Evaluation of a stable Gonadotropin-Releasing Hormone analog in mice for the treatment of endocrine disorders and prostate cancer

Gonadotropin-releasing hormone (GnRH) receptor agonists have wide clinical applications including the treatment of prostate cancer and endocrine disorders. However, such agonists are characterized by poor pharmacokinetic properties, often requiring repeated administration or special formulations. Therefore, the development of novel peptide analogs with enhanced in vivo stability could potentially provide therapeutic alternatives. The pharmacological evaluation of a bioactive peptide [Des-Gly10,Tyr5(OMe),D-Leu 6,Aze-NHEt9]GnRH, analog 1, is presented herein and compared with leuprolide. Peptide stability was evaluated using mouse kidney membrane preparations, followed by a liquid chromatography-tandem mass spectrometry-based approach that afforded identification and quantification of its major metabolites. The analog was significantly more stable in vitro in comparison with leuprolide. In vitro and in vivo stability results correlated well, encouraging us to develop a clinically relevant pharmacokinetic mouse model, which facilitated efficacy measurements using testosterone as a biomarker. Analog 1, an agonist of the GnRH receptor with a binding affinity in the nanomolar range, caused testosterone release in mice that was acutely dose-dependent, an effect blocked by the GnRH receptor antagonist cetrorelix. Repeated dosing studies in mice demonstrated that analog 1 was well tolerated and had potency similar to that of leuprolide, based on plasma and testis testosterone reduction and histopathological findings. Analog 1 also shared with leuprolide similar significant antiproliferative activity on androgen-dependent prostate cancer (LNCaP) cells. On the basis of pharmacokinetic advantages, we expect that analog 1 or analogs based on this new design will be therapeutically advantageous for the treatment of cancer and endocrine disorders. Copyrigh

A Novel Role of Peripheral Corticotropin-Releasing Hormone (CRH) on Dermal Fibroblasts

Corticotropin-releasing hormone, or factor, (CRH or CRF) exerts important biological effects in multiple peripheral tissues via paracrine/autocrine actions. The aim of our study was to assess the effects of endogenous CRH in the biology of mouse and human skin fibroblasts, the primary cell type involved in wound healing. We show expression of CRH and its receptors in primary fibroblasts, and we demonstrate the functionality of fibroblast CRH receptors by induction of cAMP. Fibroblasts genetically deficient in Crh (Crh−/−) had higher proliferation and migration rates and compromised production of IL-6 and TGF-β1 compared to the wildtype (Crh+/+) cells. Human primary cultures of foreskin fibroblasts exposed to the CRF1 antagonist antalarmin recapitulated the findings in the Crh−/− cells, exhibiting altered proliferative and migratory behavior and suppressed production of IL-6. In conclusion, our findings show an important role of fibroblast-expressed CRH in the proliferation, migration, and cytokine production of these cells, processes associated with the skin response to injury. Our data suggest that the immunomodulatory effects of CRH may include an important, albeit not explored yet, role in epidermal tissue remodeling and regeneration and maintenance of tissue homeostasis

Molecular Basis of Ligand Dissociation in β-Adrenergic Receptors

The important and diverse biological functions of β-adrenergic receptors (βARs) have promoted the search for compounds to stimulate or inhibit their activity. In this regard, unraveling the molecular basis of ligand binding/unbinding events is essential to understand the pharmacological properties of these G protein-coupled receptors. In this study, we use the steered molecular dynamics simulation method to describe, in atomic detail, the unbinding process of two inverse agonists, which have been recently co-crystallized with β1 and β2ARs subtypes, along four different channels. Our results indicate that this type of compounds likely accesses the orthosteric binding site of βARs from the extracellular water environment. Importantly, reconstruction of forces and energies from the simulations of the dissociation process suggests, for the first time, the presence of secondary binding sites located in the extracellular loops 2 and 3 and transmembrane helix 7, where ligands are transiently retained by electrostatic and Van der Waals interactions. Comparison of the residues that form these new transient allosteric binding sites in both βARs subtypes reveals the importance of non-conserved electrostatic interactions as well as conserved aromatic contacts in the early steps of the binding process

Μελέτη της δομής και λειτουργίας του τύπου 1 υποδοχέα του εκλυτικού παράγοντα της κορτικοτροπίνης

The subfamily B of G-protein-coupled receptors (GPCRs) consists of receptors that bind peptides, such as secretin, vasoactive intestinal peptide, glucagon, corticotropin-releasing hormone (CRF), which play a fundamental role in body function. These receptors, like all GPCRs, are plasma membrane proteins sharing a common structural motif of seven membrane-spanning domains (TMs), which have been shown to bind small molecules, such as antalarmin, a non-peptide antagonist of the type 1 receptor for CRF (CRF1). This leads to the hypothesis that similar to family A, rhodopsin-like, GPCRs, the TMs of subfamily B GPCRs form a water-accessible crevice, the binding-site crevice, which extends from the extracellular surface of the receptor into the plane of the membrane. The surface of this crevice is formed not only by residues that can contact small molecules but also by residues that may play a structural role and affect binding indirectly. However, the lack of considerable structural information for the family B GPCRs precludes the support of this hypothesis. To test this hypothesis we started obtaining structural information about subfamily B GPCRs, using as prototype the CRF1 and determining its ability to react with the charged, hydrophilic, lipophobic, sulfhydryl-specific methanethiosulfonate (MTS) derivative, MTSethylammonium (MTSEA). The reaction of MTSEA with CRF1 was tested by its ability to irreversibly inhibit [125I]-sauvagine binding to receptor. Reaction of MTSEA with CRF1 inhibited [125I]-sauvagine binding to CRF1. These results, in conjunction with that the antagonist antalarmin protected against irreversible inhibition by the MTSEA, suggest that MTSEA reacted with the sulfhydryl of one or more cysteines of CRF1. To identify the susceptible cysteine(s), we mutated to serine, one at a time, the four endogenous cysteine residues, Cys128, Cys211, Cys233 and Cys364, which are located in the first (TM1), third (TM3), fourth (TM4) and seventh (TM7) membrane spanning segment of CRF1. In contrast to Cys128 mutation, substitution of Cys211, Cys233 and Cys364 by serine decreased the susceptibility of sauvagine binding to irreversible inhibition by MTSEA. These results suggest that Cys211, Cys233 and Cys364 are exposed in the binding-site crevice of CRF1, and their reaction with MTSEA decreased sauvagine binding to receptor. Subsequently we mutated, one at a time, 18 residues (engineered Cys) of the third membrane spanning segment (TM3) of CRF1 to cysteine, and tested the accessibilities of the engineered Cys for reaction with the MTSEA. Six of the mutant receptors reacted with MTSEA, suggesting that the side chains of these residues are exposed in the binding-site crevice, of CRF1. The pattern of accessibility was consistent with an alpha-helical conformation. In contrast to membrane spanning segments of CRF1 which have been proposed to bind the small non-peptide antagonists, the extracellular amino-terminal regions and the three extracellular loops of CRF1 interact with the larger peptides belonging to CRF family, such as CRF and sauvagine. In specific previous studies have been shown that upon binding of sauvagine to CRF1, the amino-terminal portion of the peptide lies near Lys257 in the receptor?s second XI extracellular loop (EL2). To test the hypothesis that EL2 residues play a role in the binding of sauvagine to CRF1 we carried out an alanine-scanning mutagenesis study to determine the functional role of EL2 residues (Leu251 to Val266). Only the W259A, F260A, and W259A/F260A mutations reduced the binding affinity and potency of sauvagine. In contrast, these mutations did not seem to significantly alter the overall receptor conformation, in that they left unchanged the affinities of the ligands astressin and antalarmin that have been suggested to bind to different regions of CRF1. The W259A, F260A, and W259A/ F260A mutations also decreased the affinity of the endogenous ligand, CRF, implying that these residues may play a common important role in the binding of different peptides belonging to CRF family. Parallel amino acid deletions of the two peptides produced ligands with various affinities for wild-type CRF1 compared with the W259A, F260A, and W259A/F260A mutants, supporting the interaction between the amino-terminal residues 8 to 10 of sauvagine and the corresponding region in CRF with EL2 of CRF1. This is the first time that a specific region of CRF1 has been implicated in detailed interactions between the receptor and the amino-terminal portion of peptides belonging to the CRF famil

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)[Pro1, d-Leu6, BABA10] 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)[Pro1, d-Leu6, BABA10] 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)[Pro1, d-Leu6, BABA10] LHRH render this analogue a promising platform for future rational drug design studies towards the development of non-peptide LHRH mimetics. [Display omitted] ► We designed cyclic peptide analogues of LH-RH hormone with crucial substitutions. ► The cyclic analogues were more stable in proteolysis compared to linear counterparts. ► The rationally designed cyclic analogues were studied in binding assay. ► We report the testosterone release of more potent cyclic analogue in mice

Structural-Functional of the Analysis of the Third Transmembrane Domain of the Corticotropin-releasing Factor Type 1

Backround: The molecular mechanisms underlying activation of CRF1 receptor (CRF1R) were elusive. Results: We determined specific residues in the transmembrane domains (TMs) of CRF1R that are critical for receptor activation. Conclusion: A possible “transmission switch” involving TM interactions is important for CRF1R activation. Significance: This knowledge may aid in the development of nonpeptide CRF1R antagonists for use in stress-related disorder

IL-6 and TGF-β1 production from Crh+/+ and Crh−/− fibroblasts.

<p>Primary dermal fibroblasts isolated from <i>Crh+/+</i> and <i>Crh−/−</i> mice were cultured in 24-well plates (10⁵ cells/well). Supernatants were collected and analyzed by ELISA for IL-6 (a) or TGF-β1 (b). * represents statistical difference (P<0.05) between genotypes (n = 3 wells/condition/experiment, at least 3 independent experiments).</p