Fahad Al-Obeidi

α-melanotropin: The minimal active sequence in the lizard skin bioassay

alpha-Melanotropin (alpha-melanocyte-stimulating hormone, alpha-MSH) is a tridecapeptide, Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2. The minimal sequence of alpha-MSH required for agonism in the lizard (Anolis carolinensis) skin bioassay was determined to be Ac-His-Phe-Arg-Trp-NH2 (Ac-alpha-MSH6-9-NH2). Smaller fragments of this sequence (Ac-alpha-MSH6-8-NH2, Ac-alpha-MSH6-7-NH2, Ac-alpha-MSH7-9-NH2, and Ac-alpha-MSH7-8-NH2) were devoid of melanotropic activity. The tetrapeptide, Ac-alpha-MSH7-10-NH2, was also inactive, thus again demonstrating the importance of His at position 6 for minimal activity. The important potentiating amino acids were found to be Met-4, Lys-11, and Pro-12, since Ac-alpha-MSH4-10-NH2 was about 100 times more potent than Ac-alpha-MSH5-10-NH2, and Ac-[Nle4]-alpha-MSH4-11-NH2 was about 40 times more potent than Ac-alpha-MSH4-10-NH2 or Ac-[Nle4]-alpha-MSH4-10-NH2. Ac-alpha-MSH4-12-NH2 and Ac-[Nle4]-alpha-MSH4-12-NH2 were equipotent and about six times more potent than alpha-MSH. Since [Nle4]-alpha-MSH and Ac-[Nle4]-alpha-MSH4-13-NH2 were both equipotent but about sixfold less active than Ac-[Nle4]-alpha-MSH4-12-NH2, it is clear that valine at position 13 does not contribute to the potency of alpha-MSH, except possibly in a negative way. The minimal message sequence for equipotency to alpha-MSH appears to be Ac-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-NH2, since the analog, Ac-[Nle4]-alpha-MSH4-11-NH2, was as active as the native hormone. Ser-1, Tyr-2, Ser-3, Glu-5, and Val-13 are not important for melanotropic potency since Ac-alpha-MSH4-12-NH2 was more potent than alpha-MSH, and Ac-alpha-MSH5-10-NH2 and Ac-alpha-MSH6-10-NH2 were equipotent, being about 4,000 times less active than alpha-MSH.

Design, synthesis, and conformation of superpotent and prolonged acting melanotropins.

a-Melanotropin (a-MSH, Ac-Ser-Tyr-Ser-Met-Glus-His-Phe-Arg-T~-GlyloLys-Pro-Val-NH,) has a long and storied history, being one of the first peptide hormones to be studied as a crude extract from the pituitary and to be isolated in pure form and have its structure determined. It was early recognized to have essentially the same N-terminal tridecapeptide sequence as adrenocorticotropic hormone (ACTH, corticotropin) except that the N-terminal was acetylated in the case of a-MSH but not in the case of ACTH, indicating that their biosyntheses were different. Subsequently it was discovered that a-MSH and ACTH were derived from the same gene, currently referred to as proopiomelanocortin (POMC), of which much more is discussed elsewhere in the proceedings of this meeting. ACTH does have weak melanotropinlike activities in certain a-MSH-specific assays such as the frog skin bioassay, but the acetyl group is important to maximize the biological potency of the native a-MSH (a-MSH is about 100 times more potent than ACTH in the classical pigmentary assays). It is now understood that though a-MSH was originally discovered as a peptide hormone, affecting pigmentation in peripheral tissues especially the skin, there is much anatomical and pharmacological evidence that a-MSH is found in the brain where it and/or ACTH and its fragments have a growing list of proposed neurotransmitter, neuromodulatory, neuroimmunological, behavioral, and other biological activities. Much more is said elsewhere in the proceedings of this meeting regarding these sites of action of this remarkable hormone. This paper, which is devoted to the chemistry and structure-biological activity relationships, will focus entirely on a-MSHs activities at peripheral receptors responsible for pigmentation, since this is the primary receptor for which extensive structure-biological activity studies have been done. Structure-activity studies on a-MSH began immediately with its isolation and

α-Melanocyte stimulating hormone message and inhibitory sequences: Comparative structure-activity studies on melanocytes

We investigated the structure-activity relationships of alpha-MSH (alpha-melanocyte stimulating hormone) fragment derivatives of the generic formulae Ac-alpha-MSH(x-13)-NH2 and Ac-alpha-MSH(6-x)-NH2. The minimal C-terminal sequences required for melanotropic activity were 8-13 and 7-13, respectively, in the frog and lizard skin bioassays. The Arg8-Trp9 sequence appears to be a fundamental component of the minimal message sequences found to date such as alpha-MSH(6-9), alpha-MSH(8-13) and alpha-MSH(7-13). We discovered that Ac-alpha-MSH(7-10)-NH2 was a weak and selective alpha-MSH antagonist on the lizard skin bioassay. Analysis of alpha-MSH(7-10) analogues of the generic formula Ac-Xaa-Arg-Trp-Yaa-NH2 led to Ac-[D-Trp7,D-Phe10]alpha-MSH(7-10)-NH2, a moderately potent, specific and competitive inhibitor of alpha-MSH in both the frog and the lizard skin bioassays.

The effects of structure-conformation modifications of melanotropin analogs on learning and memory: D-amino acid substituted linear and cyclic analogs

Alpha-MSH has a wide variety of putative biological activities in addition to its classical melanocyte dispersing activity. Since each of these activities appears to be mediated by a discrete receptor, this peptide is an excellent candidate for exploring conformational restrictions which determine the chemical-physical basis for hormone action on specific activities. Experiments One and Two evaluated several cyclic and linear analogs of alpha-MSH on retrieval of memory during the reactivation of memory for a passive avoidance response following hypothermia-induced amnesia. Three of the cyclic analogs appear to have enhanced the peptides ability to serve as a reactivation agent. One of the linear Nle4,D-Phe7 analogs antagonized whereas three others enhanced reactivation. The D-Phe7 substitution in cyclic analogs did not affect reactivation. Another group of animals were trained on a step-through passive avoidance task and tested 25 days later. The cyclic analog enhanced memory whereas the D-Phe7 analog and alpha-MSH had no effect. Finally, two analogs were tested on a black-white discrimination. Although the cyclic analog had no effect on either acquisition or reversal of this learning, the Nle4,D-Phe7 analog significantly impaired reversal learning. The results from these preliminary studies suggest that structural modifications of alpha-MSH do alter its potency and pattern of actions in learning and memory situations.

Toxicologic studies of a superpotent α-melanotropin, [Nle4, D-Phe7]α-MSH

SummaryA toxicology study was performed in mice given a superpotent α melanocyte stimulating hormone (MSH) analog. This 13 amino acid derivative, [Nle4, D-Phe7]α-MSH or NDP-MSH, is a melanotropin which is very slowly biodegraded in vivo and is active at 1/1,000 the concentration of natural α-MSH. Mice were administered up to 2 mg/kg of the analog daily and weekly over 4 or 12 weeks by both topical application (in 90% DMSO) or by IP injections (in physiologic saline). At the end of this period, no toxic effects were observed in various organs, on hematologic indices, or on weight gain. A slight increase in triglyceride and platelet levels were noted in mice given the analog weekly for 12 weeks. There was no evidence of an effect on behavior nor ACTH-like endocrine actions such as elevated serum cortisol levels. Transdermal drug delivery studies performed in vitro showed reproducible diffusion of the NDP-MSH analog through full-thickness mouse skin. Approximately 0.002% to 0.05% of a 10−4M preparation was transdermally delivered using a DMSO/water solution or a PEG/alcohol cream base, respectively. This superpotent analog is now entering a Phase I clinical trial with possible therapeutic applications for the treatment of hypomelanotic disorders such as vitiligo and for pharmacologic tanning without the need for sunlight exposure.

Transdermal delivery of a melanotropic peptide hormone analogue

We previously reported that topical application of [Nle4,D-Phe7]alpha-MSH, a superpotent analogue of alpha-melanocyte stimulating hormone, to mice induces a darkening of follicular melanocytes throughout the skin. We now report that the melanotropin analogue can be delivered across mouse but not rat skin in an in vitro model system. Passage of the analogue from the topically applied vehicle (polyethylene glycol) across the skin into a subcutaneous receiving vessel was demonstrated by both bioassay as well as by radioimmunoassay. The bioassay data demonstrate that percutaneous absorption of the melanotropin did not result in loss of biological activity of the peptide. The differential penetration of the peptide across rodent skin reveals that one cannot predict percutaneous absorption of a substance across the stratum corneum from studies on a single species. The present results are the first to demonstrate, by direct quantitative measurements, that a bioactive peptide can be delivered across the vertebrate integument in vitro. These studies point out the potential of a topically applied melanotropin for tanning of the skin and possibly for treatment of certain hypopigmentary disorders.

Strategies for cyclizations of novel peptides on solid supports

Synthetic strategies and methods for the preparation of cyclic peptides on solid supports are very important for peptide synthesis in general, and for the preparation of conformationally constrained biologically active peptides. In this paper we report synthetic methods for the preparation on solid supports of the cyclic and bicyclic biologically active peptides, Ac-[Nle4, Asp5, d-Phe, Lys10]α-melanotropin(4–10)-NH2, [Lys17, Lys18, Asp21]glucagon-NH2, [β-Mpa1, Glu4, Cys6, Lys8]oxytocin and cyclo[GlyTrpNMeNleAspPhe] (cyclic CCK-5 analogue).

Conformational Constraints in the Design of Receptor Selective Peptides: Conformational Analysis and Molecular Dynamics

Efforts to understand the relationships between the three dimensional structure and topography of a peptide hormone or neuropeptide and its biological activities have been difficult due to the many conformations generally available to these natural products, and the complexity of the hormone-receptor interaction which involves at least three different conformational states in the course of information transfer (Hruby 1981a,b; Hruby and Hadley, 1986; Hruby et al., 1983). A major advance in this area has come with the development of conformational constraints, for example via cyclization (Kessler, 1982; Hruby, 1982, 1985), and their use in the design of more receptor selective, potent, and prolonged acting peptide analogues. A major goal in this area is the design of conformationally constrained analogues with stable conformations and high receptor specificity that can be used as templates to assess the topographical features which are important for ligand-receptor interaction and for transduction to produce a biological response. Strategies for accomplishing these goals are urgently needed. In this paper we will discuss two strategies which we believe can be utilized generally, but at different times, in the course of developing insights into the “biologically active” conformation for a peptide hormone or neurotransmitter.

Aspects of the design of conformationally constrained peptides

Publisher Summary This chapter discusses different aspects of the design of conformationally constrained peptides. It also describes the use of nuclear magnetic resonance (NMR)-generated parameters for static and dynamic structures in conjunction with modern theoretical calculations as a general strategy for the design of bioactive and selective peptides. The general approach to conformational analysis of peptides in solution includes the following steps: (1) the absolute assignment of NMR signals to the peptide components (residues); (2) the extraction of various NMR parameters—for example, coupling constants, chemical shifts, nuclear Overhauser effect (NOE) signals, and relaxation times—that can be used in conformational studies; (3) the construction of models based on distance and/or dihedral angle information; and (4) the optimization and refinement of the proposed models by using various techniques, for example, distance geometry (DG), molecular mechanics energy minimization, and molecular dynamics with augmentations to include NMR-derived constraints. The comparison of theoretically derived results with other experiments is necessary to prove or disprove the initial assumption of the conformational distribution. The properties of peptides vary with salt concentration and pH. Binding affinities and conformations in solution are among the most important properties of peptides that determine activity and, ultimately, potency.