Spray congealed solid lipid microparticles as a sustained release delivery system for Gonadorelin [6-D-Phe]: Production, optimization and in vitro release behavior.

Sustained release lipid microparticles for a potential veterinary application were produced by the means of spray congealing using saturated triglycerides with respective surfactants. The spray congealing process was optimized using unloaded and loaded microparticles, revealing the highest impact of the spray flow on material loss. Yield could be optimized by increasing the spray flow as well as a reduction of the melt temperature from 90 to 75 °C. For the delivery system developed in this study, a release of around 15 days was targeted. The release profile was in first hand determined with the use of model substances (aspartame and tryptophan), before incorporating the decapeptide Gonadorelin [6-D-Phe]. Release could be controlled between 2 and 28 d, which was dependent on stability of microparticles upon incubation, type and concentration of emulsifier, as well as the used triglyceride. Differential scanning calorimetry and X-ray powder diffraction confirmed the crystallization behavior of C14 and C16-triglycerides in combination with various emulsifiers in different modification without impact on release.

Evaluation of a New 99mTc-labeled GnRH Analogue as a Possible Imaging Agent for Prostate Cancer Detection.

INTRODUCTION: Prostate cancer is a serious threat to men's health so it is necessary to develop technics for early detection of this malignancy. The purpose of this research was the evaluation of a new99mTc-labeled GnRH analogue as an imaging probe for tumor targeting of prostate cancer. METHODS: 99mTc-labeled-DLys6-GnRH analogue was prepared based on HYNIC as a chelating agent and tricine/ EDDA as coligands for labeling with 99mTc. HYNIC was coupled to epsilon amino group of DLys6 through aminobutyric acid (GABA) as a linker. Radiochemical purity and stability in normal saline and serum, were determined by TLC and HPLC methods. Furthermore, calculation of protein-binding and partition coefficient constant were carried out for 99mTc labeled peptide. The cellular experiments including receptor binding specificity and affinity were studied using three prostate cancer cell lines LN-CaP, DU-145 and PC-3. Finally, the animal assessment and SPECT imaging of radiolabeled GnRH analogue were evaluated on normal mice and nude mice bearing LN-CaP tumor. RESULTS: The GnRH conjugate was labeled with high radiochemical purity (~97%). The radiolabeled peptide showed efficient stability in the presence of normal saline and human serum. The in vitro cellular assays on three prostate cancer cell lines indicated that the radiotracer was bound to LN-CaP cells with higher affinity compared to DU-145 and PC-3 cells. The Kd values of 99mTc- HYNIC (tricine/ EDDA)-Gaba-D-Lys6GnRH were 89.39±26.71, 93.57±30.49 and107.3±18.82 in LN-CaP, PC-3 and DU-145 cells respectively. The biodistribution studies in normal mice and LN-CaP tumor-bearing nude mice showed similar results including rapid blood clearance and low radioactivity accumulation in non-target organs. High kidney uptake proved that the main excretion route of radiopeptide was through the urinary system. The tumor uptake was 1.72±0.45 %ID/g at 1h p.i. decreasing to 0.70±0.06%ID/g at 4h p.i. for 99mTc-HYNIC-Gaba-D-Lys6GnRH. The maximum tumor/ muscle ratio was 2.30 at 1h p.i. Pre-saturation of receptor using an excess of unlabeled peptide revealed that the tumor uptake was receptor mediated. The results of the SPECT image of LN-CaP tumor were in agreement with the biodistribution data. CONCLUSION: Based on this study, we suggest LN-CaP as a favorable cell line for in vivo studies on GnRH analogues. Moreover, this report shows that 99mTc-HYNIC (tricine/EDDA)-Gaba-D-Lys6GnRH may be a suitable candidate for further evaluation of prostate cancer.

Novel Crizotinib-GnRH Conjugates Revealed the Significance of Lysosomal Trapping in GnRH-Based Drug Delivery Systems.

Several promising anti-cancer drug-GnRH (gonadotropin-releasing hormone) conjugates have been developed in the last two decades, although none of them have been approved for clinical use yet. Crizotinib is an effective multi-target kinase inhibitor, approved against anaplastic lymphoma kinase (ALK)- or ROS proto-oncogene 1 (ROS-1)-positive non-small cell lung carcinoma (NSCLC); however, its application is accompanied by serious side effects. In order to deliver crizotinib selectively into the tumor cells, we synthesized novel crizotinib analogues and conjugated them to a [d-Lys6]-GnRH-I targeting peptide. Our most prominent crizotinib-GnRH conjugates, the amide-bond-containing [d-Lys6(crizotinib*)]-GnRH-I and the ester-bond-containing [d-Lys6(MJ55*)]-GnRH-I, were able to bind to GnRH-receptor (GnRHR) and exert a potent c-Met kinase inhibitory effect. The efficacy of compounds was tested on the MET-amplified and GnRHR-expressing EBC-1 NSCLC cells. In vitro pharmacological profiling led to the conclusion that that crizotinib-GnRH conjugates are transported directly into lysosomes, where the membrane permeability of crizotinib is diminished. As a consequence of GnRHR-mediated endocytosis, GnRH-conjugated crizotinib bypasses its molecular targets-the ATP-binding site of RTKs- and is sequestered in the lysosomes. These results explained the lower efficacy of crizotinib-GnRH conjugates in EBC-1 cells, and led to the conclusion that drug escape from the lysosomes is a major challenge in the development of clinically relevant anti-cancer drug-GnRH conjugates.

Synthesis, in vitro stability, and antiproliferative effect of d-cysteine modified GnRH-doxorubicin conjugates.

Overexpression of gonadotropin-releasing hormone (GnRH) receptor in many tumors but not in normal tissues makes it possible to use GnRH analogs as targeting peptides for selective delivery of cytotoxic agents, which may help to enhance the uptake of anticancer drugs by cancer cells and reduce toxicity to normal cells. The GnRH analogs [d-Cys6 , desGly10 , Pro9 -NH2 ]-GnRH, [d-Cys6 , desGly10 , Pro9 -NHEt]-GnRH, and [d-Cys6 , α-aza-Gly10 -NH2 ]-GnRH were conjugated with doxorubicin (Dox), respectively, through N-succinimidyl-3-maleimidopropionate as a linker to afford three new GnRH-Dox conjugates. The metabolic stability of these conjugates in human serum was determined by RP-HPLC. The antiproliferative activity of the conjugates was examined in GnRH receptor-positive MCF-7 human breast cancer cell line by MTT assay. The three GnRH-Dox conjugates showed improved metabolic stability in human serum in comparison with AN-152. The antiproliferative effect of conjugate II ([d-Cys6 , desGly10 , Pro9 -NHEt]-GnRH-Dox) on MCF-7 cells was higher than that of conjugate I ([d-Cys6 , desGly10 , Pro9 -NH2 ]-GnRH-Dox) and conjugate III ([d-Cys6 , α-aza-Gly10 -NH2 ]-GnRH-Dox), and the cytotoxicity of conjugate II against GnRH receptor-negative 3T3 mouse embryo fibroblast cells was decreased in comparison with free Dox. GnRH receptor inhibition test suggested that the antiproliferative activity of conjugate II might be due to the cellular uptake mediated by the targeting binding of [d-Cys6 -des-Gly10 -Pro9 -NHEt]-GnRH to GnRH receptors. Our study indicates that targeting delivery of conjugate II mediated by [d-Cys6 -des-Gly10 -Pro9 -NHEt]-GnRH is a promising strategy for chemotherapy of tumors that overexpress GnRH receptors.

Synthesis, characterization and systematic comparison of FITC-labelled GnRH-I, -II and -III analogues on various tumour cells.

Targeted tumour therapy is the focus of recent cancer research. Gonadotropin-releasing hormone (GnRH) analogues are able to deliver anticancer agents selectively into tumour cells, which highly express GnRH receptors. However, the effectiveness of different analogues as targeting moiety in drug delivery systems is rarely compared, and the investigated types of cancer are also limited. Therefore, we prepared selectively labelled, fluorescent derivatives of GnRH-I, -II and -III analogues, which were successfully used for drug targeting. In this manuscript, we investigated these analogues' solubility, stability and passive membrane permeability and compared their cellular uptake by various cancer cells. We found that these labelled GnRH conjugates provide great detectability, without undesired cytotoxicity and passive membrane permeability. The introduced experiments with these conjugates proved their reliable tracking, quantification and comparison. Cellular uptake efficiency was studied on human breast, colon, pancreas and prostate cancer cells (MCF-7, HT-29, BxPC-3, LNCaP) and on dog kidney cells (Madin-Darby canine kidney). Each of the three conjugates was taken up by GnRH-I receptor-expressing cells, but the different cells preferred different analogues. Furthermore, we demonstrated for the first time the high cell surface expression of GnRH-I receptors and the effective cellular uptake of GnRH analogues on human pharynx tumour (Detroit-562) cells. In summary, our presented results detail that the introduced conjugates could be innovative tools for the examination of the GnRH-based drug delivery systems on various cells and offer novel information about these peptides. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Synthesis and in vitro anti-cancer evaluation of luteinizing hormone-releasing hormone-conjugated peptide.

Luteinizing hormone-releasing hormone (LHRH) is a decapeptide hormone released from the hypothalamus and shows high affinity binding to the LHRH receptors. It is reported that several cancer cells also express LHRH receptors such as breast, ovarian, prostatic, bladder and others. In this study, we linked B1, an anti-cancer peptide, to LHRH and its analogs to improve the activity against cancer cells with LHRH receptor. Biological evaluation revealed that TB1, the peptide contains triptorelin sequence, present favorable anti-cancer activity as well as plasma stability. Further investigations disclosed that TB1 trigger apoptosis by activating the mitochondria-cytochrome c-caspase apoptotic pathway, it also exhibited the anti-migratory effect on cancer cells.

A novel synthetic luteinizing hormone-releasing hormone (LHRH) analogue coupled with modified ß-cyclodextrin: insight into its intramolecular interactions.

BACKGROUND: Cyclodextrins (CDs) in combination with therapeutic proteins and other bioactive compounds have been proposed as candidates that show enhanced chemical and enzymatic stability, better absorption, slower plasma clearance and improved dose-response curves or immunogenicity. As a result, an important number of therapeutic complexes between cyclodextrins and bioactive compounds capable to control several diseases have been developed. RESULTS: In this article, the synthesis and the structural study of a conjugate between a luteinizing hormone-releasing hormone (LHRH) analogue, related to the treatment of hormone dependent cancer and fertility, and modified ß-cyclodextrin residue are presented. The results show that both the phenyl group of tyrosine (Tyr) as well as the indole group of tryptophan (Trp) can be encapsulated inside the cyclodextrin cavity. Solution NMR experiments provide evidence that these interactions take place intramolecularly and not intermolecularly. CONCLUSIONS: The study of a LHRH analogue conjugated with modified ß-cyclodextrin via high field NMR and MD experiments revealed the existence of intramolecular interactions that could lead to an improved drug delivery. GENERAL SIGNIFICANCE: NMR in combination with MD simulation is of great value for a successful rational design of peptide-cyclodextrin conjugates showing stability against enzymatic proteolysis and a better pharmacological profile.

Protein expression profile of HT-29 human colon cancer cells after treatment with a cytotoxic daunorubicin-GnRH-III derivative bioconjugate.

Targeted delivery of chemotherapeutic agents is a new approach for the treatment of cancer, which provides increased selectivity and decreased systemic toxicity. We have recently developed a promising drug delivery system, in which the anticancer drug daunorubicin (Dau) was attached via oxime bond to a gonadotropin-releasing hormone-III (GnRH-III) derivative used as a targeting moiety (Glp-His-Trp-Lys(Ac)-His-Asp-Trp-Lys(Da  = Aoa)-Pro-Gly-NH2; Glp = pyroglutamic acid, Ac = acetyl; Aoa = aminooxyacetyl). This bioconjugate exerted in vitro cytostatic/cytotoxic effect on human breast, prostate and colon cancer cells, as well as significant in vivo tumor growth inhibitory effect on colon carcinoma bearing mice. In our previous studies, H-Lys(Dau = Aoa)-OH was identified as the smallest metabolite produced in the presence of rat liver lysosomal homogenate, which was able to bind to DNA in vitro. To get a deeper insight into the mechanism of action of the bioconjugate, changes in the protein expression profile of HT-29 human colon cancer cells after treatment with the bioconjugate or free daunorubicin were investigated by mass spectrometry-based proteomics. Our results indicate that several metabolism-related proteins, molecular chaperons and proteins involved in signaling are differently expressed after targeted chemotherapeutic treatment, leading to the conclusion that the bioconjugate exerts its cytotoxic action by interfering with multiple intracellular processes.

Synthesis and in vitro evaluation of glycosyl derivatives of luteinizing hormone-releasing hormone (LHRH).

Luteinizing hormone-releasing hormone (LHRH) analogues are used extensively for the treatment of various hormone-dependent diseases. However, none of the currently marketed derivatives can be administered orally. Modification of peptide sequences by attachment of carbohydrate moieties is a promising strategy that may increase the metabolic stability of the target peptide and enhance its transport across cell membranes, subsequently improving peptide bioavailability. In this study, either the N- or C-terminus of the LHRH peptide was altered by attachment of carbohydrate moieties. Caco-2 cells were chosen as an in vitro model to investigate both the permeability and stability of the new LHRH analogues. Our findings show that conjugating sugar moieties to the N-terminus of the LHRH peptide significantly increased both permeability and metabolic stability of most of the modified LHRH derivatives.

Structure-activity studies of lGnRH-III through rational amino acid substitution and NMR conformational studies.

Lamprey gonadotropin-releasing hormone type III (lGnRH-III) is an isoform of GnRH isolated from the sea lamprey (Petromyzon marinus) with negligible endocrine activity in mammalian systems. Data concerning the superior direct anticancer activity of lGnRH-III have been published, raising questions on the structure-activity relationship. We synthesized 21 lGnRH-III analogs with rational amino acid substitutions and studied their effect on PC3 and LNCaP prostate cancer cell proliferation. Our results question the importance of the acidic charge of Asp6 for the antiproliferative activity and indicate the significance of the stereochemistry of Trp in positions 3 and 7. Furthermore, conjugation of an acetyl-group to the side chain of Lys8 or side chain cyclization of amino acids 1-8 increased the antiproliferative activity of lGnRH-III demonstrating that the proposed salt bridge between Asp6 and Lys8 is not crucial. Conformational studies of lGnRH-III were performed through NMR spectroscopy, and the solution structure of GnRH-I was solved. In solution, lGnRH-III adopts an extended backbone conformation in contrast to the well-defined ß-turn conformation of GnRH-I.

Rationally designed cyclic analogues of luteinizing hormone-releasing hormone: enhanced enzymatic stability and biological properties.

This article describes the rational design, synthesis and pharmacological properties of amide-linked cyclic analogues of Luteinizing Hormone-Releasing Hormone (LHRH) with substitutions at positions 1 (Pro), 6 (D-Leu/D-Trp), 9 (Aze) and 10 (BABA/Acp). These LHRH analogues fulfil the conformational requirements that are known in the literature (bend in the 5-8 segment) to be essential for receptor recognition and activation. Although, they are characterised by an overall low binding affinity to the LHRH-I receptor, the cyclic analogues that were studied and especially the cyclo(1-10)[Pro(1), D-Leu(6), BABA(10)] LHRH, exhibit a profoundly enhanced in vitro and in vivo stability and improved pharmacokinetics in comparison with their linear counterpart and leuprolide. Upon receptor binding, cyclo(1-10)[Pro(1), D-Leu(6), BABA(10)] LHRH causes testosterone release in C57/B16 mice (in vivo efficacy) that is comparable to that of leuprolide. Testosterone release is an acutely dose dependent effect that is blocked by the LHRH-I receptor antagonist, cetrorelix. The pharmacokinetic advantages and efficacy of cyclo(1-10)[Pro(1), D-Leu(6), BABA(10)] LHRH render this analogue a promising platform for future rational drug design studies towards the development of non-peptide LHRH mimetics.

Enhanced cellular uptake and in vitro antitumor activity of short-chain fatty acid acylated daunorubicin-GnRH-III bioconjugates.

Here we report on the synthesis and biochemical characterization (enzymatic stability, cellular uptake, in vitro antitumor activity, membrane interaction and GnRH-receptor binding affinity) of novel short-chain fatty acid (SCFA) acylated daunorubicin-GnRH-III bioconjugates, which may serve as drug delivery systems for targeted cancer chemotherapy. Ser in position 4 of GnRH-III was replaced by Lys, followed by the acylation of its ε-amino group with various fatty acids. SCFAs are potentially chemoprotective agents by suppressing the growth of cancer cells and therefore may enhance the antitumor activity of the bioconjugates. We found that all synthesized bioconjugates had high cytostatic effect in vitro, were stable in cell culture medium for 6 h and degraded in the presence of rat liver lysosomal homogenate leading to the formation of an oxime bond-linked daunorubicin-Lys as the smallest active metabolite. In the presence of α-chymotrypsin, all compounds were digested, the degradation rate strongly depending on the type of fatty acid. The bioconjugate containing Lys(nBu) in position 4 was taken up most efficiently by the cancer cells and exerted higher in vitro cytostatic effect than the previously developed GnRH-III((4)Lys(Ac), (8)Lys(Dau = Aoa)) or the parent GnRH-III(Dau = Aoa) bioconjugate. Our results could be explained by the increased binding affinity of the newly developed compound containing Lys(nBu) to the GnRH receptors.

[Synthesis of GnRH analogs and their application in targeted gene delivery systems].

A set of GnRH analogues containing nuclear localization signal (NLS) of SV-40 virus large T-antigen have been synthesized using solid phase peptide synthesis and chemical ligation technique. Selective chemical ligation was achieved as a result of hydrazone formation in the course of interaction between NLS hydrazide and GnRH analog modified by pyruvic acid. The efficiency of synthesized peptide carriers was demonstrated in experiments with human cancer cells transfected by reporter luciferase and beta-galactosidase genes or suicide HSV-1 thymidine kinase gene. It was shown that selectivity of action on cancer cells can be achieved as a result of peptide/DNA complex penetration through the cell membrane by GnRH receptor-mediated endocytosis pathway.

Fast conventional Fmoc solid-phase peptide synthesis: a comparative study of different activators.

The ability to speed up conventional Fmoc solid-phase peptide synthesis (SPPS) has many advantages including increased productivity. One way to speed up conventional Fmoc SPPS is the choice of activator. Recently, several new activators have been introduced into the market, and they were evaluated along with some older activators for their ability to synthesize a range of peptides with shorter and longer reaction times. It was found that HDMC, PyClock, COMU, HCTU, and HATU worked well at shorter reaction times (2 × 1 min), but PyOxim and TFFH only worked well at longer reaction times. The performance of PyBOP at shorter reaction times was poor only for more difficult sequences. These results are important for selecting an appropriate activator for fast SPPS applications.

Paclitaxel conjugation with the analog of the gonadotropin-releasing hormone as a targeting moiety.

A new targeted conjugates in which paclitaxel was used as a cytostatic compound and an analog of the gonadotropin-releasing hormone (GnRH) as a targeting moiety were synthesized. The molecule of the peptide hormone GnRH was modified to allow its connection to paclitaxel via spacer. The conjugates were prepared as prodrugs using 2'-hydroxyl group of paclitaxel. 4-Maleimidobutyric acid and chloroacetic acid served as spacers. The structures of the prepared derivatives were analysed by NMR and HR-MS. The conjugates MP264 and MP265 were chosen and their antiproliferative effect was tested in the breast cancer cell line MCF-7 using the MTT test of cell viability and neutral red uptake test. In MCF-7 cells, conjugate MP265 showed higher antiproliferative effect than paclitaxel alone. Receptor saturation tests showed that the unconjugated peptide analog of GnRH decreased efficacy of conjugate MP265 in concentration- and time-dependent manner. In conclusion, the paclitaxel conjugate with the analog of GnRH exhibited targeted antiproliferative effect for which its further testing will be implemented.

A modular approach to assembly of totally synthetic self-adjuvanting lipopeptide-based vaccines allows conformational epitope building.

The technology described here allows the chemical synthesis of vaccines requiring correctly folded epitopes and that contain difficult or long peptide sequences. The final self-adjuvanting product promotes strong humoral and/or cell-mediated immunity. A module containing common components of the vaccine (T helper cell epitope and the adjuvanting lipid moiety S-[2,3-bis(palmitoyloxy)propyl]cysteine) was assembled to enable a plug and play approach to vaccine assembly. The inclusion within the module of a chemical group with chemical properties complementary and orthogonal to a chemical group present in the target epitope allowed chemoselective ligation of the two vaccine components. The heat-stable enterotoxin of enterotoxigenic Escherichia coli that requires strict conformational integrity for biological activity and the reproductive hormone luteinizing hormone-releasing hormone were used as the target epitopes for the antibody vaccines. An epitope from the acid polymerase of influenza virus was used to assemble a CD8(+) T cell vaccine. Evaluation of each vaccine candidate in animals demonstrated the feasibility of the approach and that the type of immune response required, viz. antibody or cytotoxic T lymphocyte, dictates the nature of the chemical linkage between the module and target epitope. The use of a thioether bond between the module and target epitope had little or no adverse effect on antibody responses, whereas the use of a disulfide bond between the module and target epitope almost completely abrogated the antibody response. In contrast, better cytotoxic T lymphocyte responses were obtained when a disulfide bond was used.

[GnRH analogues containing SV-40 virus T-antigen nuclear localization sequence].

To improve the efficiency of anticancer drugs due to their delivery to intracellular targets a set of GnRH analogues containing nuclear localization signal (NLS) of SV-40 virus large T-antigen have been synthesized. NLS was attached to the parent molecule via ε-amino group of D-Lysine in position 1 or 6 of peptide sequence using orthogonal protection strategy. The biological activity studies revealed that incorporation of NLS moiety significantly increases cytotoxic activity of palmitoyl-containing GnRH analogues in vitro. The influence of tested peptides on tumor cells does not accompanied by the destruction of cell membrane, as confirmed in experiments with normal fibroblasts, used as a control.

The single-shot GnRH immunocontraceptive vaccine (GonaCon) in white-tailed deer: comparison of several GnRH preparations.

PROBLEM: An effective, single-injection, multi-year, GnRH contraceptive agent is needed to control reproduction in overabundant white-tailed deer populations. METHOD OF STUDY: Two GnRH conjugates, GonaCon (GnRH-KLH) and GonaCon-B (GnRH-blue protein), were prepared in emulsion form as one-injection and two-injection immunocontraceptive vaccine formulations. In addition, the GnRH-KLH protein conjugate was lyophilized and suspended in AdjuVac adjuvant to produce a fifth vaccine formulation. Each formulation was administered to a group of five captive adult female white-tailed deer. Reproductive performance of treated female deer was monitored for 5 years to determine the comparative efficacy of the various treatments. RESULTS: The longevity of the contraceptive response (2-5 years) was strongly influenced by the design of the conjugate antigen, the adjuvant used, and the delivery form of the vaccine. CONCLUSION: One-injection and two-injection formulations of GonaCon and GonaCon-B produced multi-year contraception in adult female white-tailed deer. GonaCon-B provided a longer lasting contraceptive effect.

Structure-activity study on the LH- and FSH-releasing and anticancer effects of gonadotropin-releasing hormone (GnRH)-III analogs.

UNLABELLED: GnRH-III was reported to have selective FSH-releasing activity in rats and significant anticancer potency on human breast cancer cells. To improve either of these effects, 14 analogs were synthesized and investigated for FSH/LH stimulation and breast cancer inhibition. Analogs with single amino acid changes in positions 5-7 or 10 showed small or no difference in the FSH- or LH-releasing activity compared with GnRH-III but their anticancer potency decreased significantly. Modification of the terminal amino acids, side chain cyclization at the 6-8 regions, or combined amino acid changes at positions 4, 6 and/or 8 resulted in the decrease of both effects. Gonadotropin-releasing activity of Arg(8)-GnRH-III was improved 3-11-fold. A copolymer conjugate of GnRH-III showed 2-3-fold anticancer activity while losing endocrine potency. CONCLUSION: The activation of GnRH-receptors on pituitary and breast cancer cells requires a specific structure and/or conformation that makes possible to improve the anticancer selectivity of GnRH analogs.

Design, synthesis, and molecular modeling of a novel amide-linked cyclic GnRH analogue cyclo(4-9)[Lys4,D-Trp6,Glu9]GnRH: stimulation of gonadotropin gene expression.

This report describes the rational design, synthesis, and pharmacological properties of an amide-linked cyclic analogue of gonadotropin-releasing hormone (GnRH) namely Cyclo(4-9)[Lys(4),d-Trp(6),Glu(9)]GnRH. The conformationally restricted analogue is characterized by reduced flexibility of the peptide strand due to the introduction of a beta-turn mimetic through 4,9 residue amide cyclization. The cyclic analogue was found to stimulate gonadotropin gene expression in the goldfish pituitary with similar potency compared to two native forms of GnRH. Simulation studies based on ROE connectivities in linear GnRH and potency of cyclic analogue supports the His(2), Trp(3), Tyr(5) clustering considered important for triggering receptor activation.