The CRF receptor: structure, function and potential for therapeutic intervention

Introduction

Corticotropin releasing factor (CRF) or hormone (CRH) is one of several neurohormones synthesized by specific hypothalamic nuclei in the brain and released into the portal system which bathes the anterior pituitary [1]. It also has marked CNS effects by acting at higher centers in the brain, particularly cortical regions where there is a widespread distribution of CRF neurons. The fundamental role of CRF is to prepare the organism for an appropriate response to various stressors such as physical trauma, insults to the immune system and social interactions [2]. It is the hyper- or hyposensitivity of the system that can lead to human pathologies such as anxiety, depression and feeding disorders [3].

Although its biological activity had been known from the early 1950’s, it was not until 1981 that CRF was isolated and purified from sheep hypothalamus and subsequently synthesized as a 41 amino acid peptide [4]. Now, CRFs from many species have been sequenced and the family of functional, naturally occurring peptides includes sauvagine (frog), urotensin I (fish) and a novel mammalian form of urotensin I called ‘urocortin’. CRF mediates its actions through high affinity binding sites in various tissues now known to be one of several variants of CRF receptors [5]. An additional high-affinity binding site is provided for by the soluble CRF-binding protein (CRF-BP) which is expressed predominantly in the brain and pituitary and is presumed to modulate the ability of CRF to activate the hypothalamic-pituitary-adrenocortical (HPA) axis as well as CRF-mediated neurotransmission by acting as a molecular sponge [6]. Radiolabeled versions of natural peptides, or close analogues have been used in radioligand binding and receptor autoradiographic studies, to study the distribution of CRF receptors and their role(s) in physiological processes. Two distinct sub-types of receptor encoded by separate genes have been described to date, CRF₁ and CRF₂, and they belong to the Class-B subdivision within the G-protein coupled receptor (GPCR) super family which also includes receptors for secretin, vasoactive intestinal peptide (VIP), calcitonin and glucagon amongst others. CRF₁ receptors positively regulate the accumulation of cAMP in response to CRF in both heterologously expressed systems and native tissues from brain and periphery and are therefore coupled to Gs as the major signal transduction mechanism [7,8]. The CRF₂ receptor has also been shown to couple through Gs and stimulate the production of cAMP but thus far only in heterologously expressed cell lines containing the human, rat or mouse forms [9-11]. To date there has been no report of the signaling characteristics of any of the isoforms of the CRF₂ receptor in any native tissue.

In this review we will present the structure-activity relationship of the CRF peptide agonists and antagonists as well as small molecule CRF₁ antagonists and how they interact with the two receptors. Since this review does not claim to be exhaustive, we apologize to those investigators whose work was not cited. Several other recent reviews which refer to such work and which complement the current article have appeared over the last two years [12-16]

Peptide Ligands for CRF Receptors

At the dawning of the Neuroendocrinology era, a substance subsequently termed corticotropin-releasing factor (CRF) present in the hypothalamus was proposed to be the primary regulator of adrenocorticotrophic hormone (ACTH) in the pituitary [17]. Once the elusive CRF molecule was identified, it engendered a plethora of studies to unravel the physiology and pharmacology of the CRF system. We now know that the CRF system in mammals encompasses at least two different ligands (CRF and urocortin), two receptors (CRF₁ and CRF₂) with functional splice variants for both and a binding protein [18].

While this manuscript was in press a novel selective CRF₂ receptor agoinst was cloned, characterized and termed ‘urocortin-II’ (Reyes et al. Proc. Natl. Acad. Sci. USA, 2001, 98, 2843). The selectivity and distribution of this novel mammalian ligand will offer new insights into the functional role of these molecules in stress-related physiologic or pathophysiologic conditions.

Primary Structure and Conformation of the CRF Peptide Family

When the structure of ovine hypothalamic CRF was first elucidated, the first surprising discovery was that its amino acid sequence is closely related to two other peptides, sauvagine and urotensin-I, which were identified at about the same time. Sauvagine was isolated from a skin extract of the South American tree frog Phyllomedusa sauvegei [19] while urotensin-I was isolated from the urophysis extract of the sucker (teleost) fish Catostomus commersoni [20]. When assayed in primary culture of rat anterior pituitary cells, all three peptides showed the same potency to release ACTH [21]. Additional hypothalamic CRF molecules were later identified from rat [22], human [23], goat [24], cow [25], pig [26], suckerfish [27] and xenopus [28] by chemical methods or cDNA cloning of their mRNA precursors. Because of the existence of urotensin-I as well as authentic CRF in the fish, this prompted many laboratories to search for the presence of other CRF-like molecules in mammals. In 1995 such a molecule was finally isolated when an urotensin-like molecule, now called urocortin, was characterized in the Edinger-Westphal nucleus of the rat brain [29]. The human homolog of urocortin was also identified from cloning of its mRNA from a human brain library [30]. Among the sub-family of hypothalamic CRF peptides, the absolute amino acid sequence homology is > 80%. Homology of rat/human CRF, which has the identical amino acid sequence, to other family members such as urotensin-I, urocortin and sauvagine are much less, ranging from 35 – 63% (see Table 1). Overall, homology of the CRF peptide family is more conserved at the N-terminus and C-terminus with identical amino acids at positions 4, 7, 9, 10, 15, 16, 31, 34 and 35, whereas the central portion of the molecule from residues 21 – 28 appears to be more variable. The conformation of several members of the CRF peptide family has been determined by Chou-Fasman theoretical prediction [31] as well as experimentally by CD measure-ments [32,33] and NMR analysis [33,34]. In aqueous solution the peptide exists largely as a random coil but in increasing concentrations of trifluoroethanol the peptide backbone tends to form one central a-helical secondary structure from residues 6/7 – 31/32 with a possible turn at residues 32 – 35 [33]. The N- and C-terminal regions of the peptide are not structured but there is a possibility for the C-terminal residues 36 – 41 of r/hCRF to form another helix. Therefore, it has been postulated that binding of CRF to its cell membrane receptor would transform the conformation of CRF to a a-helical form [35], which presumably is the biologically active form of the peptide. This hypothesis was substantiated by the higher affinity of a-helical CRF, in which a-helical preferring amino acids were substituted into the 8 - 32 region to enhance the a-helical structure of ovine CRF [36].

Structure – Activity Relationship of CRF Pep-tide Analogs

Since ovine CRF (oCRF) was the first mammalian CRF that was characterized, most of the early structure-activity studies carried out were based on this molecule. To locate the minimal amino acid sequence that possesses the full intrinsic activity, two series of deletion peptides were prepared, one starting from the N-terminus and the other from the C-terminus [36]. Deletion of the first three N-terminal amino acids did not diminish the intrinsic activity and potency of the resulting analogs on their ability to stimulate ACTH release from rat anterior pituitary cells compared to the parent oCRF. Further successive deletions of the residues from the N-terminal resulted in a gradual loss of potency but the resulting peptides maintained full intrinsic activity until deletion of the 8^th residue (leucine) because the resulting oCRF(9-41)NH₂ retains <1% the intrinsic activity of oCRF. This finding indicates that the 4 – 8 residues are important for activation of the receptor. However, oCRF(9-41)NH₂ still binds the rat pituitary CRF receptor with good affinity and incorporation of the a-helical preferring amino acids into the oCRF(9-41) sequence yielded the first useful antagonist of CRF, a-helical-CRF(9-41)NH₂[36]. By contrast, deletion of the C-terminal amino acids resulted in rapid loss of potency and intrinsic activity [37]. As a matter of fact, changing the C-terminal of oCRF to the corresponding carboxylic acid or deletion of just two C-terminal residues to yield oCRF(1-39)NH₂ rendered the resulting molecules inactive, losing 99.9% of the activity in comparison with oCRF [37]. Further deletion of the amino acids from the C-terminal were carried out with urotensin-I in another study [38], which showed that the resulting urotensin-I fragments still retained some minimal residual intrinsic activity until the fragment urotensin-I(1-19)NH₂ was reached, suggesting that the biologically active core of the molecule lies in the 1 - 19 sequence of urotensin-I.

Table 1. Amino Acid Sequences of CRF-Related Peptides

In an attempt to determine which amino acid side-chains of oCRF are most sensitive to alteration with respect to receptor binding and activation, three series of oCRF analogs were prepared and assayed by using rat anterior pituitary cells and rat brain membrane preparations. The first series consisted of substituting a single L-alanine residue into each position of the 5 - 40 sequence of oCRF [37]. Loss of potency was found in those analogs, in which alanine replaced the hydrophobic residues at the N-terminal 5 – 19 region of oCRF; and a gain of potency was noted in those analogs in which alanine replaced the hydrophilic residues at positions 22, 32, and 33. Of the 18 alanine-substituted analogs in the C-terminal 20 – 41 half of the molecule, 13 analogs had equal or higher potency than oCRF. In a related publication [39], a polyalanine substituted r/hCRF, with replacement of the native residues at positions 25, 26, 29, 32, 33, 39, 40 and 41 by alanine, exhibited similar activity as r/hCRF, confirming that the side-chain functional groups in those substituted residues have no bearing on activation of the receptor. In the second series of oCRF-derived peptides, each residue was replaced by a similar proteinogenic amino acid to maintain a minimal change of character at each position [40]. The analogs were assayed for their receptor binding affinity using [¹²⁵I]Tyr^o-oCRF with a rat brain membrane preparation. The results showed that amino acid residues in the N-terminal 5 – 17 region were most sensitive to alteration with loss of binding affinity, whereas residues in the non-conserved C-terminal 21 – 29, 32 – 33 and 36 – 41 regions could be replaced without significant loss of receptor affinity. Similar potency results as those found in the single alanine-substituted series were obtained in the ACTH-release assay using rat anterior pituitary cells for the proteinogenic amino acid-substituted series [40]. These data suggest that the side-chains in residues 5 – 19 are critical for receptor binding and activation, while the side-chains in residues of the C-terminal half are more important for binding structure conservation than for activation. In the third series, each residue from positions 5 – 41 of oCRF was replaced by the corresponding D-amino acid and the resulting analogs assayed for their ACTH-releasing potency [41]. The results showed that most of the analogs suffered a loss of activity except for the D-Phe¹² and D-Glu²⁰ substitutions. In addition, none of the analogs could antagonize the oCRF-induced ACTH release in rat pituitary cells. However, incorporation of the D-Phe¹²-substitution into several N-terminal deleted fragments of oCRF was able to enhance the antagonistic potency of the resulting analogs. This substitution together with replacement of the two methionine residues at positions 21 and 38 of h/rCRF by norleucine resulted in a very potent antagonist, [D-Phe¹², Nle^21,38]h/rCRF(12-41)NH₂, which is 15 times more active than a-helical-CRF(9-41)NH₂[36]. This antagonist was employed subsequently as a standard in many studies.

To further increase the potency of [D-Phe¹², Nle^21,38]h/rCRF(12-41)NH_2,selected alanine and leucine residues in the 12 - 41 region were replaced individually by their respective C^a-methyl derivatives in order to maximize the a-helical potential of the molecule [42]. The reason behind this strategy stems from the fact that the design of the first potent CRF antagonist, a-helical-CRF(9-41)NH₂, was based on the assumption that the preferred biologically active form of the molecule assumed an a-helical structure in residues 8 - 32. These a-helix inducing amino acids could also prevent degradation of the peptide by peptidases. The mono-substituted C^a-methyl analogs were found to be 2 – 0.25 times as potent as [D-Phe¹², Nle^21,38]h/rCRF(12-41)NH₂in vitro but, interestingly, one analog, [D-Phe¹², Nle^21,38, (C^a-Me)Leu³⁷]h/rCRF(12-41)NH₂,was found to be more potent and longer acting than the parent compound in two in vivo assays, measuring ACTH release after intravenous administration to adrenalectomized rats and reversal of stress-induced delay in gastric emptying in the rat after intracisternal administration [42].

In another approach to develop more potent antagonists by restricting the molecule to a a-helical conformation, a series of cyclic analogs formed by incorporating a glutamic acid and a lysine/ornithine into the sequence, followed by closure of the incorporated side-chain carboxylic acid and amino moieties to form a lactam ring was prepared [43,44]. These lactam ring-restricted analogs were tested for their ability to inhibit the oCRF-induced release of ACTH by rat anterior pituitary cells. The results revealed that an i – (i+3) bridge, consisting of the cyclo(Glu-Xaa-Xaa-Lys) scaffold gave the most potent molecules, such as cyclo(20-23)[D-Phe¹², Glu²⁰, Nle^21,38, Lys²³] h/rCRF(12-41)NH₂ and cyclo(30-33)[D-Phe¹², Nle^21,28, Glu³⁰, Lys³³]h/rCRF(12-41)NH₂[45], which are 2- and 32-fold more potent than the standard [D-Phe¹², Nle^21,38]h/rCRF(12-41)NH₂, respectively. The latter compound designated as astressin (see Table 1) also exhibited negligible intrinsic activity in vivo and low binding affinity to the CRF-binding protein but high affinity (Ki, 2 nM) to the cloned CRF₁ receptor [45].

The lactam ring scaffold was also introduced into a series of CRF agonists derived from the parent molecule [Pro⁴, D-Phe¹², Nle^21,38]Ac-h/rCRF(4-41)NH₂[46]. Acetylation of the N-terminal generally increases potency by a factor of 2 – 3 fold with no effect on intrinsic activity. In addition, it was determined that introduction of a lactam ring at the Glu³⁰-Xaa-Xbb-Lys³³ region of the CRF molecule greatly enhanced its agonist activity in comparison with the corresponding linear analog when the sequence is progressively shortened from h/rCRF(4-41) to h/rCRF(8-41) [46]. Further deletion of the 8^th residue leucine in h/rCRF resulted in total loss of intrinsic activity, suggesting that residue 8 is critical for activation of the receptor. This is consistent with the original finding that deletion of the first 8 residues in oCRF resulted in an antagonist. Additional improvement of agonistic and antagonistic activity was found by incorporating a D-amino acid at the 3^rd position of the lactam ring cyclo(Glu-Xaa-D-Xbb-Lys) [47] and, together with the introduction of a (C^a-Me)Leu at position 27, resulted in cyclo(30-33)[D-Phe¹², Nle^21,38, (C^a-Me)Leu²⁷, Glu³⁰, D-His³², Lys³³]Ac-h/rCRF(9-41)NH₂ and cyclo(30-33)[D-Phe¹², Nle^21,38, (C^a-Me)Leu²⁷, Glu³⁰, Lys³³]Ac-h/rCRF(9-41)NH₂. These two CRF antagonists are more potent than astressin at reversing intracisternal CRF-induced and abdominal surgery-induced delay of gastric emptying in conscious rats [46]. Also, since C^a-methylation at other positions may favor a bioactive conformation while also preventing degradation and/or elimination, introduction of another (C^a-Me)Leu into the design of antagonists to yield cyclo(30-33)[D-Phe¹², Nle^21,38, (C^a-Me)Leu^27,40, Glu³⁰, Lys³³]Ac-h/rCRF(9-41)NH₂ resulted in the most efficacious antagonist to date and this compound was designated as astressin B [48].

So far, all of the CRF peptide antagonists synthesized were “mixed antagonists” with similar affinity and potency towards the two CRF receptors. However, recently it was reported that truncation of the first ten N-terminal amino acids of sauvagine and with substitution of the leucine and glutamic acid at the 11^th and 12^th positions by D-phenylalanine and histidine, respectively, resulted in a potent antagonist, [D-Phe¹¹,His¹²] sauva-gine(11-40)NH₂, exhibiting a 100-fold selectivity for CRF₂_b over CRF₁ [49]. This CRF₂-selective antagonist was named antisauvagine-30 (Table 1) and used in many studies to explore the pharmacology of CRF₂ [50].

Up to now, the design of potent CRF peptide analogs was based on the assumption that interaction of the CRF peptide with its receptor required that the linear CRF peptide sequence existed in a a-helical conformation, involving residues 6/7 – 31/32 with a fixed distance between the N- and C-terminal regions of the peptide for binding with two separate receptor sites. Recently, however, this concept was challenged [39]. While the existence of two receptor binding sites for CRF, comprising N-terminal residues 1 – 20 and C-terminal residues 34 – 41, was substantiated in their studies, they also showed that the connection of the N- and C-terminal regions need not be restricted in a a-helical conformation dictated by the peptide backbone. Instead, connection of the two binding sites in the peptide ligand by highly flexible linkers resulted in CRF analogs with full, albeit weak agonistic activity independent of linker length. They proposed that the a-helical conformation serves only to restrict the relative orientation of the two binding sites in CRF to enhance the potency of the peptide rather than maintaining a fixed distance between them. This concept could point to a new way for the design of novel, shortened peptide analogs for ligand-receptor interaction studies.

Structure and function of CRF receptors

The Major Sub-Types - CRF₁ and CRF₂

It is now known that CRF exerts its actions by interaction with one of two distinct subtypes of G-protein coupled receptors (GPCRs), each being encoded by separate genes, and which fall into the second category (‘Class B’) of this gene superfamily [18]. Various forms of these two CRF receptors arise from different splice modifications and also differ both in their anatomical location and their response to peptide ligands (Table 2). As represented by the traditional ‘snake plot’ [51] (Fig. 1), amino acid changes between the two major sub-types are spread throughout all domains but with particular diversion within the extracellular regions thought to be the initial peptide ligand binding region prior to receptor activation. The most striking sequence similarity occurs between transmembrane domains 5 and 6 and the connecting intracellular loop as anticipated by the two receptors’ common signal transduction mechanism. The most notable difference in their behavior to natural peptide agonists, is the lower binding and potency of CRF for CRF₂ receptors with the affinity for CRF₁ being at least 10-fold higher [52]. Mammalian CRF₁ is nonselective for hypothalamic CRF isolated from all species, sauvagine, urotensin-I and urocortin [53]. All of these peptides bind the CRF₁ receptor subtype with similar affinity and are equipotent in their ability to stimulate intracellular cAMP production [54]. Xenopus CRF₁ is the only ligand-selective receptor reported [55], which binds r/hCRF, xCRF, urotensin-I and urocortin with higher affinity than oCRF and sauvagine. By contrast, CRF₂ from all species binds urocortin, urotensin-I and sauvagine with much higher affinity than human and ovine CRF [53]. Considering that urocortin is expressed in brain regions that overlap with CRF₂ expression, it has been postulated that urocortin is one of the natural ligands for CRF₂ receptors whereas CRF is the natural ligand for the CRF₁ subtype [29].

Splice Variants of CRF-R

There is sporadic evidence for a splice variants of the CRF₁ with the first report for a ‘CRF₁_b’ accompanying the original cloning of CRF₁ from a human corticotropic tumor library [56]. This receptor had a 29 amino acid insert into the first intracellular loop (see Figure 2), and had both reduced affinity and efficacy in its interaction with CRF. A second splice variant (CRF₁_g) exists in human hypothalamus (and elsewhere) in which the exon encoding the mid-region of the N-terminus has been deleted [57]. However this receptor fails to bind CRF with high affinity and requires heroic concentrations of CRF to elevate cAMP in COS-1 cells (EC₅₀ 300 nM; c.f. CRF₁, EC₅₀ = 0.55 nM). Another CRF₁ variant (‘CRF₁_d’) has also been identified in human pregnant myometrium and fetal membranes arising from an exon deletion normally encoding a 14 amino acid sequence towards the C-terminus of the putative 7^th helical region [58]. Although binding of CRF to this variant remained essentially unchanged when compared to CRF₁_a the receptor was only poorly coupled to G_s so that CRF behaved as a weak, partial agonist. Whilst there is uncertainty about the physiological significance of these splice variants, these data may give some insight intodownstream from the 7^th helical domain are important for signal transduction whereas the N-terminal domain may be more concerned with formation of the first ‘collision complex’ between ligand and receptor.

Table 2. Characteristics of Human and Rat CRF Receptor Subtypes

Also, for Gs-linked GPCRs in general, agonist-dependent desensitization has been shown to involve phosphorylation of residues, typically Ser or Thr, in the C-terminal domain and/or IC3 – a process which for hCRF₁ seems to be driven by a G-protein receptor kinase (GRK3) rather than PKA or calmodulin pathway [59]. Whether or not any of these isoforms exist in vivo rather than simply products of RT-PCR remains to be established since the necessary mRNA localization experiments have yet to be reported.

Fig (1). Snake plot representation of CRF receptors showing the sub-type differences and the various splice forms of the CRF₂ isoform. (Where differences occur, CRF₁ residues are to the right and CRF₂ residues to the left).

Splice variants of CRF₂, on the other hand, are more widespread both in terms of tissue distribution and across species. In comparison with the first CRF₂ to be cloned (CRF₂_a) [52], two of the other three forms have the N-terminal 34 residues replaced by a new 54 (CRF₂_b) or 20 (CRF₂_g) amino acid sequence respectively. Human CRF₂_b was shown to prefer urocortin binding to sauvagine (5-fold) and then CRF (30-fold) again suggesting that urocortin may be the natural agonist [52,60]. The role of the receptor N-terminus is again apparent given that urocortin bound 10 times more tightly to the CRF₂_b variant. A severely truncated form of CRF₂_a, CRF₂_a-tr, from rat amygdala, thalamus and hypothalamus where levels of mRNA for CRF₁ and CRF₂_a are undetectable, is composed of the N-terminal 236 amino acids terminating mid-way up the fourth transmembrane domain [61]. This receptor bound rCRF with identical affinity to CRF₂_a but not sauvagine or urocortin and did not cause accumulation of cAMP. For CRF₂, this points to a dominant role for the N-terminal region and first extracellular loop in binding CRF (again, not activation) to the receptor and suggests that the preferred ligand, urocortin, has additional productive interactions with other extracellular elements. CRF₂_b and CRF₂_g are expressed in completely different brain regions [62] and are therefore also distinct from CRF₂_a. Nevertheless, the pharmacological profile of all three isoforms is similar since urocortin preference is maintained throughout.

Fig. (2). Topology of CRF receptors. A, Domain definition; B, Function of various domains suggested by studies with CRF receptor splice variants.

From studies with the various isoforms of the CRF receptor alone, the following may be concluded: First, the N-terminal domain together with the first extracellular loop (Fig. 2) form the initial ligand recognition site producing a complex likened above to the ‘collision’ complex between an enzyme and its substrate prior to development of the transition state. The C-terminus in concert with the third intracellular loop is the key element required for signal transduction. Finally, extracellular domains 2 and 3, at least in the CRF₂ receptor probably provide additional binding sites for urocortin. This supports the idea that the family of natural CRF peptide agonists may have different, albeit overlapping, binding sites.

Mutational Studies with CRF Receptors

All mutational studies reported to date have relied upon hydropathy profiles to predict the putative helical, transmembrane domains of the receptor [51]. Such plots, whilst representing a ‘good start’, do not accurately predict such domains for the (bovine) rhodopsin protein, the details of which can now be seen from the recently published crystal structure [63]. With the assumption that rhodopsin truly is an adequate homology model on which to base predictions for GPCRs, both the position and the length of each near-helical domain has to be revised – in turn, this has some impact of interpretation of both chimeric and single point mutational studies with CRF receptors (Table 3). In particular, transmembrane domains 1, 2, 3 and 6 will have to be significantly lengthened in line with the observed values in rhodopsin. Another finding is the role of the ubiquitous disulphide bridge (Cys110-Cys187 in rhodopsin), one of several ‘fingerprints’ within the GPCR superfamily, between the second and third extracellular loops. In rhodopsin, Cys110 is close to the extracellular surface of TM3 and places the EC2 loop in such a position to impede access of ligands to the helical bundle. It is this molecular feature, the Cys-Cys disulphide bond, that is a key ‘landmark’ in CRF receptor structure since others, such as the D(E)-R-Y sequence at the intracellular boundary of TM3, are apparently missing from type B GPCRs in general. A closer look at type B receptors reveals that a D(E)-R-X counterpart may exist if one assumes that a glutamic acid residue towards the cytosolic boundary of TM3 (E-209 in CRF₁) and an arginine at the N-terminus of TM2 (R-151 in CRF₁), both conserved throughout the class B sub category, are spatially positioned to fulfill this role.

Table 3. Location of the Various Domains of CRF₁ Compared with Bovine Rhodopsin

Studies with several GPCRs have demonstrated that the integrity of conserved cysteine residues is essential for ligand binding (extracellular cysteines) [64] and receptor expression, activation and internalization (intracellular and trans-membrane cysteines) [65,66]. Particularly important are the two cysteines in the first and second EC loops (C188 and C258 in CRF₁) which, with only a few exceptions, is conserved throughout the GPCR superfamily forming a disulphide bond (C110-C187 for rhodopsin) clearly visible in the crystal structure of rhodopsin [63]. For rhodopsin, and for the GPCRs having a short N-terminal domain such as the GPCRs for monoamines, only this extracellular S-S bond is conserved and clearly has a profound effect on the accessibility of the helical domains to small molecule ligands since it places the EC2 at the extracellular surface of and, indeed partially penetrates the helical bundle as noted above. As will be highlighted later, since many small molecule antagonists of CRF₁ are highly lipophilic, perhaps a more favored approach requires first dissolution of such ligands in the lipid membrane surrounding the receptor followed by receptor occupancy. This same disulphide bridge in the CRF₁ may be implied from both single and double mutational studies [67]. High affinity CRF binding was not observed in the C188S, C258A or C188S/C258A mutants although the double mutant remained fully coupled to G_s possibly suggesting that a reorganization of disulphide bonds is a necessary step towards receptor activation. Although not definitive, it was proposed that the pattern of disulphide bonds for the CRF₁ is C44-C102, C68-C87 and C188-C258.

Fig. (3). Chimeric forms of CRF₁ with CRF₂. Shaded areas are the CRF₂ sequence. (EC₅₀ value for CRF-induced cAMP accumulation).

Chimeric constructs of CRF₁ and CRF₂_a have been studied in order to pin point regions that are differentially involved in agonist binding given the 10-100 fold preference of CRF₁ over CRF₂ for CRF. Since it had been shown that there is good correlation between potency (as measured by EC₅₀ for stimulation of cAMP) and affinity (as measured by K_i in competition binding experi-ments), it was assumed that changes in EC₅₀ for the chimeric proteins corresponded to changes in binding [68]. Beginning with the wild type CRF₁ (EC₅₀ = 0.16 nM) and progressively increasing its CRF₂ composition (Fig. 3), caused a 15-fold shift in EC₅₀ at the stage of the R₁243R₂ chimera and a second jump at the level of the R₁166R₂ chimera to mimic the behavior of wild type CRF₂ (EC₅₀ = 58 nM). From this and other point mutation data, it was concluded that two regions of the receptor are important for r/hCRF binding to CRF₁ – the junction of the second EC loop with TM5 and, secondly, the first EC loop. Data recorded for the His189 mutant are superfluous since it has now been shown in many species that both CRF₁ and CRF₂ receptors have a conserved Arg at this position [69]. Note that this residue is critically placed adjacent to the cysteine (C188) known to be an important structural determinant as described above. The same two regions also affected the binding of urocortin and sauvagine, albeit to different extents. In addition, a third distinct locus, Asp254 in EC2, was identified to be important for sauvagine but not r/hCRF or urocortin binding [70]. Thus, the three ligands, r/hCRF, urocortin and sauvagine, not only interact with a different set of regions in CRF₁ and CRF₂ receptors but also differently with some of the same regions. These data could at least in part account for the much higher affinity of CRF₂ for urocortin and sauvagine compared with r/hCRF. However, since the reciprocal mutations, where the corresponding critical amino acids of CRF₂ were mutated to the amino acids of CRF₁, were not carried out, the decrease in binding/activation observed in the CRF₁ mutants could result from changes in receptor conformation rather than loss of ligand contact in the mutants. Therefore, the issue whether the EC1 and EC2 domains in CRF₁ receptors are important for CRF peptide agonist binding and activation remains to be confirmed.

Fig. (4). Structures of NBI 27914 (left) and Antalarmin (right)

In the same study [70], two residues that were implicated in binding of the non-peptide pyrimidine antagonist, NBI 27914 (Fig. 4) but, importantly, not r/hCRF, were identified. Since this molecule is highly selective for CRF₁ receptor (K_i = 3.5 nM) over CRF₂ (K_i = > 1000 nM), point mutations were made to CRF₁ to the corresponding CRF₂ residues to give a series of mutant receptors, which had approximately the same EC₅₀ for sauvagine-induced stimulation of cAMP as the native receptor. The two point mutations within the helical domains, H199V (TM3) and M276I (TM5), caused greater than 100-fold reduction in NBI 27914 binding suggesting that these (and other as yet undetermined) residues are involved in small molecule antagonist binding. Perhaps coincidentally, an un-optimized model of the receptor based on rhodopsin structure predicts a separation of around 15 Å between these two residues approximately the same distance which separates the two lipophilic regions in NBI 27914 (the trichlorophenyl ring and the di-alkylamine).

Fig. (5). Chimeric CRF₁ with GHRH receptors. Shaded areas correspond to the contribution to the chimera of the GHRH receptor sequence. Thus wild type rCRF₁ is shown as the first graphic. K_i values for homologous displacement of Astressin (A) or Urocortin (U) are listed below each schematic. -- appears, where specific binding was too low to measure accurately.

To further probe the ligand-binding domains residing in CRF₁, several studies involving construction of chimeric receptors that incorporate amino acid sequences from other class B G-protein coupled receptors into the CRF₁ background or vice versa have been conducted [71-74]. For the most part, hydropathy profiles were used to predict the TM domains, and hence the regions corresponding to the NT and EC loops need to be interpreted with the obvious limitations discussed above. Thus, these ‘loop’ regions may contain several residues now thought to be part of the helical bundle. The results obtained showed that the N-terminal domain of CRF₁ was critical for binding to all CRF peptides and this observation is in agreement with the finding in other class B G-protein coupled receptors [75-77]. An exhaustive range of chimeras constructed with rat growth hormone-releasing hormone (or factor) receptor (rGHRHR) and rCRF₁ was tested for binding against the radiolabeled peptides assuming that the agonist, urocortin, and the peptide antagonist, astressin, maintained both their binding affinity and (for urocortin) efficacy when chemically altered during the radiolabeling process [71]. Thus replacement of NT domain in the rCRF₁ with the corresponding N-terminal domain of rGHRH receptor resulted in a chimeric receptor that did not bind radiolabeled [Tyr⁰]-urocortin nor [D-Tyr¹]-astressin (Fig. 5). The complementary chimera, in which the N-terminal domain of rGRF receptor was replaced by the corresponding N-terminal domain of rCRF₁ receptor, bound the labeled urocortin and astressin with only a 5-fold decrease in affinity compared to the wild-type rCRF₁. Moreover, the chimera, in which the N-terminal domain of the activin IIB receptor (a Ser/Thr kinase growth factor receptor with only one transmembrane domain) was replaced by the N-terminal EC1 domain of rCRF₁, was able to bind astressin (K_i = 3.5 nM) but not urocortin with similar affinity as rCRF₁ again suggesting that urocortin has ancillary binding sites with other EC domains. The presence or absence of individual EC loops, or combinations of two or all three in addition to the NT caused a maximal 10-fold change in affinity indicating that no key determinants for binding exist outside the NT domain.. Unfortunately, whether any of these receptors maintained function was not reported.

These findings were substantiated in another study, which demonstrated that the soluble N-terminal domain of rCRF₁ expressed in E. coli bound oCRF specifically, albeit with low affinity (IC₅₀ = 6.8 mM) [78]. Receptor constructs with successive elongation of the N-terminal domain towards the C-terminus of rCRF₁ receptors when expressed in transfected cells, bound oCRF specifically but still with micromolar affinity. Only after the TM7 domain was added was the affinity significantly improved (IC₅₀ = 61 nM) as well as the ability to again stimulate cAMP accumulation by oCRF in the transfected cells. These results indicate that the region responsible for binding of the CRF peptides, both agonists and antagonists, to CRF₁ is located in the N-terminal domain of the receptor. Moreover, for high affinity binding and activation by CRF agonistic peptides the EC3 domain in addition to the N-terminal domain is required. The EC3 domain probably interacts with the N-terminal residues 1 – 11 of CRF to transduce the activation signal since CRF peptide antagonists lacking those residues are unable to activate the receptor even though they exhibit high binding affinity [49]. Interestingly, there was little difference in the EC₅₀ values between mutants having either none or 46 amino acids deleted from the C-terminus suggesting that the region immediately adjacent to TM7 is the vital region for interaction with G-protein.

By replacing the putative EC3 of rCRF₁ with the corresponding loops from either the PACAP or glucagon receptors, it was shown that this region is involved in both binding and activation. Accordingly, both mutant receptors failed to bind oCRF with high affinity (K_D ~ 2 mM) and both were uncoupled from cAMP production. Furthermore, the amino acid residues in the EC3 domain responsible for receptor activation were tentatively identified as Tyr³⁴⁶, Phe³⁴⁷ and Asn³⁴⁸ in CRF₁ [73]. The mutant rCRF₁ receptor with all three amino acids changed to Ala displayed reduced binding (K_D = 64 nM) and oCRF behaved as a weak partial agonist (EC₅₀ = 32 nM; 15% efficacy; c.f. with wild type receptor, EC₅₀ = 0.3 nM). To determine the amino acid residues in the N-terminal domain of CRF₁ receptors responsible for binding to the CRF peptides, mutant receptors were constructed [74] by replacing specific residues in the hCRF₁ with amino acids from the corresponding positions in the N-terminal domain of human vasoactive intestinal peptide (VIP) receptor type-2. Two regions in the NT domain of hCRF₁, one mapped to residues 43 – 50 and the second from residues 76 – 84, were found to be important for binding of CRF peptide agonists and antagonists as well as activation by CRF peptide agonists. The second region was also found to be responsible for determining the CRF peptide ligand selectivity of Xenopus CRF₁ receptors [72].

The strategy based on construction of chimeric receptors derived from human and Xenopus CRF₁ and CRF₂ was also used to elucidate the ligand-binding domains residing in CRF₂ [79]. Chimeric receptors, in which the N-terminal domain of either human or Xenopus CRF₂ replaced the NT domain of hCRF₁, bound all CRF peptides non-selectively with high affinity, whereas chimeric receptors, in which the NT domain of either the hCRF₂_a or xCRF₂ replaced the NT domain of xCRF₁, bound the CRF peptides with significantly lower affinity. Chimeric receptors, incorporating the NT domain of xCRF₁ linked to either hCRF₂ or xCRF₂, showed a similar pharmacological profile as the two parent CRF₂ proteins, indicating that the peptide ligand-selective domains in CRF₂ reside in EC1, EC2 and EC3. Chimeric receptors incorporating the NT domain of xCRF₁ linked to either hCRF₂ or xCRF₂ exhibited a novel pharmacological profile. This chimeric receptor bound r/hCRF, urocortin and sauvagine with higher affinity than oCRF. Furthermore, when three or five residues in the NT domain of xCRF₁ (Gln⁷⁶, Gly⁸¹and Val⁸³ or Gln⁷⁶, Gly⁸¹, Val⁸³, His⁸⁸ and Leu⁸⁹, respectively) were introduced into chimeric receptors bearing the N-terminal domain of hCRF₁ linked to xCRF₂, the same novel pharmacological profile was observed. These data indicate that a compensatory mechanism exists in at least two different EC domains, NT and EC1/EC2/EC3, for formation of the binding pocket to activate xCRF₁ and xCRF₂. Further mutational studies should shed more light on the interaction of the CRF peptide ligands with their receptors.

For CRF peptide agonists, the essential function of the N-terminal domain in the CRF receptor is to capture the CRF peptide ligand at its C-terminal region, followed by proper orientation of the ligand’s N-terminal region to activate the receptor at the EC domains in the seven transmembrane region. This concept was validated by preparation of a constitutively activated hCRF₁ involving a tethered CRF peptide reminiscent of the thrombin receptor system [80]. Thus a CRF₁ chimera, in which the N-terminal domain (approximately) corresponding to residues 1 – 111 of hCRF₁ was replaced by the N-terminal r/hCRF(1-16) sequence, had a 25-fold increase in ‘basal’ levels of cAMP production and a 20-fold increase over wild type receptors where the basal levels were determined in the presence of 10 mM of the non-peptide, CRF₁ selective antagonist, antalarmin (Fig. 4). As would be expected from a model which predicts a different binding site for peptide (NT and EC domains) and non-peptide (TM regions predominantly) antagonists, the peptide antagonist, astressin, did not inhibit constitutive activation – astressin clearly was not able to compete with the intramolecular tethered ligand. Furthermore, when the same leucine at position 8 of CRF that is critical for potency at the wild type receptor (Leu8Ala is 300-fold less potent than CRF; see above) is mutated to alanine in a tethered peptide, the resulting protein is no longer constitutively active even though the receptor itself could still be activated with urocortin. These results are truly remarkable since it can only be by chance that the tethered peptide could pick out the correct conformation to activate the receptor – a conformation which is presumably induced only after the usual initial binding event has taken place through interaction of the ligand with the NT of the wild type receptor. It is likely that, by exploring the separation between CRF(1-16) and the receptor, an even higher level of constitutive activity will be discovered. This finding is in agreement with the results obtained from the study involving a series of successive deletion of the amino acids at the N-terminal of oCRF [36].

Interaction of non-peptide, small molecules to CRF receptors

During last 10 years several non-peptide ligands have emerged as potent CRF₁ receptor antagonists. Despite the large number of patents disclosing these ligands, few publications describing structure-activity relationships have been reported. On the other hand, little is known about non-peptide ligands for CRF₂ receptor antagonists, CRF-BP receptor antagonists and CRF₁ / CRF₂ receptor ligands. To simplify the discussion, ligands are presented in order of increasing complexity of the central core feature.

Monocyclic Series

Five Membered Rings

The first reported non-peptide, small molecule CRF₁ receptor antagonists were a series of oxopyrazoline thiocyanates [81], represented by structure 1, which have weak binding affinity (IC₅₀ values in the range of 3-70 mM). Since then, several series of monocyclic five-member ring systems have been developed with perhaps the most potent being based on a thiazole core. All preferred compounds [82,83] also contained a propyl group on the side chain. This propyl group might be playing a role in positioning the nitrogen (hydrogen bond acceptor) for optimal binding affinity. Furthermore, it also appears that a bulky side chain, such as dicyclopropyl or quinoline, is necessary for optimal binding affinity (compare compounds 2 (K_i 10 nM) and 3 (binding IC₅₀ 80 nM) to 4 (K_i 370 nM) [84]). SAR also established the need for a twisted orientation between the planes of the phenyl ring and thiazole ring for high binding affinity.

Other five-membered rings have been described in patent applications with limited biological data [85-88]. Preferred compounds listed in such patents would suggest that an ethyl substituent (structure 5, no biological data) adjacent to the pyrazole nitrogen (hydrogen bonding acceptor) or its bioisostere, thio-methyl, is optimal for higher affinity (compare compounds 6 K_i = 15 nM and 7 K_i = 11nM with compound 8 K_i = 1500 nM). It should be noted that whilst several of these compounds also contain a basic nitrogen (i.e. partially protonated at physiological pH) on the side chain, it is unlikely that this feature contributes to a productive electrostatic interaction with a cationic counter ion on the receptor .

Recently a Japanese patent [89] described highly lipophilic thiazoles (represented by structure 9) as CRF₂ receptor antagonists albeit with no biological data. Most of the compounds claimed in the patent also contained a basic nitrogen on the side chain. This close similarity between the CRF₁ and CRF₂ receptor antagonist core structures as well as the close homology between both receptors postulate that the discovery of dual CRF₁ /CRF₂ antagonists is on the horizon.

Six-Membered Rings

Using different approaches, two research groups independently developed 2-anilinopy-rimidines and triazines as CRF receptor antagonists. Optimization of a screening lead [90] (Structure 10, K_i 5700 nM) generated SAR which assisted in the definition of a pharmacophore model similar to one previously proposed [91]. Similar classes of compounds were also discovered based initially on SAR studies in the triazole series [85] which again required the presence of bulky side chains for optimal binding affinity. To overcome this, newer molecules were designed where a carbon or nitrogen spacer was inserted between the triazole core structure and the aryl group and subsequent ring expansion led to the triazines (see Fig. 6).

STRUCTURES 1 TO 8

Fig. (6). Design of pyrimidine and triazine series.

From SAR studies involving changes to the 4-position in the 2-anilinopyrimidines, and in contrast to the five membered ring, the size of the side chain is not a critical factor for optimal activity. Structurally diverse substituents (compounds 11 [90], 12 [92] with K_i values of 2 nM and 3.1 nM respectively, and 13 [93] with IC₅₀ of 22 nM) are well tolerated as long as no strongly basic (compound 14 [94], K_i > 1000nM) or acidic (compound 15 [94] K_i > 1000nM) moieties are introduced into the side chain.

STRUCTURE 9 TO 15

The SAR studies [90] of the lower aromatic ring revealed the importance of steric bulk, polarity and basicity constraints on substitution of the 4-position on the phenyl ring. It was also established that medium sized lipophilic groups at the ortho position and a hydrogen bond acceptor are necessary for optimal binding affinity [91]. Again, in contrast to five-membered ring antagonists, a methyl group is the optimal substituent at the 6-position of the pyrimidines. Structure activity relationships of triazines were mostly parallel to those for the corresponding pyrimidines [90] (Fig. 7).

Fig. (7). Binding affinity of pyrimidine and its analog triazine

Several potent pyrimidine and triazine analogs have emerged from these studies. These compounds however proved to be too lipophilic for further development. Unfortunately, attempting to improve their pharmacokinetic profiles, by introducing polar groups into the side chain or aryl ring, led to decreases in binding affinity [95,96]. For instance, compound SA627 has good oral bioavailability in dogs (20%, 5 mg/Kg, iv/po) but with only moderate binding affinity at rat CRF₁ receptors (K_i 38 nM). Optimization of the pyrimidine series [91] led to NBI 27914, a selective CRF₁ receptor antagonist with a Ki value of 2 nM and this has become a valuable tool for pharmacological experiments. It has also assisted in the prediction of the conformation necessary for high binding affinity with the trichlorophenyl ring orthogonal to the heterocyclic aromatic ring (represented by NBI 27914 in Fig. 8). This antagonist inhibits CRF-mediated increases in adenylate cyclase activity and ACTH release from cultured rat anterior pituitary cells (EC₅₀ = 150 nM and 70 nM respectively) [97].

Fig. (8). Proposed conformation for high binding affinity [91] represented by NBI 27914.

Recently compounds CRA1000 and CRA1001, selective and competitive CRF₁ receptor antagonists with IC₅₀ values of 20 and 22 nM respectively, were reported to exhibit potent anxiolytic and antidepressant-like properties in various experimental animal models [98] with minimal orally effective doses in the 3 – 10 mg/kg range.

Fused Bicyclic Systems

The discovery of bicyclic CRF₁ antagonists evolved from the SAR studies of monocyclic six-membered rings, the investigation into conformational preferences and barrier to rotation of 2-anilinopyrimidine using semi-empirical methods, X-ray crystallography and variable temperature NMR spectroscopy [99]. Several structurally different bicyclic cores have been developed and together these show that, when the nitrogen-required hydrogen bond acceptor (in this case the ring nitrogen) is part of a five-membered ring, the SAR closely resembles that of the monocyclic five-membered ring systems. For instance, SAR of 6-arylpurines (Fig. 9) showed that an ethyl group was strongly preferred and branched alkyl side chains are required [100,101] exactly as had been observed with monocyclic pyrazoles (compound 5),

Fig. (9). Summary of SAR of 6-arylpurines. [Adapted from Ref. 100].

Several potent compounds have been generated from study of 6-arylpurine [96], but as potential therapeutics, suffered from high lipophilicity (compound 18, K_i = 1.81 nM; clogP = 6.2). Further optimization [101] of the side chain and aryl group did not significantly improve the pharmacokinetic profiles. However, several compounds (using formulation) exhibited rat behavior efficacy in the situational anxiety model. For instance compound 19 (K_i 0.93 nM) had an approximate ED₅₀ > 1.5 mg/Kg in this model with 73% reduction in latency relative to control. Other variants of 5,6-membered ring systems have been described without biological data. Structures 20 [102] and 21 [99] have been reported as CRF₁ antagonists. The first 5,5- member ring system is a series of imidazolo(4,5-c)pyrazole [103,104], compound 22 has shown high binding affinity for CRF₁ ( K_i 4 nM).

When the nitrogen is part of a six-membered ring within a bicyclic system (see Fig. 10) , the overall SAR was parallel, but not identical, to the corresponding monocyclics and was not affected by changes of the core structure as long as the peripheral groups are held in the correct orientation. Thiazolo-(4,5-d)pyrimidinones 23 [105], 8-arylquinolines 24 [106], purinones 25 [92], 26 [107] and triazolopyrimidines 27 [108] have been disclosed as CRF antagonists with hCRF₁ K_i values of 9.4 nM, 0.6 nM, 9.7 nM, 5.0 nM and 3.3 nM respectively. Pyrrolopyrimidines 28 [109] and thienopyrimidines 29 [110] have also been reported without data.

Only limited data has been reported which describes the influence of substituents on the fused bicyclic core on binding affinity and this has created a ‘dogma’ which should be periodically challenged particularly when the bicycle is amenable to combinatorial expansion. - methyl group (compound 30, K_i = 1.3 nM) with S-methyl (compound 31, K_i = 0.45 nM) was without significant effect, whereas the hydrophilic methyl sulfone (compound 32, K_i = 1000 nM) showed weaker binding [111]. Either because of the bulk, or because of its positive charge, addition of dimethylaminomethyl led to the inactive compound 33 but this avenue for exploration deserves further attention. Surprisingly, a butyl substituent at the 8-position of the imidazopyridine [112] ring system only diminished binding affinity (compound 35, K_i 22 nM) by 6-fold compared to methyl (compound 34, K_i 3.5 nM). Not unexpectedly however, when the methyl group of purinone 36, (K_i 5nM) was converted to cyclopropylmethyl, activity decreased more than 20-fold (compound 37, K_i = 117 nM) [107]; here again simultaneous changes both at this position and the tertiary amine would be beneficial since it is argued that these two substituents compete for the same lipophilic binding site.

The bicyclic pyrazolopyrimidine template has been the subject of an extensive SAR studies [111,113,114] and many orally active compounds have been identified. Both NBI 30545 [111] and DMP904 [114] have high hCRF₁ affinity (K_i = 2.8 nM and 1 nM respectively) and reasonable pharmacokinetic profiles (oral bioavailability: 10% (rat) and 31% (dog) respectively). Both compounds display good behavioral activity in animal models for anxiety such as the rat situational anxiety test and the rat elevated plus maze model. DMP696 [115] is a potent hCRF₁ receptor antagonist (K_i = 1.7 nM, inhibition of adenylate cyclase IC₅₀ = 82 nM). This compound

Fig. (10). Representative examples of different core structure of fused bicyclic systems.

has excellent oral bioavailabilities in rats and dogs (37% and 50% respectively) and exhibits good activity in rat situational anxiety models (MED = 3 mg/kg po). DMP696 reduced stereotypical mouth movements (lip smacking) in rhesus monkeys using the human intruder paradigm by 50% at 21 mg/kg when given orally [115].

STRUCTURE 33 TO 40

Fig. (11). Pharmacophore model generated from SAR studies of 3-(3-pyridyl)pyrazolo[1,5-a]pyrimidines. [Adapted from Ref. 117].

A search for more water-soluble pyrazolopyrimidines has led to the replacement of the substituted phenyl ring by pyridine to afford 3-(2-pyridyl)pyrazolo[1,5-a]pyrimidines [116] and 3-(3-pyridyl)pyrazolo[1,5-a]pyrimidines [117] as exemplified by structures 38, 39 and 40 which bind to hCRF₁ with K_i values of 2.6 nM, 10 nM, and 1.2 nM respectively. From this effort emerged R121919, a potent, selective CRF₁ receptor antagonist ( CRF₁: K_i = 2.8 nM; CRF₂: K_i = 2000 nM) in vitro with an excellent pharmacokinetic profile in rat following oral administration. In addition, this compound potently inhibits CRF-stimulated cAMP accumulation from cells expressing the human CRF₁ receptor (IC₅₀ = 26 nM) and inhibits CRF-stimulated ACTH production from cultured rat anterior pituitary cells (EC₅₀ = 28 nM). Oral administration of R121919 (10 mg/kg) in rat generates high brain levels (brain to plasma ratio > 1) with moderate half-life and good oral bioavailability (>10%). R121919 also demonstrated dose and time-dependent CRF₁ receptor occupancy following oral treatment concomitant with the levels of drug measured in whole brain (full inhibition at 20 mg/kg in dog brain) [118]. From detailed SAR studies across the pyrazolo[1,5-a]pyrimidine series and others, a pharmacophore model introduced above for monocyclic systems was reinforced (Fig. 11).

Fused Tricyclic Systems

To date, two types of tricyclic compounds have been described without biological data. The first, represented by structure 41 [119] and 42 [120], is an extension of fused bicyclic systems, where the position of side chain and bottom aromatic ring occupy the same region of space as in the fused bicyclic systems. One could anticipate no significant change in SARs. However, the second type, represented by structure 43 [121] and 44 [122], has a more constrained side chain and SAR differences are expected and will clearly be of interest to more critically define the topology of this lipophilic site in particular and of the receptor binding site in general.

Polycyclic Systems

A series of derivatives of oxo-7H-benzo(e)perimidine-4-carboxylic acid [123], represented by structure 46 (CRF₁ K_i = 110 nM, CRF₂_b K_i = 20 nM), were reported as dual CRF₁ / CRF₂ receptor antagonists. Compound 46 antagonized CRF-stimulated cAMP formation and CRF-stimulated ACTH release from rat pituitary in vivo. This class of antagonists does not easily fit with the pharmacophore model described for CRF₁ receptor antagonists [91,117,124] and may therefore have distinguished a novel binding site for further optimization; hopefully such optimization may lead to significantly less lipophilic antagonists

Acyclic Systems

Recently a patent application [125] described N-benzimidazolylmethy- and N-indolylmethyl-benzamides, represented by structure 47, as CRF₁ receptor antagonists with binding affinity in the range of 0.5 nM to 10 mM. One way to view this class of compounds is to consider the amide group to be the core structure with the oxygen of amide group is playing the role of hydrogen bond acceptor. If so, this would represent a diverse starting point for new CRF₁ antagonists.

STRUCTURE 41 TO 48

STRUCTURES 49 TO 50

The majority of these CRF₁ ligands described above can be divided into three major regions and are consistent with a single, classical pharmacophore model (see Figure 11): a) The core structure which in the majority of cases, consists of an planar heterocyclic system containing a critical nitrogen atom as a hydrogen bond acceptor. Adjacent to this core is a region of bulk intolerance where unproductive interactions with the receptor severely limit the range of acceptable substituents at this locus. b) An aryl ring which lies (almost) orthogonal to the plane of the central core. c) A lipophilic side chain remote from feature (b) which is limited by the substituent pattern within the core structure. Since these ligands lack structural diversity, there is little scope for comprehensive and detailed studies into the nature of binding sites of CRF₁.

There has been only limited use of positron emission tomographic (PET) and photoactivatable agents in the study of CRF receptors. This is somewhat surprising given the wealth of structural and localization information which is currently available. Several fluorinated analogs of antalarmin (48, X = F, K_i= 2.5 nM) were made as a prelude to ¹⁸F-labeling experiments and were shown [126] to have affinity equivalent to the parent compound (e.g. compound 48, X= F, K_i= 3.5 nM). Attempts were made to prepare a less lipophilic antalarmin analogue whilst still maintaining potency at CRF₁ receptors in order to improve both water solubility and bioavailability but without success. Peptide photoactivatable ligands based on oCRF (agonist) and astressin (antagonist) have also been prepared [127,128], but little useful structural information has been reported to date. In the first study [¹²⁵I]Tyr⁰ oCRF was derivatized with a diazirine group and then radiolabeled with [¹²⁵I] to give ovine ‘photoCRF’ 49, which behaved, prior to photolysis, as a full agonist at the rat CRF₁ receptor with equivalent EC₅₀ to oCRF (0.4 and 0.5 nM respectively). Photolysis at 360 nm gave a single isolatable CRF protein having a m.w. consistent with the addition of one covalently attached ‘photoCRF’ most likely through the intermediacy of a reactive carbene. Unfortunately, details of the point of attachment to the receptor were not disclosed. Similar experiments with the antagonist 50 generated a compound which bound with a higher affinity than oCRF (K_D = 0.5 and 3.0 respectively) although the new ligand had a Hill coefficient of 0.53 (oCRF, 0.9) possibly indicating multiple binding sites for the former. In addition this molecule covalently tagged the receptor after photolysis. The receptor was protected from photoaffinity labeling by both photo ligands in the presence oCRF indicating a level of specificity during the labeling experiments.

The role of CRF receptors in human disease – potential for therapeutic intervention

The CRF₁, CRF₂ receptors and the CRF-binding protein represent the end-target regulators of the actions of CRF and its related peptides [129,130]. The distribution of these proteins has been well characterized and shown to have a discrete localization within the central nervous system and peripheral tissues [129]. This discrete distribution and the availability of alternative endogenous ligands such as urocortin [130] define an intricate and complex system and suggest a considerable degree of functional diversity. With the ongoing discovery of selective tools for these proteins, their role in disease states can be better understood. Until such time as these molecular tools become available, hypotheses on the therapeutic potential of CRF receptor antagonists will be generated by preclinical animal model research. Some of the major therapeutic applications of compounds interacting with the CRF system are outlined in the following sections.

Neuropsychiatric Disorders

A number of clinical and preclinical studies have determined that dysregulation of the hypothalamic-pituitary-adrenal axis (HPA) plays a major role in the etiology of depression or anxiety-related disorders. Furthermore, a number of investigators have described a significant positive correlation between measures of brain CRF activation and the incidence and severity of these affective and anxiety disorders. For example, the affective disorder, melancholic depression, is characterized by diminished density of brain CRF receptors, diminished sensitivity of the HPA-axis to CRF challenge and elevated cerebrospinal fluid levels of CRF all of which point to overactivation of brain CRF systems. Similarly, anxiety disorders such as post-traumatic stress disorder are also characterized by apparent overabundance of brain CRF as revealed by abnormally high levels of CRF in cerebrospinal fluid [131] although HPA-axis reactivity, in contrast to melancholic depression, appears to be diminished in post-traumatic stress disorder [132]. The degree to which affective and anxiety disorders together constitute a class of neuropsychiatric disorders with a common element of CRF system overactivation has been the driving force behind the numerous drug discovery programs focused on the identification of molecules that can block the actions of this neurohormone.

Depression and Anxiety

The primary role of CRF is the stimulation of pituitary adrenocorticotropic hormone and the subsequent adrenal regulation of glucocorticoids. Hypersecretion of CRF in the brain has been hypothesized to underlie the hypercortisolemia and symptomatology seen in major depression [133]. A number of observations suggest that CRF systems function abnormally in depressed patients. It has been observed that patients with major depression are hypercortisolemic and exhibit an abnormal response in the dexamethasone suppression test [134]. Coincidentally, cerebrospinal fluid levels of CRF are significantly elevated in depressed patients [135] and these can be normalized by electroconvulsive therapy and correlate well with clinical improvement [136]. Therefore, a significant positive correlation exists between CRF concentrations in the cerebrospinal fluid and the degree of insensitivity to dexamethasone suppression of plasma cortisol in depressed individuals [137]. Concomitant with the increased levels of CRF found in the central nervous system, there is an observed decrease in the CRF receptors (CRF₁) in the cerebral cortex of suicide victims compared to normal [135]. This in fact strengthens the argument that CRF plays a major role in these disorders since the biological system seems to be attempting to regulate itself, in effect, trying to functionally decrease the activity of CRF. These data along with the observation of improvement in clinical outcome following ECT and subsequent lowering of CRF levels, are consistent with the hypothesis that CRF is hypersecreted in major depression and compounds specifically targeting the blockade of CRF function will prove therapeutically beneficial.

In view of the data above suggesting a role for CRF in depression, the hypothesis has been put forth that antidepressants may produce their therapeutic effects, in part, by decreasing CRF secretion. An increase in CRF binding sites, presumably to compensate for chronic suppression of CRF secretion, is observed in some brain regions such as the brain stem in rats treated chronically with tricyclic antidepressants such as imipramine [138]. Recently, in a mouse model of depression (tail suspension test ) induced by YM643, a consensus interferon-alpha (IFN-alpha), both imipramine and the selective CRF₁ receptor antagonist CP 154526 could decrease the immobility measure in this depression model [139] corroborating the data described above. In addition, mild chronic stress models have mimicked the elevations in CRF in the central nervous system and are beginning to identify the specific structural components within the brain that may be the most critical in the manifestation of these symptoms [140]. CRF has also been postulated to have an important role in the locus coeruleus of the brain stem. In a recent detailed microdialysis study, microinfusion of the non-peptide CRF₁ receptor antagonist CP 154526 suppressed the stimulation of noradrenaline from the locus coeruleus during mild handling stress [141]. This supports and extends much earlier observations where concentrations of CRF have been shown to be selectively increased in the locus coeruleus following application of acute or chronic stress [142] and validate the endeavor for identification of selective CRF₁ receptor antagonists. Given the major involvement of the brain noradrenergic system, in particular in the locus coeruleus in depression and the effects of CRF to activate noradrenergic neurons in this brain region [143], it is possible that current antidepressants may function by ultimately suppressing CRF secretion in the locus coeruleus, resulting in the observed increase in brain stem CRF binding sites.

In addition to the role that has been proposed for CRF in major depressive disorders, preclinical data in rats demonstrating effects of CRF administration on several behavioral changes characteristic of anxiogenic compounds [144] have led to the suggestion that CRF may also be involved in anxiety-related disorders. For example, a role for CRF in panic disorder has been suggested by observations of blunted ACTH responses to intravenously administered CRF relative to control subjects [145]. The blunted ACTH response to CRF in panic disorder patients most likely reflects a process occurring at or above the hypothalamus, resulting in excess secretion of endogenous CRF. Similarly, anxiety disorders such as post-traumatic stress disorder are characterized by overabundance of brain CRF as revealed by abnormally high levels of CRF in cerebrospinal fluid [131]. The brain region thought to mediate the symptomatology exhibited in these anxiety-related syndromes is the amygdala. The amygdala is a critical temporal lobe structure involved in the expression of anxiety and stress responses. Within the amygdala, the basolateral nucleus in particular, is thought to be involved in the transmission of the signals in these disorders. Urocortin, which has affinity for both the CRF₁ and the CRF₂ receptor subtypes, when microinjected into the basolateral nucleus directly, produces anxiogenic-like behavior as assessed by a social interaction test [146]. Furthermore, repetitive administration of subthreshold doses of urocortin results in a 'priming' phenomenon which sensitizes animals to intravenous sodium lactate, a panicogenic agent in susceptible human patients [146]. The ability of CRF peptide infusion in animal models of emotionality to produce anxiogenic-like behaviors, fear and neuronal hyperexcitability [147,148] provide critical evidence for a role for CRF overabundance in human clinical anxiety. This hypothesis is strengthened again by the fact that these peptide induced effects can be blocked by administration of either peptide or non-peptide antagonists at CRF receptors [149,150].

Further support for the hypothesis linking the brain stress-axis with anxiety disorders comes from neurochemical, endocrine and receptor binding data documenting interactions between CRF and benzodiazepine anxiolytics. Acute administration of the triazolobenzodiazepines, alprazolam and adinazolam, results in increased hypothalamic concentrations of CRF while decreasing the concentrations of CRF in other brain regions, including locus coeruleus, amygdala, pyriform cortex and cingulate cortex [151]. Of particular interest is the finding that the two triazolobenzodiazepines exert effects on CRF concentrations in the locus coeruleus and hypothalamus that are opposite to CRF changes seen after stress. Chronic administration of diazepam, alprazolam, or adinazolam in rats results in significant decreases in CRF receptors in the frontal cerebral cortex and hippocampus and there is a trend for CRF receptors to be decreased in other brain areas and increased in the anterior pituitary [138]. The latter data demonstrating increases in CRF receptor concentrations support the hypothesis for effects of the benzodiazepines to inhibit CRF release which in turn modulate the receptors. Further support for this hypothesis is provided by potent in vitro effects of benzodiazepines [152] to inhibit hypothalamic CRF release. Conversely, the reduced concentrations of CRF in the other brain regions described above following in vivo administration of the benzodiazepines may relate to increased release of CRF, which would be expected to decrease receptors in regions like the frontal cortex and hippocampus. With the discovery of the second mammalian agonist in the CRF system, urocortin, a recent study has demonstrated that chronic benzodiazepine treatment has inverse effects on the regulation of CRF and urocortin raising the intriguing possibility that these systems act in concert in a much more complex manner to manifest a complex disorder such as anxiety. However, in view of the evidence described above suggesting that hypersecretion of CRF may underlie some of the symptomatology seen in affective disorders and anxiety-related disorders, it stands to reason that CRF receptor antagonists may be useful in the treatment of these disorders. Thus, a CRF antagonist may be a useful antidepressant, anxiolytic or anti-stress drug.

Substance Abuse

Stressful aspects of drug exposure in the drug-naive organism may produce activation of brain CRF systems [153]. In addition, the acute reinforcing effects of drugs of abuse seem to involve similar neurotransmitter systems, such as dopamine, opioid peptides, serotonin, GABA, and glutamate, as the effects of withdrawal from these drugs. Withdrawal from drugs of abuse is associated with subjective symptoms of negative affect, such as dysphoria, depression, irritability and anxiety [154]. Several lines of evidence from preclinical and clinical studies support the importance of stress and negative mood in perpetuating drug use and relapse. For example, in laboratory animals, exposure to stressors has been shown to increase self-administration of amphetamines [155], morphine [156] and cocaine [157]. Brief footshock stress (a clear activator of the HPA) also results in reinstatement of drug-seeking behavior in heroin-experienced, drug-free animals and in cocaine-experienced, drug-free animals [156]. Furthermore clinical surveys of drug abusers and alcoholics report that these individuals frequently cite psychological distress and negative mood states as reasons for relapse to drug use [158]. In fact, a positive relationship between internal negative mood states and drug craving has been demonstrated in laboratory studies with alcoholics, opiate abusers and smokers while demonstrating that external drug cues provided no additional increases in craving [159]. Although the relationship between stress and cocaine use has only recently been investigated, these findings have led to the development of the hypothesis that drug abusers may often use drugs to blunt or self-medicate negative mood states. The CRF system is an ideal candidate for possible intervention since acute withdrawal is accompanied by recruitment of the brain stress axis whose key modulator is CRF and most drugs of abuse, such as cocaine, have a robust effect on the HPA axis [160]. The extension of the function of CRF is that stress, via CRF increases physiological measures as well as neuroendocrine activity, as assessed by beta-endorphin, ACTH and cortisol levels, and increases craving in cocaine abusers. Again, CRF receptor antagonists may well be powerful mitigators of this response and offering a potential therapy to those already in treatment for substance abuse.

Seizure Disorders

CRF has been implicated in playing a major role in the manifestation of seizures and in general contributing to a neurodegenerative process, particularly in the developing brain. Coupled with the evidence that stress stimulates the expression of CRF in several limbic regions of the brain, CRF neurons are strategically placed in the developing hippocampus to effect the excitability of pyramidal cells causing stress-induced, sometimes severe seizures. CRF administered into the cerebral ventricles of rats during the first postnatal week causes a specific and stereotyped behavior sequence followed by 'limbic'-type seizures [161]. Human infantile spasms are an age-specific seizure syndrome of infancy. Interestingly, this disorder responds favorably to hormonal manipulation using either adrenocorticotropic hormone (ACTH) or glucocorticoids implicating the CRF system and its dysregulation in the manifestation of this disorder [162]. Peptide tools have provided much of the initial evidence for a role and therapeutic potential of CRF and CRF receptor antagonists respectively. For example in kainic acid-induced excitotoxic seizures, astressin, the non-selective CRF₁ and CRF₂ receptor antagonist administered intracerebroventricularly, was observed to reduce the damage in hippocampal neurons by over 80%. More importantly, this protection could be observed as much as 10 minutes after the excitotoxin exposure suggesting a promising therapeutic potential of antagonists without prior anticipation of a neurological insult [163]. Astressin however, having equal affinity for the CRF₁ and CRF₂ receptor subtypes cannot discriminate between the two subtypes thus not allowing a clear anatomical role of the receptor systems to be established. In the amygdala, both subtypes of the known CRF receptors (CRF₁ and CRF₂) are expressed and are thought to be the origin of CRF-induced seizures [164]. With the initial discovery of selective tools for the CRF receptor subtypes, it has been shown that the CRF₁ receptor is the most likely mechanism through which CRF exerts its activity in producing seizures [165]. NBI 27914, a selective non-peptide CRF₁ receptor antagonist [91] was tested for its ability to abolish CRF-induced seizures, in comparison to non-selective inhibitory peptide analogues of CRF. Pretreatment with NBI 27914 increased the latency and decreased the duration of CRF-induced seizures in a dose-dependent manner. Higher doses of NBI 27914 blocked the behavioral seizures and prevented epileptic discharges in concurrent electroencephalograms recorded from the amygdala. Thus, these data indicate that activation of expression of CRF constitutes an important mechanism for generating developmentally regulated, triggered seizures, with considerable clinical relevance and that the use of CRF₁ receptor antagonists may well provide a novel therapy for this stress-related neurodegenerative disorder [165]

Somatic Disorders

Inflammation

Many correlates have been drawn and examined with CRF, the stress axis and effects on the immune system. Intracerebroventricular administration of CRF suppresses the immune system either directly through the sympathetic nervous system or indirectly through the systemic activation of glucocorticoids [166]. In direct models of acute inflammation, immunon-eutralization of CRF systemically has been shown to directly block the inflammatory response to aseptic carrageenan-induced inflammation in Sprague-Dawley rats [167]. Further studies using the selective CRF receptor antagonist Antalarmin showed a significant inhibition of both CRF-stimulated ACTH release and carrageenan-induced subcutaneous inflammation in rats [168]. These studies consistently suggest that CRF plays a role in the inflammatory process and that neutralization of its effects, either by removal of the hormone itself or blockade of the CRF₁ receptor can attenuate this process. As a possible explanation for the proinflammatory effects of CRF, it has been shown that CRF or urocortin induces skin mast cell degranulation and increased vascular permeability [169,170]. Furthermore, acute immobilization stress was found to trigger mast cell degranulation and this could be almost completely attenuated by pretreatment of the animals with intraperitoneally administered CRF antiserum; again identifying CRF as one of the major factors associated with a stress-induced pathological effect [171]. In this same study however, blockade of Substance P or neurotensin also decreased the stress-induced degranulation to the same extent. This study therefore suggests that multiple treatment paradigms may be possible for inflammatory skin disorders; however, further work will be required to identify the specificity of this stress-induced result [171]. While it is clear that the role of CRF in inflammation is only now becoming better understood, it is tempting to speculate that since CRF is the primary mediator of the stress response, any syndromes or conditions for which stress exacerbates or worsens the symptomatology will benefit from a modulation of the CRF system. Inasmuch as there is no one single mediator of the complex stress system the CRF system does however offer the best potential for the development of novel therapeutics for diseases and disorders currently identified as “stress” disorders.

As a final point on the role of CRF in inflammatory syndromes, the enigmatic condition of inflammatory bowel syndrome has been characterized as yet another stress disorder. Preclinical data have established that CRF acts centrally to mimic similar changes in gastrointestinal (GI) motor function as those induced by stress [172]. These effects include inhibition of gastric motility and emptying, and acceleration of colonic motility and transit time [173-177]. These effects are mediated through the CRF receptor subtypes since peptide antagonists have been shown to block the GI responses to either CRF or stress [178-181]. Psychological and physical stressors (such as surgical stress) are known to have significant motor effects including gastric stasis [182]. As of yet, the CRF receptor subtype selectivity of these responses is not well understood; however, with the ongoing discovery of selective tools for the receptor subtypes, there may be the opportunity to identify novel therapeutics for these syndromes.

Reproduction

There is increasing evidence from a variety of studies which suggests that CRF and its receptors play an important role in the reproductive functions of humans. Corticotropin-releasing factor (CRF) is produced in the placenta where it has a paracrine effect within placenta, and myometrium as well as having independent endocrine effects on both the mother and fetus [183]. The major effects that have been described for CRF in the reproductive system are potent local regulation of myometrial contractility and of prostaglandin release [184] leading to the evidence for a role in parturition. The receptors for CRF, both the CRF₁ and CRF₂ subtypes, have also been localized and quantified by both mRNA studies for the message and radioligand binding studies to identify the active protein [185,186]. In addition, these receptors have been characterized in terms of their second messenger profiles and found, with a few exceptions to couple to cAMP similar to other CRF receptors reported in brain, pituitary or spleen [187]. Where this local CRF regulatory system differs from these other tissues in human seems to be the unique role that has been elucidated for the CRF binding protein (CRF-BP), a soluble protein that is thought to modulate or regulate the actions of CRF for its receptor in various stages of pregnancy. Although the CRF-BP also exists in brain, its role in the pathology of any disease states is far less well understood outside a hypothesized role in the memory and cognitive pathology associated with Alzheimer’s disease [188,189]. In the reproductive system, CRF-BP modulates the paracrine effects of both CRF and urocortin [190,191].

Above and beyond the clearly established effects of CRF on the endocrine system, CRF acts directly on the placenta to influence the secretion of placental ACTH [192], has effects on the placental blood vasculature [193], stimulates the production of prostaglandins [194], and has an action on fetal adrenal gland to stimulate the production of the steroid DHEA-S [195]. In nonpregnant women, plasma CRF levels are low and these become higher during the first and second trimesters of pregnancy. A clear increase is evident at term and when CRF-BP levels decrease suggesting a key role for CRF in coordinating the smooth transition from a uterine state of relaxation to one of contraction thus initiating labor [196]. Pathologically, women with preterm labor show high CRF and low CRF-BP levels, supporting an involvement of this pathway in mechanism of parturition and suggesting that inappropriately high CRF levels may contribute to the etiology of preterm labor. Similarly, high levels of CRF have been shown in umbilical cord plasma of growth-retarded fetuses [197]. Levels can reach up to 5 times higher than in the normal fetal circulation and may arise from chronic fetal stress. Growth retarded fetuses are at a much higher risk for perinatal morbidity and mortality usually due to uteroplacental insufficiency [197]. The hypothesis that CRF antagonists may be beneficial in the treatment of preterm labor has been examined in an animal model of delayed parturition in sheep. Pregnant ewes received infusions into a fetal vein of either vehicle or the CRF₁ receptor antagonist antalarmin over 10 days. Fetuses infused with vehicle were delivered sooner than antalarmin-infused animals and the fetal ACTH and cortisol rise, which characterized the vehicle-treated group, did not occur in antalarmin-infused sheep. These data show that at least in sheep, CRF₁ antagonism in the fetus can delay the onset of parturition [198].

Thus, the CRF system including CRF itself, urocortin, the multiple subtypes of receptors and binding protein, are well distributed in placenta and decidual membranes and act in a complex regulatory manner in human pregnancy. Pathological conditions such as those described above offer a tremendous potential for novel therapies for these disorders.

Outlook

Despite an abundance of studies on the CRF system, we have yet to see useful drugs emerge from detailed clinical evaluation. To date there has been only one report of a preliminary clinical study where, in 20 depressed patients treated with a selective CRF₁ receptor non-peptide antagonist [200], a significant reduction in depression and anxiety scores was observed, albeit with an open label protocol. Nevertheless, with the system increasingly being defined, there is little doubt that new ligands selective for the two receptor sub-types will become available within the next 1-2 years which will yield exciting new therapeutic opportunities for some of the diseases listed above.

ABBREVIATIONS

CRF (CRH) = Corticotropin releasing factor (hormone)

GPCR = G-protein coupled receptor

HPA = Hypothalamic-pituitary-adrenocortical axis

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