Douglas J. Staples

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.

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.

Discovery of an α-melanotropin antagonist effective in vivo

Abstract A hybrid analogue, H-His- d -Arg-Ala-Trp- d -Phe-Lys-NH 2 , was designed based upon the primary structures of a growth hormone-releasing peptide analogue, [His 1 ,Lys 6 ]GHRP, and the MSH fragment, Ac-α-MSH(6–11)-NH 2 . In vitro studies demonstrated the α-MSH antagonistic efficacy of the analogue in the lizards Sceloporus jarrovii and Urosaurus ornatus . In live white background-adapted S. jarrovii previously injected with the antagonist (10 nmol/5 g b.wt.), maximal skin darkening induced by α-MSH was reduced to 50%. In white background-adapted U. ornatus , previous injection of the analogue (1 nmol/5 g b.wt.) totally abolished the response to α-MSH and depressed to 50% the maximal response elicited by the superpotent MSH analogue, [Nle 4 , d -Phe 7 ]α-MSH.

Peptidomimetic inhibitors of human immunodeficiency virus protease (HIV-PR): Design, enzyme binding and selectivity, antiviral efficacy, and cell permeability properties

Abstract The structure-activity relationships and pharmacophore modeling aspects of a series of HIV PR inhibitors modified at the N- and/or C-terminus of the dipeptide isostere ChaΨ[CH(OH)CH2]Val (Cha, cyclohexylalanine) are reported. The HIV PR binding affinity-selectivity (vs. human renin, pepsin, and cathepsins-D and E), antiviral efficacy (HIV-1/vVK-1 infected CV-1 cells) and cellular permeabilities (Caco-2) are noted.

[Norleucine3,6]-Substituted Cholecystokinin Octapeptide Analogues

Cholecystokinin octapeptide (CCK-8, Fig. 1) is a neurogastric peptide hormone and neurotransmitter that possesses multiple biological activities (Table I). Cholecystokinin octapeptide is one of several molecular variants (e.g., CCK-39, CCK33, CCK-12, and CCK-8) existing within a family of CCK peptides that have been identified in both the central and peripheral nervous systems as well as in the gastrointestinal tract. The specific details related to the discovery, distribution, biosynthesis, metabolism, biological activities in vitro and in vivo, and mechanisms of action of CCK peptides have been excellently reviewed recently (Mutt, 1980; Kelley and Dodd, 1981: Williams, 1982; Morley, 1982; Beinfeld, 1983; Dockray, 1983).

Design and Structure/Conformation-Activity Studies of a Prototypic Corticotropin-Releasing Factor (CRF) Antagonist: Multiple Alanine Substitutions of CRF12-41

The known physiological role(s) and proposed pathophysiological properties of the neuroendocrine peptide CRF have been previously described (for review, see 1), and CRF has been shown to exert a variety of CNS-mediated effects on behavior2,3, cardiovascular system4,5, reproduction6,7, gastrointestinal secretion8,9, motility10, and transit11. Of particular significance is that CRF may, therefore, be involved in stress stimuli-induced activation of neural/humoral pathways leading towards anxiety and depressive disorders (e.g.,depiession, panic and anorexia nervosa). Nevertheless, the molecular pharmacology and mechanisms which are involved in stress-induced behavioral, endocrine and metabolic activities are not well defined. The discovery and development of potent CRF antagonists may provide key molecular probes to investigate the biological activities of endogenous CRF in animal models as well as for studying the molecular pharmacology of CRF-receptor interactions. Such studies have been reported12,13 and have been primarily based upon synthetic modification of CRF; yet the emergence of a high affinity analog of low molecular mass (i.e., small peptide or peptidomimetic) has remained elusive to date. Nevertheless, studies14-18 on the blockade of endogenous CRF using CRF antiserum or prototypic CRF antagonists have probed the possible role that endogenous CRF may have on the effects of stress in different animal models. Of noteworthy contribution to such CRF research has been both structure-activity and structure-conformation studies12,13,19,20 to investigate CRFreceptor binding and functional properties (agonism/antagonism). These studies have culminated in the identification of prototypic CRF antagonists (or partial agonists) which were modified fragment analogs of the native peptide. Specifically, compound I (Fig. 1) has been advanced13 as a significant lead towards the development of high affinity CRF receptor antagonists. In this report we describe analogs of I to further explore the role of side-chain functionlization in CRF receptor binding using a strategy of multiple (iterative) Ala substitution with a particular focus on the central domain of this CRF analog corresponding to CRF22-31 In addition, the structure-conformation properties of these analogs were investigated by circular dichroism spectroscopy.

Exploiting the Molecular Template of Angiotensinogen in the Discovery and Design of Peptidyl, Pseudopeptidyl and Peptidemimetic Inhibitors of Human Renin: A Structure-Activity Perspective

The design of potent and pharmacologically effective, substrate-related inhibitors of renin has been the subject of intensive pharmaceutical discovery research for about one decade. Milestone achievements in synthetic tailoring of fragment analogs of angiotensinogen (ANG; Figure 1) have been documented in terms of identifying renin inhibitors of subnanomolar potency, sustained in vivo hypotensive activity, stability towards proteolytic degradation, and, more recently, oral bioavailability and decreased systemic clearance.1 By chemical modification of ANG-based derivatives, structure-activity analysis, and computer-assisted molecular modeling of peptidyl, pseudopeptidyl and peptidemimetic inhibitors using 3-D structural models of human renin, there currently exists a rather sophisticated wealth of information of relevance to the “rational” design of prototypic renin-targeted cardiovascular therapeutic agents. Such efforts have bridged biochemistry, medicinal chemistry, computational and biophysical chemistry, and in vivo pharmacology including, in a few cases, clinical evaluation in humans.