ABSTRACT
Recently, we demonstrated that the corticotropin releasing factor 2 receptor agonist, urocortin 2, demonstrated anti-atrophy effects in rodent skeletal muscle atrophy models. Compared to other CRF2R agonists however, the in vivo pharmacological potency of urocortin 2 is poor when it is administered by continuous subcutaneous infusion. Therefore, we attempted to modify the structure of urocortin 2 to improve in vivo efficacy when administered by subcutaneous infusion. By substituting amino acid residues in the linker region of urocortin 2 (residues 22-32), we have demonstrated improved in vivo potency without altering selectivity, probably through reduced CRFBP binding. In addition, attempts to shorten urocortin 2 generally resulted in inactive peptides, demonstrating that the 38 amino acid urocortin 2 peptide is the minimal pharmacophore.
Subject(s)
Corticotropin-Releasing Hormone/chemistry , Peptides/chemistry , Amino Acid Sequence , Animals , Atrophy , Cell Line , Cell Line, Tumor , Humans , Male , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Protein Binding , Protein Structure, Tertiary , Rats , UrocortinsABSTRACT
The corticotropin release factor 2 receptor (CRF2R) has many biological activities including modulation of the stress response. Recently, we have demonstrated that CRF2R activation functions to prevent skeletal muscle wasting resulting from a variety of physiological stimuli. Thus we are interested in identifying CRF2R selective agonists with optimal pharmacological properties for use in treating muscle wasting diseases. Several CRF2R agonists are known including the frog peptide sauvagine (Svg), which display superior pharmacological properties compared to other CRF2R agonists. Unfortunately sauvagine is a nonselective CRFR agonist, thus making it of less utility due to side effects resulting from corticotropin release factor 1 receptor (CRF1R) activation. Because our initial modifications of Svg at position 11 improved CRF2R selectivity, we investigated the role of amino acids at positions 12 and 13 in Svg. We observed that phenylalanine, leucine, isoleucine, threonine, glutamine, histidine, and tyrosine at the 12th position were the strongest promoters of CRF2R selectivity whereas phenylalanine, glutamine, trytophane, tyrosine, valine, isoleucine, leucine, and 2-naphthylalanine were the preferred residues at the 13th position. Selective sauvagine peptides demonstrated improved antiatrophy effects in a mouse-casting model when compared to sauvagine itself. Thus, we demonstrate that the CRF2R selectivity can be improved by optimizing amino acids at positions 12 and 13 (all with proline at position 11) and that the selective sauvagine analogues demonstrate better in vivo efficacy than sauvagine itself.
Subject(s)
Muscular Disorders, Atrophic/drug therapy , Peptides/chemistry , Peptides/pharmacology , Receptors, Corticotropin-Releasing Hormone/drug effects , Amino Acid Substitution , Amphibian Proteins , Animals , Disease Models, Animal , Drug Evaluation, Preclinical/methods , Mice , Peptide Hormones , Receptors, Corticotropin-Releasing Hormone/agonists , Structure-Activity Relationship , Tibia/drug effectsABSTRACT
Corticotropin releasing factor 2 receptor selective analogs of the amphibian peptide sauvagine, a member of the corticotropin releasing factor (CRF) peptide family, have therapeutic potential for the treatment of skeletal muscle atrophy. Previously, we demonstrated that [P11X12X13]Svg peptides have improved CRF2R selectivity, although not to the level of CRF2R selective hormones such as urocortin 2 and urocortin 3. Since we also demonstrated a potential for improvement in selectivity of sauvagine by modifications of residues 35 and 39, we investigated substitutions of these amino acids in selected [P11X12X13]Svg peptides. We have observed that substitution of Arg35 in sauvagine to Ala35 (the amino acid found in all CRF2R selective agonists), increased the selectivity of [P11, X12, X13]Svg analogs. In contrast, substitution of Asp39 in sauvagine to Ala39 (also the amino acid found in all CRF2R selective agonists) did not further increase the selectivity of [P11, X12, X13, A35]Svg analogs. Thus, the residues 35 along with 11, 12, and 13 in sauvagine represent important sites for improving CRF2R selectivity.
Subject(s)
Peptides/chemistry , Peptides/pharmacology , Receptors, Corticotropin-Releasing Hormone/agonists , Alanine/genetics , Amino Acid Sequence , Amino Acid Substitution , Amphibian Proteins , Animals , Asparagine/genetics , Binding Sites , Cells, Cultured , Humans , Mice , Molecular Sequence Data , Peptide Hormones , Peptides/genetics , RatsABSTRACT
The corticotropin-releasing factor (CRF) peptide family is an important target in pharmaceutical research. The CRF system consists of two receptors, corticotropin releasing factor receptor 1 (CRF1R) and corticotropin releasing factor receptor 2 (CRF2R), a nonreceptor binding protein, and the peptide agonists of these receptors. The recent discovery of the CRF2R selective peptide agonists, UCN2, UCN3 and URP, prompted investigations into the structural source of CRF1R versus CRF2R selectivity of CRF peptide family members. Data from chimeric peptides demonstrated that amino acids in the N-terminus and C-terminus of CRF, UCN1, UCN2 and Sauvagine peptide families influence CRFR selectivity. Analysis of specific amino acid residues in the N-terminus and C-terminus demonstrated that the presence of a proline at position 11 and alanine at positions 35 and 39 (hCRF numbering) decreases CRF1R activity and increases CRF2R selectivity in CRF, UCN1 and sauvagine peptides. The availability of a large group of selective and nonselective CRF receptor peptide agonists will facilitate the development of CRF receptor selective drugs.