In our search for potent and receptor-selective agonists and antagonists, we report here the results of D-amino acid substitution at each position of the short peptide gamma-melanocyte-stimulating hormone (gamma-MSH). The native gamma-MSH shows weak binding at all three receptors (i.e., the human MC3, MC4, and MC5) and a selectivity of 1-2 orders of magnitude at the MC3R over the MC4R and MC5R. Sequential replacement of each residue in the gamma-MSH sequence with the corresponding D-isomer results in analogues which mostly have weaker binding affinity than the native peptide, except for two analogues. For the DTrp(8) analogue, there is an increase in binding affinity by about 1 order of magnitude (IC(50) = 6 nM) at the MC3R compared with that of the natural molecule and an increase in selectivity for the MC3R by 2 orders of magnitude compared with the activity at the MC4R and MC5R. The DPhe(6) analogue is about 10-fold more potent (IC(50) = 8.8 nM) at the MC3R compared with the native peptide but lacks subtype selectivity. Measurement of the intracellular cAMP accumulation in human MC3R, MC4R, and MC5R revealed that the native peptide shows potent activity at the MC3R (EC(50) = 5.9 nM) and is about 50-100-fold selective at this receptor compared with the MC4R and MC5R. The DArg(10) (EC(50) = 35 nM) and DPhe(11) (EC(50) = 11 nM) analogues are selective for the MC3R by 1 and 2 orders of magnitude compared with the MC4R and MC5R, respectively. The DTrp(8) compound (EC(50) = 0.33 nM) shows about 300- and 250-fold increase in selectivity at the MC3R compared with the MC4R and MC5R, respectively. Finally, the DTyr(1) peptide is selective for the MC3R (EC(50) = 12 nM) by 40-200-fold compared with the MC4R and MC5R. In general, the trend is that D-amino acid substitutions of the aromatic residues 1, 6, 8, and 11 and the basic residue Arg(10), but not Arg(7), result in an increase in MC3R selectivity over the MC4R and MC5R and only agonist activity is observed. Thus, the key residues of gamma-MSH identified in this study include the aromatic residues 1, 6, 8, and 11 and the basic residue Arg(10) (but not Arg(7)), as important for MC3 selectivity over the MC4 and MC5 subtypes. Further, the study reveals the extreme importance of DTrp at position 8 in imparting potency and selectivity since this is the most selective analogue for the human MC3R reported thus far.
We synthesized porcine neuropeptide Y (pNPY) N-terminal fragments by solid-phase synthesis techniques and analyzed them for solution conformational properties by CD and 1H-nmr spectroscopy. The analogues pNPY1-9 and pNPY1-14 displayed CD spectra indicative of random structures and showed no evidence for induced alpha-helical structures in trifluoroethanol (TFE) up to 50%. However, the CD spectra of pNPY1-9 suggested a conformational shift in tetrahydrofuran. Although in aqueous solution the CD spectra of pNPY1-21 indicated random structures with induction of only a small percentage of alpha-helix in aqueous TFE, pNPY1-25 displayed 13% alpha-helical structure in aqueous solution that increased to 40 and 41% by the addition of TFE and methanol, respectively. The nmr spectra of pNPY1-9 and the proline region of pNPY1-25 indicated extended structures with all-trans conformers at Pro5 and Pro8 for pNPY1-9 and at Pro5, Pro8, and Pro13 for pNPY1-25; in each case the Tyr1-Pro2 amide bond was in both cis and trans conformations. However, observed nuclear Overhauser effect correlations and HN exchange experiments indicated an alpha-helical segment in pNPY1-25 initiated by Pro13 and extending from residues 14 to 25. Thus, the N-terminal polyproline region of NPY has no propensity to fold into a regular secondary structure, although Pro13 is a helix initiator, a result consistent with the proposed role of this amino acid in the NPY structural model.
N alpha-Acetyl (Ac), N-terminal deletion fragments of porcine neuropeptide Y (NPY) have been synthesized and characterized for solution conformation properties by circular dichroism and for receptor binding activity at benextramine-sensitive Y1 binding sites in rat brain cortex. Sequential deletion of Tyr1, Pro2, and Ser3 had no effect on the structural (alpha-helical content of 32.5, 30.6, and 30.7%, respectively, at 1 x 10(-5) M) or aggregation (monomer to dimer transition for N alpha-Ac-NPY3-36 and N alpha-Ac-NPY4-36) properties of NPY. In contrast, deletion of Tyr1 decreased receptor binding activity in rat brain cortex by 4-fold (IC50 = 13.0 nM versus 3.75 nM for NPY), but further deletion of Pro2-Ser3 had no additional detrimental effect on receptor binding activity relative to the desTyr1 analog. Thus, Pro2 and Ser3 do not contribute either to the stability of the NPY tertiary structure nor directly to the receptor-ligand interactions. Additional removal of N-terminal amino acids Lys4-Pro5 decreased the helical content and abolished aggregation to a dimeric form of the resultant analog, results suggesting that the residues around Pro5 are important for formation of NPY's compact, pancreatic polypeptide (PP)-fold structure. This loss in structure also correlated with a further 2-3 fold drop in receptor binding activity. These structure-activity correlations provide evidence for the importance of the PP-fold structure in the activity of NPY at Y1 receptors in rat brain cortex.
The 2-(4-nitrophenylsulfonyl)ethoxycarbonyl (Nsc) group is a new base-labile protecting group for solid-phase peptide synthesis, completely interchangeable with the fluorenylmethoxycarbonyl (Fmoc) protecting group, but with certain advantages. In this paper, we report a methodology with Nalpha-Nsc-protected amino acids for the synthesis of some melanotropins important to our research, namely, gamma-melanocyte-stimulating hormone (gamma-MSH), its [Nle3]-analogue, and a cyclic alpha-MSH/beta-MSH hybrid. We developed an efficient protocol for the synthesis of the cyclic MSH analogue that yielded this peptide in >98% purity. The gamma-MSH synthesis, which gave problems with both the Boc and Fmoc strategies, yielded the desired peptide by Nsc-chemistry but was accompanied by side products. Finally, the Nle3-gamma-MSH analogue was synthesized more efficiently using the Fmoc strategy, suggesting that Nsc-chemistry might not be the best methodology for certain sequences.
Synthesis of 2-Azido-1,N6-etheno and 2-Azido Analogues of Deoxyadenosine as Nucleotide Photoaffinity Probes.-The title compounds (VIII) and (XII) are nucleotide photoaffinity labels used extensively to characterize ribonucleotide binding sites in purified enzymes and in vitro systems (for compound (X) no yield given). -(FLAHERTY, D.; BALSE, P.; LI, K.; MOORE, B. M.; DOUGHTY, M. B.; Nucleosides Nucleotides 14 (1995) 1-2, 65-76; Dep. Med. Chem., Univ. Kans., Lawrence, KS 66045, USA; EN)
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