Structural modifications of non-mammalian gonadotropin-releasing hormone (GnRH) isoforms: design of novel GnRH analogues

ELSEVIER

Regulatory Pepfides 60 (1995) 99-115

Structural modifications of non-mammalian gonadotropin-releasing hormone (GnRH) isoforms: design of novel GnRH analogues D.A. Lovejoy a,b,*, A.Z. Corrigan a, C.So Nahomiak b, M.H. Perrin a, j. Porter a, R. Kaiser a, C. Miller a, D. Pantoja a, A.G. Craig a, R.E. Peter b, W.W. Vale a, J.E. Rivier a, N.M. Sherwood c The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, 10010 N. Torrey Pines Rd., La Jolla, CA 92037, USA b Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada c Department of Biology, University of Victoria, Victoria, BC V8W 2Y2, Canada

Received 20 April 1995; revised 15 July 1995; accepted 20 July 1995

Abstract

Three natural forms of vertebrate gonadotropin-releasing hormone (GnRH) provided the structural basis upon which to design new GnRH agonists: [HisS,Trp7,LeuS]-GnRH, dogfish (df) GnRH; [HisS,AsnS]-GnRH, catfish (cf) GnRH; and [HisS,Trp7,TyrS]-GnRH, chicken (c) GnRH-II. The synthetic peptides incorporated the position 6 dextro (D)-isomers D-arginine (D-Arg) or t)-naphthylalanine (D-Nal) in combination with an ethylamide substitution of position 10. The in vitro potencies for LH and FSH release of these analogues were assessed using static cultures of rat anterior pituitary cells. Efficacious peptides were examined for their gonadotropin-II and growth hormone releasing abilities from perifused goldfish pituitary fragments. Rat LH and FSH release was measured using homologous radioimmunoassays, whereas goldfish growth hormone and gonadotropin-II release were determined using heterologous carp hormone radioimmunoassays. The receptor binding of the most potent analogues was determined in bovine pituitary membrane preparations. Substitution of D-Nal 6 into [HisS,AsnS]-GnRH increased the potency over 2200-fold compared with the native ligand (cfGnRH) in cultured rat pituitary cells. This was equivalent to a 55-fold greater potency than that of the native mammal (m) GnRH peptide. Substitution of D-Nal 6 or D-Arg6 into dfGnRH or cGnRH-II resulted in potencies that were related to the overall hydrophobicity of the analogues. The [D-NaI6,Pro9NEt]-cfGnRH bound to the bovine membrane preparation with an affinity statistically similar to that of [D-Nal6,Pro9NEt]-mGnRH (k d = 0.40 _ 0.04 and 0.55 5:0.10 nM, respectively) in cultured rat pituitary cells. All analogues tested released the same ratio of FSH to LH. In goldfish, the analogues did not possess superagonistic activity but instead desensitized the pituitary fragments at lower analogue doses than that of the sGnRH standard suggesting differences in receptor affinity or signal transduction. Keywords: Reproduction; Evolution; Neuropeptide; Releasing factor; Hydropathy

* Corresponding author. Fax: + 1 (619) 5521546. 0167-0115/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0167-0115(95)00116-6

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D.A. Lovejoy et al. / Regulatory Peptides 60 (1995) 99-115

1. Introduction The successful application of gonadotropin-releasing hormone (GnRH) analogues in the treatment of hypogonadotropism [1] led to their exploitation in a host of related pathological conditions including precocious puberty [2] polycystic ovary syndrome [3], breast cancer [4,5], endometriosis [6] and fibroids [7]. However, in spite of the clinical successes of these analogues, a variety of side effects including hypersensitivity and the formation of ovarian cysts have been reported [8]. The primary structures of the majority of these analogues are based on the amino acid sequence of mammal GnRH. Recent evidence suggests more than one form of GnRH may be present in the mammalian and possibly human brain [9,10]. Since the initial discovery of the GnRH primary structure, [11,12] over 4000 synthetic agonists and antagonists have been synthesized for various uses. Stabilization of the /3 II turn using dextro (D)isomer substitutions in the 6th residue position in combination with a C-terminal ethylamide group can yield a particularly potent molecule [13]. Physiological events, such as enzymatic degradation, receptor binding and activation, plasma protein binding and renal clearance rates can be affected by these structural modifications on the native hormone. Structure-function and computer modeling studies of the mammalian GnRH molecule suggest that the N-terminal pyroglutamic acid, C-terminal glycine amide, tryptophan 3 and arginine 8 side chains are essential for receptor binding [14]. Yet, of the numerous naturally occurring vertebrate forms of GnRH, residues 2, 4 and 9 are conserved [15] suggesting essential structural/functional attributes to these residues. Positions 5, 7 and 8 are variable in jawed vertebrates. The position 8 residue may provide specificity to the molecule as it is the most variable [15]. In contrast, only histidine or tyrosine is found in position 5 and tryptophan or leucine is found in position 7 suggesting conserved functions with these residues. Although some of the non-mammal GnRH forms possess little or no activity in mammalian systems, others do show a significant effect. In 1992, the primary structures were reported for two new forms of GnRH; one from the brain of a dogfish shark [16] and the other from Thai catfish [17]. The structure of

dogfish GnRH ([HisS,TrpT,LeuS]-GnRH; dfGnRH) had been previously anticipated by Folkers and his associates [18] who predicted the presence of new GnRH sequences based on an examination of 5, 7 and 8 position residues of non-mammal GnRH molecules. They reported the synthetic version of dfGnRH not only released significant amounts of LH and FSH in vivo, but the ratio of LH to FSH differed from that stimulated by mammal GnRH. Like mammal GnRH, catfish GnRH ([HisS,AsnS]-GnRH; cfGnRH) possesses leucine in position 7. However, this isoform has histidine in position 5 reflecting the GnRH structure in phylogenetically older vertebrate lineages. This position appears to play a significant role in the recognition of the mammalian GnRH receptor for its ligand [13]. Chicken GnRH-II ([HisS,TrpT,TyrS]-GnRH; cGnRH-II) is present in the brain of vertebrates from most classes [ 15,19]. Furthermore, indirect evidence indicates it may be present in the insectivores, musk shrew and mole [9,10]. The insectivore and primate lineages are believed to have a common ancestor in recent phylogenetic history [20]. This report [20], in combination with the widespread occurrence of cGnRH-II suggests its presence in the primate brain. Previous studies indicate that this hormone releases significant quantities of LH in vivo in the rat [21] and sheep [22]. Indeed, in most vertebrate species, this form of GnRH tends to be one of the most potent of the naturally occurring GnRH molecules [15]. Further evidence suggests cGnRH-II as well as other forms of GnRH may play a role in the release of growth hormone in fishes and possibly other species [16,17,23-26]. The goldfish is a viable model system to examine the release of growth hormone by GnRH. These three GnRH forms (cGnRH-II, cfGnRH and dfGnRH) may provide the structural basis upon which to design new GnRH analogues for therapeutic uses in humans and other species. In continuation of our investigation of the structure-function relationships of the vertebrate GnRHs, the biological potency and receptor affinity of structurally stabilized D-isomer modified cfGnRH, dfGnRH and cGnRH-II were investigated in mammalian-based assay systems. Further, these analogues were used to determine if differential regulation of the gonadotropins would occur as a function of different

D.A. Lovejoy et al./ RegulatoryPeptides60 (1995) 99-115 GnRH sequences. The most potent analogues in the mammalian system were investigated in cultured goldfish pituitary cells to determine if this activity could be reproduced in a species widely divergent from mammals.

2. Materials and methods 2.1. Peptide synthesis The amino acid derivative tBoc-D-arginine was obtained from Bachem Inc. (Torrance, CA) and BocD-naphthylalanine was synthesized at the Southwest Foundation for Biomedical Research (contract N01HD 6-2928) and made available by the Contraceptive Development Branch, Center for Population Research, NICHD. The methylbenzhydrylamine (MBHA) resin used for the peptide synthesis was obtained as previously reported [27]. The resin bound peptides were prepared using solid-phase peptide synthesis [28] on a Beckman 990 peptide synthesizer with use of previously described protocols [29] on the MBHA resin (1-2.5 g per peptide) using tertbutyloxycarbonyl groups for N a amino protection. 2.2. Peptide purification The lyophilized crude peptide preparations were dissolved in 200 ml 0.25 M triethylammonium phosphate (TEAP, pH 2.25) and loaded onto a 5 × 30 cm preparative reverse-phase HPLC cartridge packed with Vydac C18 silica 330 ,~ pore size, 15-20 /xm particle size. The peptide was eluted with a flow rate of 100 m l / m i n on a Waters Prep 500 System using a mixture of reservoir A (TEAP, pH 2.25) and reservoir B (60% CH3CN, 40% A) with an appropriate

Table 1 Mass spectral analysesof syntheticGnRHanalogues GnRH analogue Observed m/z monoisotopomer [D-Arg 6,Pro 9NEt]-cGnRH-II 1306.5 [D-Na]6,Pro 9NEt]-cGnRH-II 1347.4 [D-Arg6,ProgNEtl-cfGnRH 1184.4 [D-Nal6,Pro9NEt]-cfGnRH 1225.3 [D-Arg6,Pro9NEt]-dfGnRH 1256.5 [D-Nal6,Pro9NE0-dfGnRH 1297.3

101

gradient (90 min) to provide a retention time of about 45 min. The collected fractions were screened by use of analytical reverse-phase HPLC under isocratic conditions: 0.1% T F A / H 2 0 at a flow rate of 2 m l / m i n (Vydac Cls column 5 ~ m , 300 A, pore size; 4.5 X 250 ram). Appropriate fractions were then combined and converted to the acetate salt by loading after dilution ( 1 / 1 ) in water on a preparative reversed-phase HPLC cartridge and eluted with a mixture of solvents A (0.05% acetic acid) and B (60% CHaCN, 40% A) with an initial gradient of 20% B (10 min) followed by a 20 min gradient to 90%. 2.3. Peptide characterization Amino acid analyses (after 4 M methanesulfonic acid hydrolysis at 110°C) for 24 h were performed on a Perkin Elmer HPLC using o-phthaldehyde postcolumn derivatization and fluorescence detection. Peptide purity was assessed by HPLC using a concentration of 0.5 /zg/5 /xl. The Vydac C18 column length was 0.21 X 15 cm. Reservoir A consisted of 0.1% TEAP and reservoir B was a mixture of 60% acetonitrile in 0.1% TEAP. Analyses were performed at 40°C at a flow rate of 0.2 m l / m i n and detected at 210 nm. Linear gradient conditions were varied to allow for an elution time of about 20 min. LSIMS mass spectra were measured with a JEOL JMS-HXll0 double focusing mass spectrometer (JEOL, Tokyo) fitted with a Cs ÷ gun. An accelerating voltage of + 10 kV and Cs ÷ gun voltage of +25 kV were employed. The sample (ca. 10 /.Lg; TFA salt; lyophilized) was added directly to a glycerol and 3-nitrobenzyl alcohol (1:1) matrix. The magnetic field spectra were calibrated using Cs (CsI), cluster peaks whilst the linked field scans at constant B / E

Calculated[M + H] ÷ monoisotopicmass (Da) 1306.62 1347.61 1184.61 1225.59 1256.64 1297.63

102

D.A. Lovejoy et al. / Regulatory Peptides 60 (1995) 99-115

ratio (product ion) were calibrated with Ultramark 1621 (PCR Inc., Gainesville, FL). The magnetic field and product ion spectra were measured at a nominal resolution of 3000.

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Fig. 1. Purification of GnRH analogues by reverse-phase HPLC. The absorhance (A = 210 nm) of two representative compounds (a, [D-Na]6,Pro9NEt]-cfGnRH; b, [D-Arg6,Pro9NEt-]cGnRH-II)are shown. Reservoir A consisted of 0.1% triethylammonium phosphate (TEAP) and reservoir B, 60% acetonitrile in 0.1% TEAP. The reservoir B gradient was adjusted to provide an elution time of about 20 min. A single sample peak is evident indicating the synthesized peptide is uncontaminated by racemization products or other peptides.

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The anterior pituitary glands were dissected from mature (12 months) male S p r a g u e - D a w l e y strain rats. The tissue was extensively rinsed in Hepes buffer, then dissociated by incubation in 0,4% collagenase and deoxyribonuclease II at 37°C for 2 h in a jacketed spinner flask followed by a subsequent incubation in 0.25% Viokase (Gibco) for 8 min. The dissociated cells were washed and plated in fl-PJ culture medium (Scientific services, Salk Institute) containing nystatin, S a t o ' s cocktail (insulin, 5 m g / 1 ; transferrin 5 m g / 1 ; parathyroid hormone, 0.5 r a g / l ;

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Fig. 2. Primary structure of GnRH analogues. Each of the peptides were prepared using solid phase synthesis (see Section 2). The analogues differed by the natural substitution of His or Tyr in position 5, and the synthetic substitution of o-arginine (D-Arg) or ~naphthylalanine (o-Nal) in position 6, and the NHCH2CH3 (ethylamide) substitution in position 10. The incorporation of D-arginine imparted greater hydrophilicity to the peptide whereas o-naphthylalanine resulted in an analogue of increased hydrophobicity. The iodinated analogue used for the receptor assays incorporated an additional modification at position 7 (a-methyl leucine, MLeu). cGnRH-II, chicken GnRH-II; dfGnRH, dogfish GnRH; cfGnRH, catfish GnRH; mGnRH, mammal GnRH; sGnRH, salmon GnRH.

D.A. Lovejoy et al. / Regulatory P eptides 60 (1995) 99-115

T3; 30 pM and fibroblast growth factor, 1 m g / l ) supplemented with 2% ( w / v ) fetal bovine serum (Hyclone). The cells were grown to a density of 5.0.106 cells in 60 mm culture dishes and were allowed to attach for 3 days at 37°C under 5% CO 2 [30]. Dilutions of the GnRH analogues were prepared using 0.05% acetic acid. The dosage range was calculated to provide zero to maximal gonadotropin secretion. The cells were pretreated with either the vehicle (0.05% acetic acid) or the GnRH analogue. The culture medium was sampled for radioimmunoassay 4 h later. Putative superagonists were assayed 3-5 times for an accurate measurement of their gonadotropin-releasing activity. Secreted LH and FSH were determined by radioimmunoassay using the materials provided by the NIDDK program containing the LH and FSH RP-2 standards. All assays were performed in triplicate. The potencies were determined relative to the response elicited by mammal GnRH and were calculated using the EDs0 from each dose-response curve. Sexually mature goldfish (Carassius auratus, Comet variety; Ozark, MO) of mixed sex were held in 100- or 300-1 aquaria at 18°C with a photoperiod of 16 h light-8 h dark. All analyses were performed during a 2-week period in November, 1991 to avoid seasonal affects. The fish were anesthetized in 0.05% tricaine methanesulfonate (MS-222). The pituitaries were dissected, minced and cultured as previously described [31]. The fragments (an equivalent of 3 pars distali) were transferred to 0.5 ml microchambers. The fragments were equilibrated overnight at 18°C in Medium 199 at a flow rate of 5 m l / h . The perifusion of pituitary fragments was based on previously established methods [26,31]. The perfusion apparatus consisted of 8 Teflon microchambers (columns) connected to an IPN Ismatec peristaltic pump. Each set of 2 columns was connected to a single reservoir of HHBSA and the GnRH analogue to be examined. The synthetic peptides were diluted to the desired concentration in 5 ml of Hanks buffered salt solution supplemented with in 25 mM Hepes and 0.1% BSA (HHBSA). Following overnight equilibration in Medium 199, perifusion of fragments with HHBSA at 15 m l / h began, After 2 h of perifusion with HHBSA, 5-min fractions (1.2 ml) from each of the 8 columns were collected. The GnRH analogue of interest was delivered as a 2-min pulse every 90

103

min. The concentrations were delivered in ascending order. The fractions were frozen at - 2 5 ° C immediately after collection. In each set of perifusions, salmon GnRH was run as a standard. Gonadotropin secretion by the goldfish pituitary fragments was measured by a heterologous radioimmunoassay using carp gonadotropin-II as both the iodinated and competing epitopes. This method has been previously validated to measure gonadotropin-II secretion in the goldfish [32]. Similarly, a carp growth hormone radioimmunoassay [23] was used to determine the secretion of goldfish growth hormone. The hormone concentration in each fraction of column perifusate was measured in duplicate.

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D.A. Lovejoy et al. / Regulatory Peptides 60 (1995) 99-115

104

tides for 2 h on ice in a final volume of 100-200/xl. The reaction was quenched by the addition of 1 ml ice-cold saline and immediately filtered through Whatman G F / C filters presoaked in 10 mM Hepes containing 1% BSA. All filters were counted in a gamma counter.

2.5. Receptor binding assay The agonist [D-Ala6,t~ MeLeuT,Pro9NEt]-GnRH was iodinated using chloramine T and purified by HPLC as previously described [33]. The assay was modified from a previously published procedure [34]. The excised anterior lobe was homogenized in 10 vol. of ice-cold 0.32 M sucrose, then centrifuged at 600 g for 5 min. The pellet was washed twice with 10 vol. 0.32 M sucrose. The resulting supernatants were combined and centrifuged at 48,000 g for 20 min. The top layer of the resulting pellet was resuspended in equal volumes of 10 mM Hepes (pH 7.6) and stored at -20°C. The radioligand (70,000100,000 cpm, ca. 20 fmol) in binding assay buffer (0.2% BSA, 10 mM Hepes pH 7.6) was incubated with plasma membrane fraction and unlabeled pep-

2.6. Statistical analyses Statistical variation between experimental groups was calculated using a one-way analyses of variance. In data sets with P < 0.05, Student-Neumann-Keuls multiple comparison tests incorporating Kramer's extension [35] were used to determine significant differences among the hormone release response within each experiment. An unpaired two-tailed Students t-test was used to assess the significance be-

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GnRH concentration (nM) Fig. 4. LH and FSH release by GnRH analogues in static cultures of rat anterior pituitary cells. LH and FSH secreted into the culture medium were measured by homologous rat gonadotropin radioimmunoassays. The mean + S.E.M. are indicated. The concentration of the secreted hormone was determined in triplicate within each assay. Each assay was performed 3 - 5 times. The ratio of LH to FSH release did not vary between analogues. See text for further description.

D.A. Lovejoy et al./Regulatory Peptides 60 (1995) 99-115

tween the means of LH and FSH response by incremental mammal GnRH doses. All statistical analyses were performed using 'InStat' 1.14 statistical software (GraphPad Software Inc., San Diego, CA, USA).

monoisotopic mass calculated for the [ M + H] + (Table 1). Linked field scans at constant B / E ratio (product ion scans) were measured for each of the synthetic analogues. Consecutive series of N-terminal fragment ions observed in the product ion scans were used to confirm the incorporation of the naphthylalanine residue and the overall sequence. The confirmed sequences of each peptide are presented in Fig. 2.

3. Results

The synthesized peptides were not significantly contaminated by either racemization products or other peptides as shown by amino acid analysis and HPLC separation (see Fig. 1) The observed m / z of the monoisotopomer compared favorably with the

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Fig. 5. Relationship between hydropathy and biological potency among GnRH isoforms. (a) Hydrophilicity of GnRH forms as defined by predicted relative retention times from a reversed-phase C ~8 HPLC column using trifluoroacetic acid and acetonitrile as the mobile phase [33]. The scale is calibrated in relative units where glycine = 0. Each GnRH value represents the sum of the retention time for each residue. (b) Predicted relative polarity of the 13II turn defined by residues 5 - 8 utilizing the scale established in [34]. The scale units are kcal/nmol and represent the free energies required for the transfer of an amino acid in an a helix from the membrane inl~dor to water. Glycine = 1. Substitution of the hydrophobic residue o-naphthylalanine into cfGnRH enhances potency much greater than the o-arginine substitution. The opposite is true for bydrophobic GnRHs where the D-arginine substitution potentiates hormone release greater than o-naphthylalanine. The ordinate scale on each graph is calibrated as the increase in biological potency over the natural L-isomer sequence (see Fig. 3).

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pituitary cells in a dose-dependent manner for concentration 0.02-5 nM (data not shown). The amount of FSH released into the culture medium was typically 50-70% that of LH released as determined by the radioimmunoassays. The dose-response curves of GnRH analoguestimulated gonadotropin release for each analogue were parallel relative to both the mammal GnRH and [D-Nal6,Pro9NEt]-mGnRH standards. The LH release potency of each GnRH analogue was compared to that of mammal GnRH (mGnRH), which was assigned a potency equal to 1. The relative potency was defined as: log [Ga/GmGnRrl] w h e r e Ga is the amount of LH released by each analogue and Gm~,a H is the amount released by mGnRH (Fig. 3). Thus, analogues with a potency greater than mGnRH possess a positive value whereas those with decreased potency had a negative value. Among the superago-

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Fig. 7. Displacement of 1251-[D-AIa6,NotMeLeuT,Pro9NEt]mGnRH from bovine pituitary cell membranes by GnRH analogues. Non-specific binding was subtracted from the total for the calculation of B / B o. O, [D-Ala6, Not MeLeuT,Pr09 NEt]-mGnRH; II, [D-NaI6,Pro9NEt]-mGnRH; O, [D-Nal 6, Pro9NEt]-cfGnRH; zx, cfGnRH.

nist analogues the interassay coefficient of variation was less than 20%. The potency of all native nonmammalian GnRHs were markedly lower than that obtained with mGnRH. Substitution of the D-isomer residues into the native molecules resulted in increased gonadotropin release for all 5 of the native analogues tested, sGnRH and cfGnRH possessed the lowest potency of all GnRH forms ( - 1 . 6 8 and 1.60, respectively). These peptides represent opposite poles of a hydrophilicity continuum among the -

Fig. 8. Gonadotropin release from perifused goldfish anterior pituitary fragments, a, b: salmon GnRH (n = 5). c, d: [~Arg6,Pro9NEt] cGnRH-II (n = 4). e, f: [a-Arg6,ProqNEt]-dfGnRH (n = 4). g, h: [D-Nal6,ProqNEt]-cfGnRH (n = 3). a, c, e and g in the left column indicate the untransformed profile of gonadotropin release from one set of perifused fragments. The GnRH was delivered as a 2-min pulse (shown by the black vertical bar) every 90 rain. Five-rain fractions (1.2 ml) were obtained. Gonadotropin was determined by using a carp gonadotropin radioimmunoassay, b, d, f, and h in the right column represent mean gonadotropin release from all columns in separate experiments. The data have been transformed as a function of the baseline gonadotropin release defined as the prepulse mean (see Section 2). The mean + S.E.M. are indicated. Statistical significance was calculated using Student-Neumann-Keuls multiple comparison test. Comparison of each dose with that of the first and lowest dose is shown by asterisks over the column. Brackets indicated comparison between column pairs. * P < 0.05; * * P < 0.01; * * * P < 0.001.

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D.A. Lovejoy et al. / Regulatory Peptides 60 (1995) 99-115

native GnRH forms. The most potent analogue within the series was [D-Nal6,Pro9NEt]-mGnRH. It stimulated the release of LH 127-fold greater than mGnRH. It was included in the assay to provide a measurement of the upper limit for GnRH analogues using the substitutions in this investigation. The high activity of [D-NaI6,Pro9NEt]-cfGnRH was surprising. Its mean relative potency of 1.75 represented a 55-fold increase over mGnRH and 2227-fold increase over that of the native (cfGnRH) form. The 3 most effective analogues were examined for their ability to release FSH from the rat cultured pituitary cells (Fig. 4). The order of potency was [ D - N a I 6 , P r o 9 N E t ] - m G n R H > [D-Nal6,Pro9NEt]c f G n R H > [D-Arg6,Pro9NEt]-cGnRH-II and reflected the same order of potency for LH release. For each of the 4 peptides the ratio of FSH to LH release ranged between 0.64 and 0.68. The differences were not significant. The role of hydropathy was apparent in the potencies of the analogues. Fig. 5 depicts the relative hydrophilicity of the 5 native forms of GnRH studies defined by: (1) the predicted retention time on a reversed-phase (C is) HPLC column [35] and (2) the free energy required to transfer an a helix of a given residue from a membrane interior to water [37]. The prediction of retention time in ' a ' correlates well (r = 0.963) with previous measurements of GnRH retention times using an alternate mobile phase and solvent gradient [38]. Substitution of o-arginine into the 6th position of cGnRH-II and dfGnRH yielded an analogue of greater potency than the substitution of D-naphthylalanine, a particularly hydrophobic residue. In contrast, the greatest potency of the hydrophilic peptides cfGnRH was produced when Dnaphthylalanine was present in position 6 compared with D-arginine. Hence, the natural GnRHs have improved potency if their relative hydropathic value

109

is around that indicated by the position of cGnRH-II and m G n R H in Fig. 5a. Similarly, if the predicted retention time of various L-isomer substituted GnRH analogues and homologues are plotted against their relative biological potencies [39] in a sheep LH assay, an optimum hydropathy value of about ' 4 0 ' is obtained when using the scale in 'a' (Fig. 6). The relationship is much more striking when the arginine is removed from the 8th position. 3.2. Receptor binding assays

The receptor binding affinities of the most potent GnRH analogue, [D-Nal6,ProgNEt]-cfGnRH and native cfGnRH were compared with two mammal GnRH analogues (Fig. 7). Competitive displacement of the iodinated analogue, t25I-[DAla6,NaMeLeuT,Pro9NEt]-mGnRH, occurred with all 4 peptides examined. The affinities of both of the mammal GnRH analogues for the bovine membrane preparations were statistically equivalent; k d = 0.40 __+ 0.04 nM and 0.42 _+ 0.04 nM for [DNal6,ProgNEt]-m GnRH and [DAla6,Na MeLeuT,Pro9NEt]-mGnRH, respectively. The [D-NaI6,Pro9NEt]-cfGnRH analogue had an affinity of 0.55 + 0.10 nM, which was not significantly different from that of the mGnRH analogues. In contrast, the affinity of the native catfish GnRH for the membrane homogenates was 7 0 0 - 1 0 0 0 fold less than the o-isomer constrained analogues (k d = 400 + 84 nM). 3.3. Goldfish gonadotropin release

The gonadotropin-II (Gth-II) release response for each pulse of GnRH was calculated relative to the baseline response. The mean of the 3 basal values immediately prior to the GnRH pulse (prepulse mean) was defined as 100%. Each peak was expressed as a

Fig. 9. Growth hormone release from perifused goldfish anterior pituitary fragments, a, b: salmon GnRH (n = 5). c, d: [o-Argf,ProgNEt]cGnRH-II (n = 4); e, f: [D-Arg6,Pro9NEt]-dfGnRH(n = 4). g, h: [D-NaI6,Pro9NEt]-cfGnRH(n = 3). a, c, e and g in the left column indicate the untransformed profile of growth hormone release from one set of perifused fragments. The GnRH was delivered as a 2-min pulse (shown by the black vertical bar) every 90 mins. Five-rain fractions (1.2 ml) were obtained. Growth hormone was determined by using a carp growth hormone radioimmunoassay, b, d, f, and h in the right column represent mean gonadotropin release from all columns in separate experiments. The data have been transformed as a function of the baseline growth hormone release defined as the prepulse mean (see Section 2). The mean + S.E.M. are indicated. Statistical significance was calculated using Student-Neumann-Keuls multiple comparison test. Comparison of each dose with that of the first and lowest dose is shown by asterisks over the column. Brackets indicated comparison between column pairs. * P < 0.05; * * P < 0.01.

110

D.A. Lovejoy et al. / Regulatory Peptides 60 (1995) 99-115

percentage above the prepulse mean. The Gth-II release response was considered to be initiated when the concentration of immunoreactive Gth-II within the perifusate fraction exceeded one standard error of the prepulse mean. The Gth-II release response was considered terminated when the values returned to within the standard error. This method of data transformation has been previously described and standardized [40]. Salmon (s) GnRH elicited a linear dose-dependent release of Gth-II by the perifused pituitary fragments over the concentrations 0.1-100 nM (Fig. 8a, b). Maximal Gth-II release-response at the 100 nM dose was 1419 + 284% above the basal release. A subsequent sGnRH pulse of 1000 nM elicited a release of 966 + 130% indicating a plateau in the Gth response. The goldfish pituitary fragments possessed decreased tolerance for high concentrations of the [D-Arg6,Pro9NEt]-cGnRH-II analogue (Fig. 8c, d) Maximal response was achieved at the 10-nM dose. The hormone response of 789 + 251% above basal levels was significantly ( P < 0.05; SNK Multiple Comparison test) greater than the amount released at 0.1 and 1 nM, but not significantly different from the Gth-II release elicited by 10 nM salmon GnRH. A subsequent inverse relationship between Gth-II release and GnRH dose was observed for doses 10-1000 nM indicating a desensitization of the pituitary fragments for this GnRH analogue. In contrast to sGnRH and [D-Arg6,Pro9NEt]-cGnRH-II, [D-Arg6,ProgNEt]-dfGnRH (Fig. 8e, f) produced the greatest Gth release at the lowest dose tested (0.1 nM) This response elicited by [D-Arg6,ProgNEt]dfGnRH was significantly greater than sGnRH ( P < 0.001), [D-Arg6,Pro9NEt]-cGnRH-II ( P < 0.01) and [D-Nal6,Pro9NEt]-cfGnRH analogue ( P < 0.05) doses, but was statistically similar to that produced by the 10 nM dose response in sGnRH (Fig. 8b) and [D-Arg6,Pro9NEt]-cGnRH-II (Fig. 8d). Stimulation of Gth-II release at 1.0 nM by [D-Arg6,Pro9NEt] dfGnRH was almost abolished; however, Gth-II release ability was recovered by the subsequent dose (10 nM) and the maximal response was restored by the 100 nM dose. The Gth-II response achieved by [D-Nal6,Pro9NEt]-cfGnRH was much weaker than that achieved by the others (Fig. 8h). Maximal response was about one-third that of the other D-isomer analogues. There were no significant (SNK multiple

comparison test) differences in the Gth-II release stimulated by any of the concentrations of this analogue.

3.4. Goldfish growth hormone release The sGnRH standard elicited a maximal GH response at 10 nm (439 + 118% prepulse mean). There were no significant differences in GH release between the concentrations of [D-Arg6,Pro9NEt]cGnRH-II (Fig. 9c, d), although at the 0.1-nM dose, a significantly ( P < 0.05; SNK multiple comparison test) greater amount of GH was released compared with that for sGnRH and [D-NaI6,Pro9NEt]-cfGnRH. The greatest GH release by [D-Arg6,ProgNEt]cGnRH-II appeared to occur at 10 nM, although high variability in the hormone release response rendered a clear trend inconclusive. Release of growth hormone by [D-Arg6,Pro9NEt]-dfGnRH was independent of dose and no significant differences of GH release occurred among the GnRH concentrations (Fig. 9e, f). The stimulation of GH at the lowest dose was significantly greater ( P < 0.05) than that by sGnRH and [D-Nal6,Pro9NEt]-cfGnRH, however. Unlike the other D-isomer analogues, [DNal6,Pro9NEt]-cfGnRH released GH in a dose-dependent manner where maximal response occurred at 1000 nM. However, it was not significantly ( P > 0.05; SNK multiple comparison test) different from the greatest GH release obtained using the other 3 analogues.

4. Discussion

In spite of the low potencies of the natural forms of non-mammalian gonadotropin-releasing hormones, substitution of a D-isomer residue into position 6 in conjunction with an ethylamide modification of the C-terminal glycine can render some peptides ([D-Nal6,ProgNEt]-catfish GnRH) particularly potent in mammalian pituitary cells. The resulting stabilization of the secondary structure and the degree of hydrophobicity imparted onto the molecule appear to be major factors that define the potency of these modified non-mammalian GnRHs. In this study, all analogues released the same proportion of FSH

D.A. Lovejoy et al. / Regulatory Peptides 60 (1995) 99-115

relative to LH suggesting differential regulation of LH and FSH is not directly dependent on the structure of GnRH. Whilst these data indicate that the vertebrate GnRH analogues have superagonistic activity in mammalian systems, paradoxically, the analogues are not more potent than the endogenous salmon GnRH peptide in goldfish pituitaries. Introduction of a D-isomer residue into position 6 in combination with the C-terminal ethylamide substitution yields increased potency among some analogues. For example, substitution of the D-naphthylalanine residue into position 6 of the native mGnRH molecule increases the potency for LH release about 127-fold over the native mGnRH molecule. This same modification produces over a 2200-fold increase in potency from that of the native cfGnRH molecule. The GnRH molecule consists of a /3 II turn around residues 5-8. GnRH analogues have been synthesized incorporating substitutions which stabilize this conformation [13]. Of the thousands of GnRH analogues synthesized, a form incorporating both a His 5 and Asn 8 appears not to have been reported. Substitution of D-naphthylalanine into position 6 of this molecule results in a particularly potent analogue. The unexpected activity of this molecule may be a function of several factors. Both D-isomer stabilization and hydrophobicity are significant in defining activity and likely play a role in the distal aspects of receptor-ligand binding, such as formation of secondary structure and interaction with the amphiphilic milieu of the plasma membrane-extracellular matrix interface. However, the presence of asparagine may be of sufficient size to mimic the arginine as found in the mammal GnRH sequence. Moreover, interaction between the side chains of the residues 5 and 8 may provide additional structural support for this molecule [41,42]. Prediction of a peptide structure interaction with the physicochemical conditions that exist between the extracellular matrix and plasma membrane interface is difficult. However, our data suggest that this interaction is partially mediated by the general hydrophobicity of the peptide. An increase in hydrophobicity may allow the peptide to partition itself near the plasma membrane milieu rather than the medium. Substitution of a D-Nal residue into position 6 of cGnRH-II or dfGnRH causes only a 16- and 7-fold increase in potency, respectively. Indeed, sub-

111

stitution of the D-arginine residue into the hydrophobic molecules produced a 103- and 54-fold increase in potency for cGnRH-II and dfGnRH, respectively, compared with the natural molecules. However, as pH strongly affects peptide polarity [36], hydrogen ion concentration could play a role in receptor-ligand interaction. In the past, a number of reports have suggested that biological potency of an analogue correlated with increased hydrophobicity [13,43]. Such analogues tended to be lipophilic and show a greater affinity with plasma proteins, thus increasing their biological half-life. Peter and his associates [44] had previously observed that the potency of sGnRH was reduced when hydrophobic D-isomer residues were substituted into position 6 and suggested a limit to the amount of hydrophobicity the peptide could possess. However, it is unclear if the preference for a particular hydropathy value represents a general condition. Recently, the hydrophilic GnRH peptides cfG nRH, lam prey (1) GnRH-I ([Tyr3,LeuS,Glu6,TrpT,LysS]-GnRH), and [D-Lys6] mGnRH were shown to be more effective at inhibiting thymidine uptake by the human hepatocarcinoma-derived cell line Hep62 than either mGnRH or sGnRH [45]. These data indicate that hydrophilic GnRH peptides may be efficacious in the appropriate tissues perhaps expressing a novel GnRH receptor subtype. The bovine pituitary was chosen as a system to compare the affinities of the analogues as it is a more convenient and abundant source of plasma membrane receptors [46] than the rat pituitary gland. In our preparations, the affinity of some analogues for the rat pituitary membrane binding sites could not be distinguished from that of the [DAla6,Na MeLeuT,ProgNEt]-mGnRH (unpublished observations). However, despite the similarity in affinity for the agonists, pharmacological differences between these receptors cannot be ignored. The data presented in this study indicate that [DNal6,Pro9NEt]-cfGnRH binds to the same population of high affinity receptors in bovine membrane homogenates as the mammalian GnRH analogues. The analogues investigated in this study did not exhibit differential release of LH or FSH. Previous reports have indicated that dfGnRH [18] and cGnRH-II [47] preferentially release FSH rather than LH in vivo relative to mGnRH. Whilst the hydropho-

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bic nature of these peptides may extend their half-life, the rapid increase in the plasma gonadotropin concentration after the administration indicates a direct effect on the hypothalamo-hypophyseal axis. Our study suggests that a putative differential effect does not occur at the gonadotrope level. Previously, Millar and his associates reported that cGnRH-II did not preferentially release FSH from cultured ovine pituitary cells [39]. The differential release of the gonadotropin noted in the previous studies [ 18,47] may be due to the actions of GnRH on the hypothalamus. For example, GnRH secretion may be stimulated by an ultrashort feedback loop due to the autocrine actions of the peptide directly on the GnRH neuron itself [48] or as a paracrine agent on norepinephrine terminals impinging on GnRH neurons [49]. Native cGnRH-II may play a significant role in the brain of eutherian mammals. The peptide is widespread among vertebrates [19,50], but in mammals, the distribution has been limited to marsupials and insectivores [9,10,19]. The insectivores share a common ancestry with primates suggesting that cGnRH-II may be present in the primate brain. cGnRH-II could not be detected in bats [10] or primates [51]. These two groups are believed to be united within a single monophyletic taxon (grand order Archonta) [20]. Therefore this gene may have been lost in the common ancestor of these two lineages. Alternately, the peptide may be expressed in concentrations too low to be detected immunologically. In this study, native cGnRH-II possessed only about 10% of the potency shown by mGnRH in cultured rat pituitary cells. Previously, cGnRH-II was reported to possess 8% of the mGnRH potency in cultured ovine cells [22]. The apparent relationship between predicted reversed-phase HPLC elution time and biological potency suggests that the partitioning effect between the hydrophilic mobile phase and hydrophobic stationary phase of the former may represent a simple model system of the interactive behavior of small peptides with plasma flow and the tissue matrices. In this respect, a dynamic perifusion system similar to that used with the goldfish pituitary fragments in the present study may represent a particularly appropriate method to investigate peptide efficacy in vivo. A teleost model system is particularly useful in the investigation of L-isomer residue substituted GnRHs

as most species are known to contain 2 or more GnRH isoforms in their brains. In this study, the pituitary fragments were refractile to increments of Gth-II release by [DArgr,Pro9NEt]-cGnRH-II analogue beyond doses of 10 nM. Moreover, the pituitary fragments were desensitized at much lower dosages of [DArg6,Pro9NEt]-cGnRH-II than that for sGnRH. One possibility is that the analogue possesses greater receptor affinity, but is less effective at stimulating signal transduction. Thus, greater numbers of receptors may be downregulated. However, Habibi and his coworkers have shown that cGnRH-II is at least as potent as sGnRH [26] suggesting that the D-isomer variant of this isoform should also be as effective. Two alternate possibilities may be offered to explain these observations. There may be a partitioning effect between new and old stores of synthesized gonadotropins. In Xenopus laevis, for example, older stores of melanotrope POMC-derived peptides are secreted by sauvagine whereas the newer synthesized pools are refractile to sauvagine stimulation [52] Alternately, there may be a difference between gonadotropin-I and gonadotropin-II release. In rat, FSH appears to be released via both constitutive and regulatory secretory pathways whereas LH is released primarily through a regulated pathway [53]. However, the viability of this hypothesis within the context of goldfish pituitary cells is beyond the scope of the present study and remains to be investigated. Nevertheless, in goldfish, none of the analogues tested showed significantly greater potency of GH or Gth-II release than sGnRH. Both cGnRH and cGnRH-II are endogenous in the goldfish brain, although immunoreactive sGnRH is predominant in the pituitary [54]. Dogfish GnRH possesses 90% sequence similarity to both goldfish GnRHs (see Fig. 1). The observation that desensitization of the Gth-II release at the lower doses by cGnRH-II and dfGnRH analogues may be a function of the histidine residue in position 5 as it is the only structural attribute shared by these analogues relative to sGnRH. Furthermore, the His 5 residue is also present in catfish GnRH analogue. However, the incomplete response elicited by this peptide may be a function of the 7th and 8th position substitution. Indeed, in goldfish gonadotropes sGnRH appears to stimulate go-

D.A. Lovejoy et a L / Regulatory Peptides 60 (1995) 99-115

nadotropin primarily by the release of intracellular Ca 2+ stores, whereas as cGnRH-II is partially dependent on extracellular Ca 2+ stores [55,56], suggesting a possible discrimination mechanism between the His 5 and Tyr 5 isoforms in goldfish gonadotropes. Generally, the goldfish GnRH receptors do not demonstrate the same degree of specificity for their ligands as do their mammalian counterparts [15]. In goldfish clear relationship between hydropathy and gonadotropin and growth hormone release among various L-isomer-substituted analogues was not observed [26]. The surprising potency of [D-Nal6,Pro9NEt] cfGnRH in cultured rat pituitary cells suggests that it may provide a structural basis for new analogues to be developed. Moreover, the ubiquitous distribution of cGnRH-II in vertebrates in combination with its potency at gonadotropin release in vitro and in vivo is suggestive of a significant physiological role in mammals. Thus GnRH-responsive pathologies may be more appropriately treated using a cGnRH-II analogue. Analogues based on non-mammalian GnRH sequences may be useful in mammalian investigations to uncover attributes of GnRH physiology previously unknown.

Acknowledgements We wish to thank K. Kunitake for excellent technical assistance with the receptor assays. We also thank two anonymous reviewers whose comments were invaluable for the preparation of the final manuscript. This research was carried out with financial assistance Post-Doctoral Fellowship from the Alberta Heritage Foundation for Medical Research (D.A.L), Natural Sciences and Engineering Research Council (R.E.P.) National Institutes of Health (HD13527) (J.E.R., W.W.V.) and the Canadian Medical Research Council (N.M.S.).

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