Topographical modification of melanotropin peptide analogs with .beta.-methyltryptophan isomers at position 9 leads to differential potencies and prolonged biological activities

Abstract

We have introduced topographical constraints at the 9 position of a superpotent cyclic alpha-melanotropin analogue, Ac-Nle4-Asp5-His6-DPhe7-Arg8-Trp9-Lys10-NH2, by incorporating a methyl group at the beta-carbon of Trp9. These studies were performed on the Trp side chain pharmacophore to identify the bioactive topography of the indole moiety with melanocortin MC1 receptors. The four beta-MeTrp9 isomers, in addition to the stereochemical controls L- and DTrp9, were used to probe differential receptor molecular recognition of the tryptophan moiety in two bioassay systems. Approximately a 460-fold difference in potency was observed between the diastereoisomeric peptides in the frog skin bioassay, with only 33- and 10-fold efficacy differences observed in binding and intracellular cAMP accumulation, respectively, on the human melanocortin receptor, hMC1R. The relative orders of potencies in the frog skin bioassay were 2R,3S > 2S,3S = 2R,3R >> 2S,3R and for the hMC1R were 2S,3S > 2R,3R > 2R,3S >> 2S,3R. Of particular interest is the ability of these topographically constrained ligands to differentially affect prolonged biological activity. The 2R,3R diastereoisomeric peptide possessed superprolonged activity, whereas the 2S,3S peptide lacked any residual activity in the frog skin bioassay. However, on the melanocortin receptor, the 2S,3S diastereoisomeric peptide maintained slow dissociation rates (t1/2 = 7 h), while the other diastereoisomeric peptides possessed dissociation t1/2 rates of ca. 2 h. These data strongly implicate ligand-receptor interactions and kinetics as contributing to the observed prolonged biological activities and clearly illustrate topographical recognition differences between these two peripheral MC1 receptors involved in skin pigmentation. This study also demonstrates that topographical modifications of pharmacophore side chain residues, in addition to identifying preferential side chain orientation, can be a useful strategy for the design of peptides to increase the duration of biological activity, relative to the native ligand.

Astressin Analogues (Corticotropin-Releasing Factor Antagonists) with Extended Duration of Action in the Rat

“…By our definition, we consider 6 and 8 to still be short-acting. In summary, while in earlier studies, we had identified the Glu 30 32 ] substitutions as being favorable, the above data indicated that acetylation of the N-terminus of CRF antagonists may also have favorable effects on potency and duration of action.…”

“…19 We therefore substituted leucines at positions 14, 15, 19, 27, and 37 of astressin and [DHis 32 ]astressin with CRMe-Leu (structures shown in Table 1). We hypothesized that this residue might block the action of endopeptidases in ways that could not be achieved with the introduction of D-residues alone.…”

“…We also noted that further deletions (residues 8-14) produced antagonists such as astressin {cyclo (30)(31)(32)(33) were ca. twice and 1/100 as potent as astressin, respectively, suggesting a putative turn that encompasses residues 30-33 (previous paper: Koerber et al J. Med.…”

“…While oCRF and hCRF have been used in the clinic, CRF antagonists | Abbreviations: IUPAC rules are used for nomenclature of peptides including one-letter codes for amino acids. Also: Ac, acetyl; ACTH, adrenocorticotropin hormone; adx, adrenalectomized; astressin, cyclo- (30)(31)(32)(33)[DPhe 12 ,Nle 21,38 ,Glu 30 ,Lys 33 ]hCRF(12-41); Boc, tert-butyloxycarbonyl; BOP, benzotriazolyloxy-tris(dimethylamino)phosphonium hexafluorophosphate; BSA, bovine serum albumin; CD, circular dichroism; CRF, corticotropin-releasing factor (o, ovine; h, human); CSF, cerebrospinal fluid; CZE, capillary zone electrophoresis; DCM, dichloromethane; DIC, diisopropylcarbodiimide; DMF, dimethylformamide; FBS, fetal bovine serum; Fmoc, 9-fluorenylmethoxycarbonyl; HBTU, O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate; HF, hydrogen fluoride; HOBt, 1-hydroxybenzotriazole; IA, intrinsic activity; ic, intracisternal; LSIMS, liquid secondary ion mass spectrometry; MBHA, 4-methylbenzhydrylamine; NMP, N-methylpyrrolidinone; NMR, nuclear magnetic resonance; OFm, O-fluorenylmethyl; SAR, structure-activity relationships; TBTU, O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate; TEAP 2.25, 4.5, and 6.5, triethylammonium phosphate, pH 2.25, 4.5, and 6.5; TFA, trifluoroacetic acid; TFE, trifluoroethanol.…”

Structure–activity studies of new melanocortin peptides containing an aromatic amino acid at the N-terminal position

“…In the past few years, several analogues of MT-II and SHU-9119, which are high affinity agonists and antagonists at the hMC3 and the hMC4 receptors were reported in literature, in which most of the modifications were made in positions 6-9 [19][20][21]24,25,40]. These modifications have suggested that amino acid residues at the 6 (His), 7 (Phe), 8 (Arg), and 9 (Trp) positions are important for molecular recognition and activation of the melanocortin receptors.…”