Pure Antagonistic Activity Research Articles

SCR-6852, an oral and highly brain-penetrating estrogen receptor degrader (SERD), effectively shrinks tumors both in intracranial and subcutaneous ER + breast cancer models

BackgroundTargeted estrogen receptor degradation has been approved to effectively treat ER + breast cancers. Due to the poor bioavailability of fulvestrant, the first generation of SERD, many efforts were made to develop oral SERDs. With the approval of Elacestrant, oral SERDs demonstrated superior efficacy than fulvestrant. However, due to the poor ability of known SERDs to penetrate the blood–brain barrier (BBB), breast cancer patients with brain metastasis cannot benefit from clinical SERDs.MethodsThe ER inhibitory effects were evaluated on ERα protein degradation, and target genes downregulation. And anti-proliferation activities were further determined in a panel of ER + breast cancer cell lines. The subcutaneous and intracranial ER + tumor models were used to evaluate the efficacy of anti-tumor effects. Brain penetrability was determined in multiple animal species.ResultsSCR-6852 is a novel SERD and currently is under early clinical evaluation. In vitro studies demonstrated that it strongly induced both wildtype and mutant ERα degradation. It potently inhibited cell proliferation in a panel of ER + breast cancer cell lines, including the cell lines containing ESR1 mutations (Y537 and D538). Furthermore, SCR-6852 exhibited pure antagonistic activities on the ERɑ signal axis identified both in vitro and in vivo. Oral administration of SCR-6852 at 10 mg/kg resulted in tumor shrinkage which was superior to Fulvestrant at 250 mg/kg, notably, in the intracranial tumor model, SCR-6852 effectively inhibited tumor growth and significantly prolonged mice survival, which correlated well with the high exposure in brains. In addition to mice, SCR-6852 also exhibited high brain penetrability in rats and dogs.ConclusionsSCR-6852 is a novel SERD with high potency in inducing ERα protein degradation and pure antagonistic activity on ERɑ signaling in vitro and in vivo. Due to the high brain penetrability, SCR-6852 could be used to treat breast patients with brain metastasis.

Elagolix sodium for the treatment of women with moderate to severe endometriosis-associated pain.

First-line medical therapies for treating pain associated with endometriosis mainly consist in combined oral contraceptives and progestins. However, some women, having persistence of symptoms, may require further therapeutic options. Among these, gonadotropin-releasing hormone (GnRH) agonists (GnRH-as) have been widely employed in the last 30 years, despite being characterized by an unfavorable safety profile. Currently, new alternative investigational options are being investigated to treat this benign chronic disease. GnRH antagonists (GnRH-ants) are innovative hormonal drugs that do not induce flare-up effects and present also a limited onset of hypoestrogenic symptoms; in fact, their pharmacological mechanism of action, which consists in pure antagonistic activity, differs from that of traditional GnRH-as. In July 2018, the U.S. Food and Drug Administration (FDA) approved elagolix sodium for the management of moderate to severe pain associated with endometriosis, after the drug showed promising efficacy and safety results in previous phase III trials. This monograph aims to provide a complete overview of the pharmacokinetics, clinical efficacy and safety of this GnRH-ant for treat¬ing patients with endometriosis.

Relugolix for the treatment of uterine fibroids.

Uterine leiomyomas represent the most common form of benign gynecological tumors affecting 20-40% of women during their life. Several therapeutic options are available for treating these patients. The use of medical treatment for myomas has largely grown in the last years, in particular for women who would refuse, postpone or are not candidates for surgery. In the last years, the clinical investigation of gonadotropin-releasing hormone (GnRH) antagonists (GnRH-ants) has emerged. This class of drugs exerts pure competitive antagonistic activity on the GnRH receptor at the pituitary gland, producing an immediate stop in the release of gonadotropins and sex steroids. Relugolix is an orally active nonpeptide GnRH-ant, recently licensed for marketing in Japan for the treatment of symptoms related to uterine myomas. Currently, several phase III clinical trials are ongoing to evaluate this molecule in this setting in the U.S. and Europe.

Lead optimization of P5U and urantide: discovery of novel potent ligands at the urotensin-II receptor.

We have optimized 1 (P5U) and urantide, two important ligands at the h-UT receptor, designing several analogues by the exchange of the Tyr9 residue with different unnatural aromatic amino acids. This study allowed us to discover novel ligands with improved activity. In particular, the replacement of the Tyr9 residue by (pCN)Phe or (pNO2)Phe within the urantide sequence led to compounds 13 (UPG-83) and 15 (UPG-95), respectively, which showed pure antagonist activity toward UT receptor in a rat aorta bioassay. More interestingly, the replacement of the Tyr9 in 1 sequence with the Btz or the (3,4-Cl)Phe residues led to superagonists 6 (UPG-100) and 10 (UPG-92) with pEC50 values at least 1.4 log higher than that of 1, being the most potent UT agonists discovered to date. Compounds 10 and 13 showed also a good stability in a serum proteolytic assay. These ligands represent new useful tools to further characterize the urotensinergic system in human physiopathology.

Discovery and Biophysical Characterization of 2-Amino-oxadiazoles as Novel Antagonists of PqsR, an Important Regulator of Pseudomonas aeruginosa Virulence

The human pathogen Pseudomonas aeruginosa employs alkyl quinolones for cell-to-cell communication. The Pseudomonas quinolone signal (PQS) regulates various virulence factors via interaction with the transcriptional regulator PqsR. Therefore, we consider the development of PqsR antagonists a novel strategy to limit the pathogenicity of P. aeruginosa. A fragment identification approach using surface plasmon resonance screening led to the discovery of chemically diverse PqsR ligands. The optimization of the most promising hit (5) resulted in the oxadiazole-2-amine 37 showing pure antagonistic activity in Escherichia coli (EC50 = 7.5 μM) and P. aeruginosa (EC50 = 38.5 μM) reporter gene assays. 37 was able to diminish the production of the PQS precursor HHQ in a PqsH-deficient P. aeruginosa mutant. The level of the major virulence factor pyocyanin was significantly reduced in wild-type P. aeruginosa. In addition, site-directed mutagenesis in combination with isothermal titration calorimetry and NMR INPHARMA experiments revealed that the identified ligands bind to the same site of PqsR by adopting different binding modes. These findings will be utilized in a future fragment-growing approach aiming at novel therapeutic options for the treatment of P. aeruginosa infections.

Applicability of selective progesterone receptor modulators in the treatment of uterine leiomyomata and their future role in the field of gynecology

Uterine leiomyomata are benign, monoclonal tumors arising from smooth muscle cells, which belong to one of the most common pathologies of the female genital system. Current pharmacotherapies (oral contraceptives, progestins, GnRH analogs) are ineffective or of limited use for long-term treatment. Although there is still much debate regarding their etiology it is very likely that progesterone and progesterone receptor play a key role in their development. Profound importance of progesterone in the female reproductive system has led to discovery of synthetic progesterone receptor ligands, which can poses the activity ranging from pure agonist activity trough mixed agonist/antagonist activity to pure antagonist activity Development of selective progesterone receptor modulators (SPRM) has created new therapeutic options and has great potential in a number of gynecologic indications. So far, ulipristal acetate has been approved for emergency contraception, mifepristone as a progesterone receptor antagonist because of the unique property of this compound for termination of pregnancy Recently the European Commission has authorized ulipristal acetate for the pre-operative treatment of uterine fibroids. Superior efficacy of ulipristal acetate versus placebo, to reduce excessive uterine bleeding and to reduce total fibroid volume prior to surgery was demonstrated. Moreover non-inferior efficacy of ulipristal acetate versus Gonadotropin Releasing Hormone (GnRH)-agonist to reduce excessive uterine bleeding prior to surgery of uterine fibroids has been documented. Ulipristal acetate is also characterized by a superior side-effect profile in comparison to leuprolide acetate in terms of serum estradiol levels and the proportion of patients with moderate-to-severe hot flashes during treatment. Regarding safety profile, except elevation of liver enzymes after telapristone and onapristone treatment, to date no serious untoward effects of other SPRM have been reported. The issue of endometrial effects of these compounds remains to be resolved, although observation that intrinsic agonist activity of SPRM prevents endometrial proliferation may suggest future use of these agents in prevention of endometrial hyperplasia. Other promising applications, including endometriosis, endometrial cancer Cushing's disease, Alzheimer disease or long-term contraception, are currently in development.

Steroid derivatives as pure antagonists of the androgen receptor

BackgroundWhile the androgens of testicular origin (representing about 50% of total androgens in men over 50 years) can be completely eliminated by surgical or medical castration with GnRH (gonadotropin-releasing hormone) agonists or antagonists, the antiandrogens currently available as blockers of androgen binding to the androgen receptor (AR), namely bicalutamide (BICA), flutamide (FLU) and nilutamide have too weak affinity to completely neutralize the other 50% of androgens made locally from dehydroepiandrosterone (DHEA) in the prostate cancer tissue by the mechanisms of intracrinology. Materials and methodsSeries of steroid derivatives having pure and potent antagonistic activity on the human and rodent AR were synthesized. Assays of AR binding and activity in carcinoma mouse Shionogi and human LNCaP cells as well as in vivo bioavailability measurements and in vivo prostate weight assays in the rat were used. ResultsThe chosen lead steroidal compound, namely EM-5854, has a 3.7-fold higher affinity than BICA for the human AR while EM-5855, an important metabolite of EM-5854, has a 94-fold higher affinity for the human AR compared to BICA. EM-5854 and EM-5855 are 14 times more potent than BICA in inhibiting androgen (R1881)-stimulated prostatic specific antigen (PSA) secretion in human prostatic carcinoma LNCaP cells in vitro. MDV3100 has a potency comparable to bicalutamide in these assays. Depending upon the oral formulation, EM-5854 is 5- to 10-times more potent than BICA to inhibit dihydrotestosterone (DHT)-stimulated ventral prostatic weight in vivo in the rat while MDV3100 has lower activity than BICA in this in vivo model. These data are supported by respective 40-fold and 105-fold higher potencies of EM-5854 and EM-5855 compared to BICA to inhibit cell proliferation in the androgen-sensitive Shionogi carcinoma cell model. ConclusionsAlthough the present preclinical results data need evaluation in clinical trials in men, combination of the data obtained in vitro in human LNCaP cells as indicator of potency in the human prostate and the data on metabolism evaluated in vivo on ventral prostate weight in the rat, could suggest the possibility of a 70- to 140-fold higher potency of EM-5854 compared to bicalutamide (Casodex) for the treatment of prostate cancer in men.

Biological properties of androgen receptor pure antagonist for treatment of castration‐resistant prostate cancer: Optimization from lead compound to CH5137291

Castration-resistant prostate cancer (CRPC) is still dependent on androgen receptor (AR) signaling. We previously reported that a novel nonsteroidal AR pure antagonist, CH4933468, which is a thiohydantoin derivative with a sulfonamide side chain, provided in vitro proof of concept but did not in vivo. We developed other derivatives, CH5137291, CH5138514, and CH5166623, and their pharmacological properties were compared with CH4933468 and bicalutamide. Agonist/antagonist activities in AR-mediated transactivation, cell proliferation against LNCaP and LNCaP-BC2, and AR translocation were evaluated. Agonist metabolite was monitored in liver microsomes and in pharmacokinetics experiments. Antitumor activities in CRPC xenograft models were examined using LNCaP-BC2 and VCaP-CRPC. All CH compounds completely inhibited AR-mediated transactivation and proliferation of LNCaP and LNCaP-BC2. In contrast bicalutamide showed a partial inhibition of AR-mediated transactivation and a proliferation of LNCaP-BC2. AR translocation to nucleus was inhibited by CH compounds, but stimulated by bicalutamide. In the LNCaP-BC2 xenograft model, however, only CH5137291 showed significant inhibition of plasma PSA level and antitumor activity. The other three CH compounds were metabolized to their core structure which had agonist activity. CH5137291 also exhibited antitumor activity in a VCaP-CRPC xenograft model, but bicalutamide did not. The molecular mechanism of the CH compounds, inhibition of AR translocation, was different from bicalutamide and this action could contribute to AR pure antagonist activity. Agonist metabolite diminished the antitumor activity of AR pure antagonist. CH5137291 exhibited antitumor activity in LNCaP-BC2 and VCaP-CRPC xenograft models, suggesting that the compound has potential for the treatment of CRPC.

Abstract A219: Universal efficacy of novel androgen receptor pure antagonist, CH5137291, against broad spectrum prostate cancer including castration-resistant prostate cancers

Abstract Background: Prostate cancer resistant to total androgen depletion therapies is termed castration-resistant prostate cancer and novel therapeutic approaches are in demand. The mechanisms of castration resistance in prostate cancer are reported to be: 1) hypersensitivity to androgen derived from overexpression of androgen receptor (AR), 2) androgen-independent activation of AR, termed “outlaw”, and 3) loss of ligand specificity derived from mutation of AR. To address this demand, we screened and developed an AR antagonist without agonist activity, a so-called AR pure antagonist, CH5137291. Methods: We compared the inhibitory effects of CH5137291 with bicalutamide on the growth of hormone-sensitive and castration-resistant prostate cancer cells. To confirm the in vivo efficacy of CH5137291, we used xenograft models. The antagonist/agonist effect of CH5137291 on AR transcriptional activity was analyzed using reporter gene assays. To elucidate the mechanisms of CH5137291, we examined the effect of CH5137291 on the protein level of nuclear AR in LNCaP-CS10 and the effect of CH5137291 on the subcellular localization of AR in LNCaP cells. Finally, the exposure of CH5137291 and its effect on serum prostate-specific antigen (PSA) level were investigated in cynomolgus monkey. Results: CH5137291 showed in vitro inhibitory effects superior to bicalutamide in all models tested including hormone-sensitive VCaP, LNCaP and castration-resistant, LNCaP-BC2 (overexpression model) and LNCaP-CS10 (outlaw model). CH5137291 treatment (10, 100 mg/kg) inhibited the tumor growth to initial levels and PSA production to lower than initial levels in castration-resistant LuCaP35V, LNCaP-BC2 and LNCaP-CS10 xenograft models. In contrast, bicalutamide (10, 100 mg/kg) did not inhibit tumor growth or PSA production in any of the xenograft models. In addition, CH5137291 exhibited prolongation of the time to progression compared with bicalutamide in the LNCaP xenograft model. In vitro functional assays clarified that CH5137291 showed pure antagonist activity against wild type, bicalutamide-resistant type (W741C), and flutamide-resistant type (T877A) AR in transcription and inhibited the nuclear translocation of all types of AR tested. CH5137291 also exhibited a dose-dependent increase in serum concentration and inhibited PSA production in cynomolgus monkey. Conclusion: Our novel AR pure antagonist CH5137291 inhibited AR nuclear translocation and showed not only superior efficacy in prostate cancers with castration-resistance acquired from AR overexpression, mutation and outlaw mechanisms but also prolonged time to progression in hormone-sensitive prostate cancer compared with bicalutamide. CH5137291 is expected to contribute to novel therapeutic approaches against a broad spectrum of prostate cancers. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A219.

Designed modification of partial agonist of ORL1 nociceptin receptor for conversion into highly potent antagonist

Nociceptin is an endogenous agonist ligand of the ORL1 (opioid receptor-like 1) receptor, and its antagonist is a potential target of therapeutics for analgesic and antineuropathy drugs. Ac-RYYRIK-NH 2 is a hexapeptide isolated from the peptide library as an antagonist that inhibits the nociceptin activities mediated through ORL1. However, the structural elements required for this antagonist activity are still indeterminate. In the present study, we evaluated the importance of the acetyl-methyl group in receptor binding and activation, examining the peptides acyl-RYYRIK-NH 2, where acyl (R-CO) possesses a series of alkyl groups, R = C n H 2 n+1 ( n = 0–5). The isovaleryl derivative with the C 4H 9 (=(CH 3) 2CHCH 2–) group was found to reveal a high receptor-binding affinity and a strong antagonist nature. This peptide achieved a primary goal of eliminating the agonist activity of Ac-RYYRIK-NH 2 and producing pure antagonist activity.

Discovery and structure–activity relationships of new steroidal compounds bearing a carboxy-terminal side chain as androgen receptor pure antagonists

Lead optimization of CH4892280 ( 4), an androgen receptor (AR) pure antagonist, was investigated. Compounds 6 and 7, which have a carboxylic acid at the end of the side chain at the position 7α of dihydrotestosterone (DHT), showed partial agonistic activities in reporter gene assay (RGA). Conversion of the steroidal core structure to 17α-methyltestosterone gave compound 14, which showed weak pure antagonistic activity. Optimization of the side chain by the insertion of a phenyl ring led to compounds 22 and 28– 30, which showed pure antagonistic activities at submicromolar concentrations. The structure–activity relationships were clarified.

Discovery of 7α-substituted dihydrotestosterones as androgen receptor pure antagonists and their structure–activity relationships

A series of 7α-substituted dihydrotestosterone derivatives were synthesized and evaluated for androgen receptor (AR) pure antagonistic activity. From reporter gene assay (RGA), the compound with a side chain containing N- n-butyl- N-methyl amide ( 19a) showed pure antagonistic activity (IC 50 = 340 nM, FI 5 > 10,000 nM), whereas known AR antagonists showed partial agonistic activities. The optimization of 19a led to compound 23 ( CH4892280), which showed more potent pure antagonistic activity (IC 50 = 190 nM, FI 5 > 10,000 nM). The SARs of tested compounds suggested that the length of the side chain and the substituents on the amide nitrogen are important for pure antagonistic activities.

Future perspectives of selective estrogen receptor modulators used alone and in combination with DHEA

Breast cancer is the most frequently diagnosed and the second cause of cancer death in women, thus making breast cancer a most feared disease. Since breast cancer metastasizes early and it is unlikely that improvements in the treatment of metastatic disease could permit a cure in most cases in the foreseeable future, it is clear that prevention is essential in order practically to eliminate deaths from breast cancer. Tamoxifen is the only selective estrogen receptor modulator (SERM) currently registered for use in breast cancer prevention; the tamoxifen versus raloxifene study should indicate the efficacy of this compound compared with raloxifene. The recent benefits of aromatase inhibitors over tamoxifen indicate the advantages of a blockade of estrogens more complete than the one achieved with tamoxifen, a SERM having some estrogenic activity in the mammary gland and an even higher estrogenic action in the uterus. However, it is unlikely that the general estrogen ablation achieved with aromatase inhibitors will be acceptable for the long-term use required for prevention. It is thus important to develop SERMs with highly potent and pure antagonistic activity in the mammary gland and uterus while possessing estrogen-like activity in tissues of particular importance for women's health, namely the bones and the cardiovascular system. However, it is expected that a SERM alone will not meet all the requirements of women's health at the postmenopause when ovarian estrogen secretion has ceased and peripheral formation of androgens and estrogens from DHEA by intracrine mechanisms is decreased by 60% or more. One possibility is to combine a SERM with DHEA, a precursor of sex steroids that permits, somewhat like SERMs, tissue-specific formation of androgens and/or estrogens according to the level of expression of the steroidogenic and steroid-inactivating enzymes. DHEA could thus compensate for the important loss of androgens that accompanies aging and could also permit sex steroid formation and action in the brain while breast cancer prevention would be achieved by the SERM.

Both N- and C-terminal transactivation functions of DNA-bound ERα are blocked by a novel synthetic estrogen ligand

Estrogen receptors (ERs) play a central role in the diverse actions of estrogen. A number of synthetic ER ligands have been generated that can modulate various ER functions. Here we show that TAS-108, representing a novel class of synthetic ER ligands, blocked both ER transactivation functions without inhibiting DNA-binding activity. A transient expression assay showed that similar to ICI182,780, TAS-108 exhibited pure antagonistic activity as it blocked both the N-terminal AF-1 and C-terminal AF-2 transactivation functions. However, unlike ICI182,780, TAS-108 promoted the recruitment of the SMRT co-repressor that abolished ER transactivation function without inhibition of the ability of ERα to bind to its target DNA. Both TAS-108 and ICI182,780 acted as antagonists for the transactivation functions of the D351Y mutant, derived from tamoxifen-resistant breast cancer cells, while estrogen and known selective estrogen receptor modulators (SERMs), 4-OH tamoxifen and raloxifene, stimulated D351Y-mediated transcription. Thus, our findings indicated that TAS-108 acts as a novel estrogen antagonist that recruits co-repressors to ERs without AF-1 activation or prevention of DNA binding. Therefore, TAS-108 may be effective against tamoxifen-resistant breast cancer via a different mechanism than that for ICI182,780.

In vitro and in vivo characterization of novel nonsteroidal progesterone receptor antagonists derived from the fungal metabolite PF1092C

We studied the pharmacological effects of novel nonsteroidal progesterone receptor antagonists CP8661 and CP8754, which were synthesized from the fungal metabolite PF1092C. CP8661 possess a tetrahydrobenzindolone skeleton and CP8754 possess a tetrahydronaphthofuranone skeleton. In binding assays for steroid receptors, CP8661 and CP8754 inhibited [ 3H]-progesterone binding to human progesterone receptors (hPR), though they are less potent than RU486. CP8661 also showed moderate affinity to rat androgen receptors (rAR), although CP8754 did not. Neither compound showed affinity to human glucocorticoid receptors (hGR) or human estrogen receptors (hER). In exogeneous and endogeneous PR-dependent enzyme expression assays using human mammary carcinoma T47D, CP8661 and CP8754 showed pure antagonistic activity. In a rabbit endometrial transformation test, CP8661 and CP8754 showed anti-progestational activity by s.c. administration in a dose-dependent manner; meanwhile, these compounds showed no progestational activity at the same dose. These results suggested that CP8661 and CP8754 are in vivo effective pure progesterone receptor antagonists and presented the possibility of synthesizing pure progesterone receptor antagonists from both tetrahydronaphthofuranone and tetrahydrobenzindolone skeletons.

Influence of Antiestrogens on the Invasiveness and Laminin Attachment of Breast Cancer Cells

Metastatic spread of breast cancer is responsible for most of the morbidity and mortality associated with this disease. Thus, it is important to identify agents with antimetastatic activity. Because invasiveness and tumor cell attachment are fundamental steps in the metastatic cascade, the major objective of the present study was to evaluate the antimetastatic potential of three antiestrogens, each with different chemical structure and mechanism of action, on breast cancer cell invasiveness and laminin attachment. The antiestrogens examined were tamoxifen, a mixed antagonist/agonist; Analog II, a cyclopropyl antiestrogen with pure antagonist activity; and ICI-182,780, a steroidal antiestrogen with pure antagonist activity. Our results indicate that MDA-MB-231 human breast cancer cells are much more invasive and have a higher affinity for laminin than do MCF-7 human breast cancer cells. All three antiestrogens, at a concentration of 10-6M, produced a reduction in MDA-MB-231 cell invasiveness, which was comparable in magnitude to their inhibition of MDA-MB-231 attachment to laminin. Evaluation of MDA-MB-231 cell morphology using scanning electron microscopy revealed the involvement of cellular pseudopodia and microvilli in the process of invasion. The results of this study suggest that antiestrogen-induced inhibition of breast cancer cell invasiveness could be due in part to a decrease in the attachment of tumor cells to laminin in the basement membrane.

Influence of Antiestrogens on the Invasiveness and Laminin Attachment of Breast Cancer Cells

Metastatic spread of breast cancer is responsible for most of the morbidity and mortality associated with this disease. Thus, it is important to identify agents with antimetastatic activity. Because invasiveness and tumor cell attachment are fundamental steps in the metastatic cascade, the major objective of the present study was to evaluate the antimetastatic potential of three antiestrogens, each with different chemical structure and mechanism of action, on breast cancer cell invasiveness and laminin attachment. The antiestrogens examined were tamoxifen, a mixed antagonist/agonist; Analog II, a cyclopropyl antiestrogen with pure antagonist activity; and ICI-182,780, a steroidal antiestrogen with pure antagonist activity. Our results indicate that MDA-MB-231 human breast cancer cells are much more invasive and have a higher affinity for laminin than do MCF-7 human breast cancer cells. All three antiestrogens, at a concentration of 10-6M, produced a reduction in MDA-MB-231 cell invasiveness, which was comparable in magnitude to their inhibition of MDA-MB-231 attachment to laminin. Evaluation of MDA-MB-231 cell morphology using scanning electron microscopy revealed the involvement of cellular pseudopodia and microvilli in the process of invasion. The results of this study suggest that antiestrogen-induced inhibition of breast cancer cell invasiveness could be due in part to a decrease in the attachment of tumor cells to laminin in the basement membrane.

Structure-activity relations of LHRH in birds.

Structural requirements in LHRH for gonadotropin-releasing activity were investigated by comparing the activity of the natural vertebrate LHRH structural variants and synthetic analogues. Substitution of Arg8 results in a loss of activity of LHRH in mammals, whilst a number of amino acid substitutions for Arg8 retain high gonadotropin-releasing activity in the chicken. Thus Arg8 of LHRH comprises an integral part of the binding site of LHRH and/or contributes towards the conformation of the binding site for the mammalian receptor while this does not pertain in the bird. The possibility that relative conformational stabilization of LHRH is important for biological activity in mammals but not birds, was supported by the demonstration that a gamma-lactam conformationally constrained analogue of LHRH was more active than LHRH in the mammalian system but equipotent in the bird. The chicken LHRH receptor is also relatively undiscriminating with regard to amino acid substitutions in positions 5 and 7. A series of LHRH analogues with pure antagonist activity in rats exhibited a spectrum of activities, from pure agonist to mixed activity and pure antagonist, in the chicken. These differences in LHRH structural requirements of the mammalian and avian receptor are reflected by a difference in molecular size of the chicken receptor (67,000) and mammalian receptor (60,000). Nevertheless, like the mammalian pituitary, the chicken pituitary does exhibit "desensitization" on prolonged exposure to LHRH.