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

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