pituitary, it is not surprising that compounds would have to have very long residence times on the receptor to effectively block the response of CRF in vivo. The data accumulated in Table 6.1 provides evidence for a link between ligand residence time, defined by receptor off-rate, and offset kinetics and insurmountable/ noncompetitive antagonism at the CRF1 receptor.
small molecules compared to the binding domains of the peptides.22 Regardless of the initial chemical starting points identified through large chemical library screening, when these molecules were ultimately modified into compounds that could be tested in the clinic, they all seem to bind to exactly the same pocket deep within the transmembrane domain of this receptor. In fact, this has now been clearly demonstrated in studies that solved the crystal structure of the CRF1 receptor bound to a typical CRF1 receptor antagonist.17 One inescapable conclusion from all these studies is that so far, this receptor has been shown to have a defined binding pocket for small molecules that can modulate its activity but more importantly, it will not tolerate structures that deviate from this well-defined pocket. As will be hypothesized below, if the current lack of progress of clinical candidates is due to the physical limitations of this particular binding site, then a complete revision of the mechanisms through which we can block this system will have to be understood and exploited.
SMALL MOLECULE NONPEPTIDE CRF1 RECEPTOR ANTAGONISTS
CLINICAL EXPERIENCE AND RELEVANCE
The discovery of small molecule receptor antagonists did not emerge from any advances in the peptide arena. Although peptide antagonists had been synthesized, their poor pharmacokinetics (oral bioavailability, rapid clearance, and minimal brain penetration) and general lack of CRF1 receptor selectivity did not yield any information useful in the development of nonpeptide blockers. Utilizing large screening libraries, many small molecule antagonists were developed and profiled for their pharmacokinetic and pharmacodynamics properties. The first disclosure of a small molecule CRF1 receptor antagonist was in 1991 and since then, numerous low-molecular weight ligands have been developed that potently bind and antagonize the CRF1 receptor (for detailed reviews see13,15,19). What is important to note is that the chemical starting points for optimization of these varied structures were as diverse as the chemical libraries amassed following years of small molecule research in the pharmaceutical industry. These structurally distinct, specific and selective CRF1 receptor antagonists were discovered through extensive screening methodologies utilizing these diverse chemical libraries and painstakingly optimized for drug-like characteristics.21 As eluded to above, these molecules were found to bind well within the transmembrane domain region of the CRF1 receptor with virtually no overlap in the binding sites of the peptides. First described in 1997, point mutation studies of the CRF1 receptor identified transmembrane domains three and five as critical points of interaction of the
The first publication of a CRF1 receptor antagonist used in the human population was a small open-label study in a group of major depression patients.40 This preliminary study energized the field and instigated a tremendous effort in the design and development of subsequent molecules. Multiple compounds were discovered and evaluated clinically for efficacy ranging from major depression, social anxiety generalized anxiety, posttraumatic stress disorder, and irritable bowel syndrome as well as studies in alcohol abuse. In these well-controlled placebo and many times comparator trials, the efficacy of a CRF1 antagonist has not been demonstrated. The clinical indications that have been examined have all been based on the following premise that CRF is the primary regulator of the stress response, and since these diseases have been reported to have a stress component in their etiology or manifestation, a CRF1 receptor blocker would be a viable therapeutic.
Central Nervous System Indications In the CNS, the stress axis has been implicated in playing a major role in depression and anxiety-related disorders and has been extensively reviewed.6,7,23 Many patients with major depression have been shown to have a perturbed HPA axis and are hypercortisolemic defined by an abnormal dexamethasone suppression test.5,18 Since it was demonstrated that CRF is the primary regulator of the stress response through the HPA
I. NEUROENDOCRINE CONTROL OF THE STRESS RESPONSE
Summary and Conclusions
axis in animals, it was a logical extension that hypersecretion or hyperactivity of CRF in brain might underlie the symptomatology seen in major depression or anxiety. Major clinical observations collectively provided overwhelming evidence that implicated the CRF system in the pathologic development of CNS diseases. For example, the first indication was the observation that the concentration of CRF was found to be significantly higher in the cerebrospinal fluid (CSF) of drug-free individuals with major depression28 and subsequently, it was found that the levels of CRF in the CSF were elevated while the CRF receptors were downregulated in the brains of suicide victims.2,27 Furthermore, this elevation could be reversed following successful treatment with electroconvulsive therapy.26 Since then, a myriad of studies have provided evidence for the role of CRF in depression and anxiety-related disorders.4,11,30 Beyond all of the animal studies where CRF1 antagonists have demonstrated efficacy in anxiety and depression behaviors, the now routinely available genetic analyses for disease states have offered a new approach in the selection of candidate populations with which to test the system.25,31,38 All of these data, evidence generated over the past three decades, created the overwhelming motivation to test this hypothesis in the clinic in a number of diseases of the CNS. To date however, no CRF1 receptor antagonist has progressed in development beyond Phase 2 proof-of-concept trials. A reevaluation of the hypothesis and specific disease-state or population is therefore warranted.
Endocrine Indications From a potential therapeutic perspective, while decades of study have focused on CNS and peripheral disorders like irritable bowel syndrome, the potential therapeutic indications at the level of the pituitary have been largely ignored. This is not entirely surprising or unexpected given the large potential patient populations with psychiatric disease, stress disorders, and immune and gastrointestinal disorders. A very small but unmet medical need does however exist for individuals whose HPA axis is chronically activated either through overexpression of hypothalamic CRF or genetic disorders that disrupt the axis. One example of such a disorder is congenital adrenal hyperplasia. This disease was first described in the 1950’s and is a complex genetic disorder in which the adrenals produce little or no cortisol. The most predominant form of this disease is a mutation in the 21-hydroxylase enzyme in the adrenal, which is directly responsible for the conversion of steroid precursors ultimately to glucocorticoids. This leads to a significant reduction in the negative feedback loop of cortisol at the level of both the hypothalamus and the pituitary, in effect causing substantial increases in both CRF and
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ACTH respectively. The elevated ACTH stimulates the adrenal steroidogenic pathways and without the ability to convert steroid precursors to cortisol, upstream precursors, such as 17α-hydroxyprogesterone, accumulate and generate high levels of androgens that are the root pathology of this disease. Current and most common treatment therapies are direct administration of glucocorticoids and are intended to not only replace the missing cortisol but also decrease the ACTH drive of androgen excess. In order to achieve this level of control, supraphysiological doses of glucocorticoids are required to effectively suppress adrenal androgens leading to issues associated with high steroid use. CRF receptor antagonists, which act at the level of the pituitary and can directly block CRF from stimulating ACTH release, may help in the overall treatment and well-being of these patients. By inhibiting the release of ACTH directly, the pressure on the adrenal to produce androgens should be lessened and administered cortisol may be reduced to physiologic replacement levels. A very recent clinical pharmacology study has been performed and shown some promising results.36
SUMMARY AND CONCLUSIONS Corticotropin-releasing factor has long been described as the primary regulator of the HPA axis and a target for the discovery of molecules that may have therapeutic utility in stress-related disorders. Despite heroic efforts in evaluating multiple CRF1 receptor antagonists in diseases such as major depressive disorder, generalized anxiety, social anxiety, posttraumatic stress disorder, irritable bowel disease, and stress in alcoholism (see www.Cinicaltrials.gov for details on numerous studies), none of the molecules examined have shown any clinical efficacy. While there may be fundamental issues with selection of the appropriate patient population, the specific design of the clinical trials, or the disease hypotheses themselves, it is evident that the tools we have currently at our disposal cannot demonstrate a therapeutic benefit in psychiatric disease. One common feature of these clinical trials (where actually measured) was that there was an effect on HPA activity. In this patient pool, there has been evidence of inhibition of ACTH release and this at least supports the utility of these agents as potential therapeutics in endocrine disorders where the primary requirement is modulation of ACTH release from the pituitary. The last three decades of intense study of this system should be examined with a critical view and the hypotheses modified to account for what the field has learned from these failures. CRF remains an important biochemical system both in the CNS and in the periphery and deserves a next-generation investigation.
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6. CORTICOTROPIN-RELEASING FACTOR AND UROCORTIN RECEPTORS
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I. NEUROENDOCRINE CONTROL OF THE STRESS RESPONSE