Stressin₁-A, a Potent Corticotropin Releasing Factor Receptor 1 (CRF₁)-Selective Peptide Agonist

Abstract

The potencies and selectivity of peptide CRF antagonists is increased through structural constraints suggesting that the resulting ligands assume distinct conformations when interacting with CRF₁ and CRF₂ receptors. To develop selective CRF receptor agonists, we have scanned the sequence -Gln-Ala-His-Ser-Asn-Arg- (residues 30–35 of [DPhe¹²,Nle^21,38]Ac-hCRF_4-41) with an i-(i + 3) bridge consisting of the Gluⁱ-Xaa-Xbb-Lysⁱ⁺³ scaffold where residues i = 30, 31 and 32. When i = 31 stressin₁-A, a potent CRF₁ receptor-selective agonist was generated. In vitro, stressin₁-A was equipotent to h/rCRF to release ACTH. Astressin₁-A showed a low nanomolar affinity for CRF₁ receptor (K_i = 1.7 nM) and greater than 100-fold selectivity versus CRF₂ receptor (K_i = 222 nM). Stressin₁-A released slightly less ACTH than oCRF in adult adrenal-intact male rats, with increased duration of action. Stressin₁-A, injected intra-peritoneally, induced pellet output (a CRF₁ receptor-mediated response) and did not influence gastric emptying and blood pressure (CRF₂ receptor-mediated responses).

Introduction

In previous reports describing the rationale used in the discovery of astressin (a potent non-selective antagonist at both CRF₁ and CRF₂ receptors) and astressin₂-B (a CRF₂ receptor-selective antagonist), we emphasized the importance of subtle structural constraints that led to the desired compounds. Whereas a Glu³⁰-Lys³³ side chain to side chain covalent lactam constraint increased affinity of linear CRF antagonists (astressin) for CRF₁ and CRF₂ receptors, ² we found that a Glu³²-Lys³⁵ side chain to side chain bridge in h/rCRF fragments and the corresponding Glu³¹-Lys³⁴ bridge in sauvagine fragments yielded potent ligands (astressin₂-B) that are highly selective for the CRF₂ receptor.³ This selectivity (>100-fold) was demonstrated using radioreceptor assays with cloned receptor cell lines and autoradiographic studies on rat brain slices. Additionally, we identified unique substitutions (CαMeLeu) that conferred on these antagonists long duration of action in vivo.⁴

Interestingly, extension of these sequences to full length CRF analogs yielded CRF₁/CRF₂ receptor (astressin-derived) agonists that were only 2- to 5-fold more potent than the ₁ parent analogues as well as agonists that had lost some of their CRF₂ receptor-selectivity. However, the discovery of urocortin 2 (Ucn 2)⁵ and urocortin 3 (Ucn 3)⁶ which are potent CRF₂ receptor-selective agonists provided the needed tools to study the physiological role of the CRF₂ receptor. Whereas a large number of studies used oCRF as a preferential CRF₁ receptor-selective ligand, we disclosed the structure of stressin₁-A¹ which was equipotent to oCRF at CRF₁ receptor and about 4 times less potent at CRF₂ receptor than oCRF, resulting in a 4-fold increase in selectivity. Independently, Tezval et al. described cortagine with about 200-fold selectivity for the CRF₁ receptor.⁷ Although any of these molecules may be used for the study of the pharmacological or physiological CRF₁-mediated activities, we hypothesized that fragments of the most potent and selective analogue might yield a potent and CRF₁ receptor-selective antagonist. It is noteworthy that nonpeptide CRF₁ receptor-selective antagonists were available at the time of the initiation of this research, yet they are not necessarily the best tools for unraveling the physiological role of the CRF₁ receptor because they do not limit their action to the peripheral or central compartments. Although one would expect that peptide CRF₁ receptor agonists administered in the periphery would not cross the blood brain barrier, it is known that centrally administered peptides may leak to the periphery.

We disclosed the structure of stressin₁-A (cyclo(31-34)[DPhe¹²,Nle^21,38,Glu³¹,Lys³⁴]Ac-hCRF_(4-41)), a CRF₁ receptor-selective agonist, at the 31^st Annual Meeting of the Society of Neuroscience held in San Diego, CA.¹ When it comes to nomenclature, we proposed to call CRF antagonists, astressins³ and use stressins for agonists with no subscript or subscript 1 or 2 (as for the antagonists) to indicate no selectivity or selectivity for either the CRF₁ receptor or the CRF₂ receptor, respectively. The essential role of the CRF₁ receptor in mediating ACTH release from the pituitary in response to stress or exogenous administration of CRF or Ucn 1 is well established using CRF₁ receptor knockout mouse model or selective nonpeptide CRF₁ receptor subtype antagonists.^8,9 In addition, convergent in vivo and in vitro studies have emphasized the role for the CRF₁ receptor in stress- and peripheral administration of CRF-induced stimulation of colonic motor function (motility, transit, defecation and diarrhea).^10,11 In contrast, in the upper gastrointestinal tract, CRF, Ucn 1, or Ucn 2 injected peripherally, exerts an inhibitory effect on ^3,10,12–14 gastric motility and transit that is mediated by CRF₂ receptors in rats and mice.

Peripheral CRF₂ receptors also play an important role in the modulation of cardiovascular function.¹⁵ Peripheral administration of Ucn 2 induced hypotension that was blocked by the selective CRF₂ receptor antagonists, astressin₂-B or anti-sauvagine-30 in rats.¹⁶ Conversely, CRF₂ receptor knockout mice displayed elevated blood pressure (BP), and did not respond to peripheral injection of Ucn 1 by change in BP.¹⁷ The expression of CRF₂, but not CRF₁ receptors in the endothelial cells of arteries, further supported a direct action of urocortins within the vascular system.^15,18

While selective CRF₂ receptor endogenous ligands, namely Ucn 2 and Ucn 3, have been characterized recently,^5,6,19 there is no evidence for the existence of endogenous CRF₁ receptor-selective ligands. Ovine CRF displays features of a preferential but not selective CRF₁ receptor agonist and Ucn 1 has equal affinity for both CRF receptors.¹⁹ Because there is increased evidence for a modulation of CRF₁ receptor-mediated actions by CRF₂ receptors,^20,21 it is important to probe the CRF signaling pathways using selective CRF₁ receptor agonists. Here we describe some of the SAR studies that led to the discovery of stressin₁-A. Additionally, we report its biological characterization in in vitro and in vivo CRF₁ receptor mediated bioassays such as the release of ACTH, and the stimulation of colonic motor function along with the absence of CRF₂ receptor-mediated biological responses on gastric emptying and BP in rats.

Results and Discussions

Synthesis and physicochemical characterization

All analogues shown in Table 1 were synthesized using the solid phase method of Merrifield on a methylbenzhydrylamine resin (MBHAR) using the Boc-strategy with orthogonal protection of the side-chains of the lysine (Fmoc) and glutamic acid (OFm) residues to be cyclized.^22–24 Main-chain assembly was mediated in most cases by diisopropylcarbodiimide (DIC). The best results were obtained when the peptide chain was assembled in its entirety prior to cleavage of the Fmoc and OFm protecting groups and when the lactam formation was mediated by TBTU or BOP. The peptides were cleaved and deprotected in HF and purified with reversed phase high performance liquid chromatography (RPHPLC). Peptides were characterized using RPHPLC, CZE and MS (Table 1).

Table 1.

Physical and biological properties of CRF analogues.

	Cpd Name	HPLCa	CZEb	MSc calc	MSc found	CRF1d (nM)	CRF2d (nM)	Relative Potencye
1	r/hCRF	>98	96	4755.5	4755.5	1.0 (0.2–4.6)	6.2 (2.0–19)	1.0
2	oCRF	99	98	4668.50	4668.5	1.2 (0.9–1.6)	52 (21–128)	1.0
3	Cortagine	96	>98	4439.35	4440.3	3.4 (2.5–4.8	102 (46–225)	0.63 (0.37–1.1)
4	cyclo(30-33)[DPhe12,Nle21,38,Glu30,Lys33]Ac-hCRF(4-41)	97	95	4440.52	4440.4	0.7 (0.2–2.0)	1.3 (0.5–3.6)	4.3 (2.5–7.8)
5	linear[DPhe12,Nle21,38,Glu30,Lys33]Ac-hCRF(4-41)	96	95	4458.54	4458.6	0.5 (0.2–1.3)	1.6 (1.5–1.8)	4.5 (2.7–7.6)
6	linear[DPhe12,Nle21,38,Glu31,Lys34]Ac-hCRF(4-41)	98	>98	4488.54	4488.5	2.7 (2.2–3.3)	252 (114–560)	1.0 (0.47–2.3)
7	cyclo(31-34)[DPhe12,Nle21,38,Glu31,Lys34]Ac-hCRF(4-41) (stressin1-A)	>98	98	4470.53	4470.3	1.7 (1.1–2.6)	222 (137–361)	1.1 (0.53–2.2)
8	linear[DPhe12,Nle21,38,MeLeu27,40,Glu31,Lys34]Ac-hCRF(4-41)	>98	98	4516.57	4516.5	2.7 (0.7–10)	261 (105–654)	1.1 (0.3–5.5)
9	cyclo(31-34)[DPhe12,Nle21,38,MeLeu27,40,Glu31,Lys34]Ac- hCRF(4-41)	97	94	4498.56	4498.3	0.8 (0.5–1.1)	47 (21–102)	2.2 (0.8–6.2) NP
10	cyclo(31-34)[DPhe12,Nle21,Glu31,Lys34]oCRF	98	94	4704.57	4704.55	66 (28.5–154)	>1000
11	cyclo(31-34)[DPhe12,Nle21,MeLeu27,Glu31,Lys34]oCRF	95	94	4718.6	4718.29	50 (18–139)	>1000
12	cyclo(31-34)[DPhe12,Nle21,Glu31,Lys34]Ac-oCRF(9-41)	95	91	3895.15	3895.13	>500	>500
13	cyclo(32-35)[DPhe12,Nle21,38,Glu32,Lys35]Ac-hCRF(4-41)	96	95	4362.45	4362.5	1.8 (0.6–5.0)	1.8 (1.2–2.7)	2.7 (1.4–5.3)
14	cyclo(32-35)[DPhe12,Nle21,38,MeLeu27,40,Glu32,Lys35]Ac- hCRF(4-41)	95	90*	4390.48	4390.6	1.3 (0.6–2.9)	1.5 (1.0–2.2)	2.8 (1.4–5.8)
15	cyclo(32-35)[DPhe12,Nle21,38,MeLeu27,40,Glu32,Lys35]Ac-hCRF(6-41)	>98	95*	4196.37	4196.4	3.9 (2.2–7.0)	3.0 (1.0–9.2)	6.1 (3.8–9.7)
16	cyclo(32-35)[DPhe12,Nle21,38,MeLeu27,40,Glu32,Lys35]Ac-hCRF(7-41)	>98	90*	4083.29	4083.3	11 (8.0–15.0)	1.6 (1.1–2.4)	0.32 (0.21–0.48)
17	cyclo(32-35)[DPhe12,Nle21,38,MeLeu27,40,Glu32,Lys35]Ac-hCRF(8-41)	>98	95*	3996.26	3996.2	15.6 (9.0–26.0)	2.0 (1.6–2.4)	0.025 (0.013–0.043)

^aPercent purity determined by HPLC using buffer system: A = TEAP (pH 2.5) and B = 60% CH₃CN/40% A with a gradient slope of 1% B/min, at flow rate of 0.2 mL/min on a Vydac C₁₈ column (0.21 × 15 cm, 5 μm particle size, 300 Å pore size). Detection at 214 nm.

^bCapillary zone electrophoresis (CZE) was done using a Beckman P/ACE System 2050 or 5500. Field strength of 15 kV at 30 °C, mobile phase: 100 mM sodium phosphate (85:15, H₂O:CH₃CN) pH 2.50, on a Supelco P175 capillary (75 μm ID × 50 cm length).

^*CZE basic; Field strength of 13 kV at 30 °C; mobile phase: 100 mM sodium borate (85:15, H₂O:CH₃CN) pH 8.50, on an Agilent μSIL-FS capillary (75 μm ID × 50 cm length). Detection at 214 nm for both systems.

^cThe observed m/z of the monoisotope compared with the calculated [M + H]⁺ monoisotopic mass.

^dThe numbers given in Table 1 reflect the inhibitory binding constants, Ki, for the analogues’ binding to the cloned hCRF1 and mCRF2β receptors derived from competitive radioligand displacement assays using the non-selective ¹²⁵I-labeled agonist [Tyr⁰,Glu¹,Nle^17]sauvagine as the radioligand. K_i values were calculated by PRISM software. Values in parentheses are 95% confidence limits.

^ePotencies are relative to ovine CRF in the in vitro rat pituitary cell culture assay, with 95% confidence limits in parentheses.

Biological characterization in vitro

Selected peptides were tested in the rat dispersed anterior pituitary cell culture assay²⁵ to determine their relative potency and in cloned receptor-based assays to determine their binding affinities for two receptors (hCRF₁ receptor and mCRF_2β receptor)²⁶ in order to determine their selectivity (Table 1). In earlier publications, we showed that deletion of the N-terminal residues (1–3)²⁷ up to (1–7)²⁸ yielded agonists with somewhat reduced relative potencies as the size of the peptide decreased. For example, whereas oCRF, Ac-oCRF and oCRF_(4-41) were equipotent in the rat dispersed anterior pituitary cell culture assay, oCRF_(6-41) and oCRF_(7-41) had 11% and 0.5% of oCRF’s potency, respectively.²⁹

SAR studies based on in vitro assays

The assay measuring the inhibition of CRF-stimulated release of ACTH from rat anterior pituitary cells in culture was instrumental in identifying astressin as a new lead for potent CRF antagonists because it was 32 times more potent than any of its predecessors [α-hel-CRF_(9-41) or [_DPhe¹²,Nle^21,38]hCRF_(12-41)].³⁰ In the same series, cyclo(26-29), cyclo(28-31) and cyclo(29-32)[_DPhe¹²,Gluⁱ,Lysⁱ⁺³,Nle^21,38]hCRF_(12-41) were ten times less potent while cyclo(24-27), cyclo(25-28), cyclo(27-30), cyclo(31-34) and cyclo(32-35)[_DPhe¹²,Gluⁱ,Lysⁱ⁺³,Nle^21,38]hCRF_(12-41) were less than 2% as potent. With the availability of receptor assays for the CRF₁ receptor and the CRF₂ receptor²⁶ and as rationalized in the introduction, the Gluⁱ, Lysⁱ⁺³ cyclic lactams spanning the [DPhe¹²,Nle^21,38]Ac-hCRF_(4-41) sequence (i = 30, 31 and 32) were tested for their affinities for the two cloned CRF receptors (Table 1). A good correlation was found between the relative in vitro potencies derived from the measure of ACTH stimulation in rat anterior pituitary cells in culture by increasing doses of the agonists and K_i at CRF₁ receptor for analogues 4–9 (Table 1). Binding affinities for the CRF₂ receptor, however, diverged significantly from binding affinities for CRF₁ in the case of both linear [DPhe¹²,Glu³¹,Lys³⁴,Nle^21,38]Ac-hCRF_(4-41) (6) and cyclo(31-34)[DPhe¹²,Glu³¹,Lys³⁴,Nle^21,38]Ac-hCRF_(4-41) (7) in that they had very high affinity for the CRF₁ receptor and low affinity for the CRF₂ receptor. This was the first indication that CRF₁ receptor-selectivity measured by the ratio of the CRF₂ receptor K_i/CRF₁ receptor K_i = 93 and 130, respectively, could be modulated by the introduction of a structural constraint similar to that used to achieve CRF₂ receptor selectivity in astressin₂-B³ and consistent with that used for the potent antagonists astressin and astressin B.^2,31

Although a cyclic constraint in 7 and 9 helped identify a region of the CRF molecule that could be altered to modulate selectivity towards CRF₁ receptor-selectivity, the fact that the corresponding linear 6 and 8 also showed similar selectivity suggested that intramolecular (ionic) forces could also induce preferential secondary structures. Introduction of CαMe-leucine (CαMeLeu) at positions 27 and 40 was particularly beneficial (conferred long duration of action) in the cases of astressin B and astressin₂-B and was well tolerated in 8 and its corresponding cyclic 9 when compared to 6 and 7 with respect to CRF₁ receptor-selectivity.

Because oCRF (2) was by itself CRF₁ receptor-selective, we hypothesized that the introduction of the Glu³¹-Xaa-Xaa-Lys³⁴ scaffold in [DPhe¹²,Nle²¹]oCRF would result in an even more CRF₁ receptor-selective analogue (10) than an analogue with the same substitution in Ac-hCRF_4-41 (4). Unexpectedly, this modification resulted in significant loss of binding affinity at CRF₁ receptor (K_i = 66 nM) and the CRF₂ receptor (K_i > 1000 nM). The additional CαMeLeu substitution of 10 to yield 11 resulted in a slight increase in CRF₁ receptor binding affinity in a manner similar to what was observed after CαMethylation of 7 to yield 9.

Analogue 12, resulting from the deletion of 8 residues at the N-terminus of 10, was hypothesized to be a potential CRF₁ receptor-selective antagonist yet was essentially inactive in both CRF₁ receptor and CRF₂ receptor binding assays.

Shifting the i-(i+3) bridge consisting of the Gluⁱ-Xaa-Xbb-Lysⁱ⁺³ scaffold in [DPhe¹²,Nle^21,38]Ac-hCRF_4-41 from positions 31–34 to 32–35 restored high binding affinity at both the CRF₁ receptor and the CRF₂ receptor (13) and CαMethylation at positions 27 and 40 was well tolerated (14). Agonists 13 and 14 were equipotent (K_i = ca 1.5 nM) at both receptors. In view of these results, we wondered whether the favorable constraints brought about by the 32–35 bridge would also favor a bioactive conformation despite further deletions at the N-terminus as we had shown for the 30–33 bridge.²² Indeed, analogues derived from 14 (missing residues 1–3 from the original CRF sequence) and missing residues 1–5 (15), 1–6 (16) and 1–7 (17) retained considerable binding affinity, yet with decreasing relative potencies (Table 1).

While this work was in progress, and after our disclosure of the structure of stressin₁-A, Tezval et al.⁷ reported the synthesis and biological characterization of cortagine, [Glu²¹,Ala⁴⁰][Svg1-12]x[human CRF14-30]x[Svg30-40], a chimera derived from 2 fragments of sauvagine (residues 1–12 and 30–40) and hCRF(residues 14–30) with two substitutions: Glu²¹ to reduce affinity to the CRF binding protein and Ala⁴⁰ to increase CRF₁ receptor selectivity. It is interesting that using two different, yet structurally based approaches, analogs of CRF were identified that showed CRF₁ receptor selectivity. In our assays however, we found that cortagine was equipotent (relative potency = 0.63) to oCRF (relative potency = 1.0) in the in vitro pituitary cell culture assay and somewhat less selective than had been reported (ratio of CRF₂ receptor K_i/CRF₁ receptor K_i = 200 compared to 30 shown in Table 1)⁷ which is not statistically different from the ratio of CRF₂ receptor K_i/CRF₁ receptor K_i = 43 for oCRF. This relatively minor difference may be attributed to the fact that both compounds have very low affinity for the CRF₂ receptor and thus, their K_is are not well defined.

In vivo biological characterization

ACTH release: As shown in Figure 2, plasma ACTH levels of vehicle-injected rats remained below 80 pg/mL at all times and showed the expected circadian variations. Both CRF and stressin₁-A induced significant (P<0.01) and dose-related increases in ACTH levels at the 10 min time point, but the pituitary response to 5.0 μg oCRF/kg (1,810 ± 239 pg ACTH/mL) was significantly larger than that measured after 5.0 μg stressin₁-A/kg (752 ± 210 pg ACTH/mL, P<0.01). Animals administered 1.0 or 5.0, but not 0.2 μg oCRF/kg still had elevated (P<0.01) ACTH values 1 h after injection, and these values had returned to control levels by 3 h. Stressin₁-A, and 9 at 1.0 and 5.0 μg/kg and 6 at 5.0 μg/kg also increased (P<0.01) ACTH concentrations 1 h after treatment. The observation that the higher dose of the three peptides maintained up-regulated (P<0.01) ACTH release at the 3 h time points suggests that their duration of action is slightly extended, compared to that of oCRF.

Figure 2.

A = Ucn 2 (hUcn 2) but not 7 (stressin₁-A) injected ip decreased gastric emptying of a viscous noncaloric meal in conscious rats. Overnight fasted rats were injected ip with saline, Ucn 2 or 7 (stressin₁-A), and 10 min later gavaged with the phenol red methylcellulose solution and 20 min later gastric emptying was monitored. Each column is the mean ± SE of 5–6 rats/group. *p < 0.05 compared with both vehicle and stressin₁-A groups. B = 7 injected ip stimulated colonic motor function in conscious rats. Non fasted rats were injected ip with saline or 7 (stressin₁-A, 10 μg/kg) and fecal pellet output and diarrhea in conscious rats was monitored. Each column represents the mean ± SE of 6 rat/group. The numbers on the bars are the number of rats with diarrhea over the total in a group. #P<0.05 vs. saline and stressin₁-A; * P<0.05 vs. saline.

Gut motor function and blood pressure

Peptides were tested in three in vivo assays that are known to be mediated by either the CRF₁ receptor or the CRF₂ receptor. These are a colonic propulsive motor function test in non fasted rat, gastric emptying of non nutrient meal in an overnight fasted rat model and arterial blood pressure measure in an overnight fasted and urethane anesthetized rat model. CRF₁ receptor activation is well established to increase colonic transit and motility^11,14 while peripheral CRF₂ activation by CRF and related family members decreases gastric emptying^10,12–14 and blood pressure¹⁶ in rodents. Because these activities are mediated by either one or the other CRF receptor (as shown), these measurements were critical in our evaluation of the peptides’ selectivity. We found that Ucn 2, but not stressin₁-A, injected ip, delayed gastric emptying in conscious rats. Stressin₁-A injected ip at 10 μg/kg had no significant effect on gastric emptying of a viscous noncaloric solution in conscious rats (51.0 ± 8.8% compared with 50.0 ± 2.5% in the vehicle group: p = 0.92 ; Figure 2A). By contrast, ip hUcn 2 given at the same dose significantly reduced gastric emptying to 26.4 ± 6.9% compared to both vehicle and stressin₁-A groups (Figure 2A). We also found that stressin₁-A injected ip stimulated colonic motor response in conscious rats. In non fasted conscious rats, stressin₁-A injected ip at 10 μg/kg significantly increased the 60 min fecal pellet output when compared to saline injected rats (8.0 ± 1.6 vs. 0.5 ± 0.5/h; Figure 2B). Time course of the cumulative effects of stressin₁-A on fecal pellet showed an increased response at 30 min that became significant at 45 and 60 min. In addition, stressin₁-A induced diarrhea in 33% of the rats whereas none of the saline injected rats had diarrhea as monitored 60 min post injection (Figure 2B). Finally, stressin₁-A, injected iv, did not modify arterial blood pressure. In urethane anesthetized rats, mean basal blood pressure was 75.9 ± 3.5 mm Hg and was not significantly modified after vehicle iv injection (77.4 ± 1.3 mm Hg; p = 0.14 vs. basal). Neither consecutive iv injections of stressin₁-A at 3, 10 or 30 μg/kg nor of cortagine 1 at 3, 10 or 30 μg/kg produced significant changes in blood pressure compared to vehicle (+2.3 ± 1.4%, p > 0.05 ; −0.1 ± 0.7%, p > 0.05; − 1.6 ± 2%, p > 0.05 for stressin₁-A and 1.4 ± 0.8%, p > 0.05; 0.1 ± 0.3%, p > 0.05; −3.2 ± 0.7%, p > 0.05 for cortagine) (data not shown).

Using one in vivo bioassay (colonic fecal expulsion and diarrhea) in which the role of CRF₁ receptors has been well established to mediate the effects of peripheral injection of CRF and Ucn 1,¹¹ and two assays (decrease in gastric emptying and blood pressure changes induced by CRF or urocortins) mediated by CRF₂ receptor, we showed that peripheral administration of stressin₁-A resulted solely in a CRF₁ receptor-mediated biological response. We previously reported that the CRF₁ receptor/CRF₂ receptor agonists, Ucn 1 or CRF,¹⁹ injected ip at 10 μg/kg stimulated colonic transit while inhibiting gastric emptying through CRF₁ and CRF₂ receptors, respectively, in rats.¹⁴ Therefore the lack of change in gastric emptying together with potent stimulation of colonic motor function induced by stressin₁-A injected ip provided in vivo evidence for the CRF₁ receptor selectivity of stressin₁-A. This is further corroborated by the lack of change in blood pressure when stressin₁-A was injected iv and by several literature reports that emphasize the specific role of CRF₂ receptors in the CRF or Ucn 1-induced drop in blood pressure.^3,16,17

To conclude, potent peptidic CRF₂ receptor agonists (urocortins 2 and 3) and antagonists (astressin₂-B, anti-sauvagine-30) have been described previously. Here we report the development of stressin₁-A and present in vitro and in vivo data providing convergent evidence that stressin₁-A is a novel potent and selective CRF₁ receptor agonist that will be very useful for characterizing biological actions mediated by the CRF₁ receptor.³² Structural studies (NMR) are presently being carried out in order to determine the conformational differences that contribute to receptor selectivity. Still missing is a potent and long acting peptide CRF₁ receptor antagonist to complete the panoply of peptide reagents that are critical for unraveling the physiological roles of the CRF₁ receptor and the CRF₂ receptor, their possible inhibitory or potentiating interactions both centrally and peripherally and ultimately, their pharmacological clinical potential.

Experimental Section

Animals

Adult male Sprague-Dawley rats (Harlan, San Diego, CA) weighing 250–300 g were housed in group cages under controlled illumination (12:12-h light-dark cycle starting at 6 AM), humidity, and temperature (21–23°C) and had free access to tap water and Purina rat chow. Protocols were approved by the respective IACUCs of the University of California Los Angeles and Veteran Affairs Greater Los Angeles Healthcare System Animal Research Committees and the Salk Institute. When required by the experiments, rats were deprived of food but had free access to tap water for ~16 h before the experiment.

Administration of peptides

Immediately before injection, stressin₁-A was dissolved in vehicle (water or saline). Human Ucn 2 and cortagine were dissolved in water. Ip and iv injections were done respectively in 0.3 mL and in 0.1 mL.

Rat dispersed pituitary cell culture assay

CRF analogues were tested for agonist activity in an in vitro assay measuring release of ACTH by collagenase dispersed rat anterior pituitary cells in culture.^27,29 For each in vitro experiment, approximately 50 anterior pituitary glands from male Sprague-Dawley rats were dissociated by collagenase treatment and plated (0.16 × 10⁶ cells/well in 48-well plates) in medium containing 2% fetal bovine serum (FBS). Three days after plating, the cells were washed three times with fresh medium containing 0.1% bovine serum albumin (BSA) and incubated for 1 h. Following the 1 h pre-incubation, the cells were washed once more and the test peptides were applied in 1 mL final volume of media. At the end of a 3 h incubation period, the media were collected and 10 μL tested for their level of ACTH using a radioimmunoassay (Nichols Institute Diagnostic).

Cloned receptor-based binding assays

The K_i values given in Table 1 reflect the affinities of the analogues for the cloned type 1 human and type 2β mouse CRF receptors. The values were derived from competitive radioligand displacement assays using the non-selective ¹²⁵I-labeled agonist [Tyr⁰,Glu¹,Nle¹⁷]-sauvagine as the radioligand and crude membrane fractions from CHO cells stably expressing the respective receptors.²⁶ Briefly, 200,000 cpm (ca. 0.5 nM) ¹²⁵I-[Tyr⁰,Glu¹,Nle¹⁷]-sauvagine were combined with increasing concentrations of peptide (0.1 nM - 1,000 nM) in 0.2 mL assay buffer (50 mM Na Hepes, pH 7.5; 10 mM MgCl₂; 2 mM EGTA; 0.1% BSA) and incubated for 90 min at 18 ºC.

Measure of gastric emptying of non nutrient meal

Gastric emptying of a non nutrient viscous meal was determined by the phenol red method as described previously in conscious over night fasted rats.³³ The non-caloric meal consisted of a viscous suspension of continuously stirred 1.5% methylcellulose (wt/vol) containing phenol red (50 mg/100 mL) given orogastrically through a stainless steel gavage tube (in 1.5-mL volume) to conscious rats. At 20 min after the administration of the solution, rats were euthanized by CO₂ inhalation. The abdominal cavity was opened, the gastroesophageal junction and the pylorus were clamped, and the stomach was isolated and rinsed in 0.9% saline. The stomach was placed into 100 mL 0.1 N NaOH and homogenized (Polytron; Brinkman Instruments, Westbury, NY). The suspension was allowed to settle for 60 min at room temperature, then 5 mL supernatant was added to 0.5 mL of 20% trichloroacetic acid (wt/vol) and centrifuged at 3,000 rpm at 4°C for 20 min. After the supernatant was mixed with 4 mL 0.5 N NaOH, the absorbance of the sample was read at 560 nm (Shimazu 260 Spectrophotometer). The absorbance of the phenol red recovered from animals euthanized immediately after gavage of the liquid meal was taken as a standard 0% emptying. The percentage of emptying during the 20-min period was calculated with the following formula: percent emptying = (1 – absorbance of test sample/absorbance of standard) × 100.

Measure of colonic propulsive motor function

In non fasted conscious rats, defecation was monitored as described previously³⁴ by counting the number of fecal pellets excreted every 15 min for 1 h. The incidence of diarrhea was assessed as percent of rats that developed watery stool from the total number of treated rats.

Measure of arterial blood pressure

Over night fasted rats were anesthetized with urethane (1.5 g/kg). Each rat was then fitted with a tracheal cannula for ventilation, a jugular vein catheter for hydration through continuous perfusion of sterile saline (0.4 mL/h) and for peptide injection, and a femoral artery catheter for BP measurement. The femoral catheter was connected to a preamplifier (model 600; Millar Instruments, Houston, TX), the output of which was subsequently amplified via a single-ended connection to the transducer amplifier. BP was acquired online at a sampling rate of 300 Hz via a Micro1401 A/D interface (Cambridge Electronic Design, Cambridge, UK) connected to a Pentium II class computer running Spike 2 (Cambridge Electronic Design) version 5 data acquisition software.

Effect of iv oCRF and stressin₁-A on ACTH release in vivo

Adrenal-intact adult (ca. 60-day-old) male Sprague-Dawley rats were implanted with iv cannulae as described earlier.²⁰ The test was conducted under minimum-stress conditions in fully awake and freely-moving animals that were not handled for either peptide injection or blood sampling. A first blood sample was obtained, immediately followed by the iv injection of the vehicle, oCRF (0.2, 1.0 or 5.0 μg/kg) or stressin₁-A (1.0 or 5.0 μg/kg). Subsequently, blood samples were obtained 10 min, as well as 1, 3, 6 and 24 h later. ACTH levels were measured in plasma with an immunoradiometric assay from DiaSorin Inc. (Stillwater, MN).

Effect of intraperitoneal hUcn 2 and stressin₁-A on gastric emptying in conscious rats

Rats were deprived of food overnight but had free access to tap water for ~16 h before the experiment. Fasted rats were injected ip (0.3 mL) with saline, Ucn 2 (10 μg/kg) or stressin₁-A (10 μg/kg). At 10 min after the ip injection, the phenol red methylcellulose solution was delivered through orogastric gavage (1.5 mL), and gastric emptying was determined 20 min later. Doses of peptides were based on previous dose-response studies showing a maximal response of peripheral injection of Ucn 2 on gastric emptying in rats and mice.^12,14

Effect of ip stressin₁-A on colonic motor response in conscious rats

Rats were injected ip with saline or stressin₁-A (10 μg/kg) and then immediately placed in individual cages to monitor the fecal pellet output and diarrhea response. These colonic responses were monitored every 15 min for 60 min after the ip injection. The dose of stressin₁-A was selected on a previous dose-response study showing maximal colonic response to CRF injected ip at 10 μg/kg.^14,34

Effect of iv stressin₁-A and cortagine on arterial blood pressure in anesthetized rats

Basal BP was monitored for 150 to 200 min using an acute catheter positioned into the femoral artery in urethane anesthetized rats (n = 3). Each animal received consecutive intrajugular injections of vehicle (distilled water), stressin₁-A at 3, 10 and 30 μg/kg, and cortagine at 3, 10 and 30 μg/kg at 15–20 min intervals. BP was recorded throughout the studies. For each treatment, mean basal BP was recorded for 5 min before and for 2 min 30 sec after the injection. Normalized values were obtained by subtracting mean basal BP from the mean BP after injection to obtain the net BP response and dividing it by the mean basal BP, yielding the percent change from the basal state.

Statistical analysis

All results are expressed as mean ± SEM. Comparison between multiple groups was performed by one-way ANOVA followed by Student Newman-Keuls multiple comparisons.

Figure 1.

Plasma ACTH levels in rats injected with the vehicle, 2 (oCRF), 6, 7 (stressin₁-A) and 9. Each point represents the mean ± SEM of 6–7 animals. **, P<0.01 vs. vehicle.

Additional abbreviations

BP

blood pressure

BOP

(benzotriazol-1-yloxy)-tris(dimethylamino)phosphonium-hexafluorophosphate

BSA

bovine serum albumin

h/rCRF

human/rat corticotropin releasing factor

oCRF

ovine CRF

CRF₁

CRF receptor 1

CRF₂

CRF receptor 2

mCRF₂

mouse CRF₂

DIC

diisopropylcarbodiimide

CZE

capillary zone electrophoresis

DCM

dichloromethane

DMF

dimethylformamide

Fmoc

9-fluorenylmethoxycarbonyl

HBTU

O-(benzotriazol-l-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate

HPLC

high performance liquid chromatography

ip

intraperitoneal

iv

intravenously

LSIMS

liquid secondary ion mass spectrometry

MeOH

methanol

MS

mass spectrometry

NMP

N-methylpyrrolidone

Ofm

O-fluorenylmethyl

RPHPLC

reversed phase high performance liquid chromatography

SAR

structure activity relationships

sc

subcutaneous

TBTU

O-(benzotriazol-l-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate

TEAP

triethylammonium phosphate

TEA

triethylamine

TFA

trifluoroacetic acid

TFE

2,2,2-trifluoroethanol

Xaa

any amino acid