Synthesis of dermorphin‐(1–4) derivatives catalyzed by proteases in organic solvents*

Abstract: In order to extend the use of proteases to organic synthesis and seek the rules of enzymatic reactions in organic media, we focused on unnatural substrates for proteases to form amide bonds. In this paper, the study of unnatural substrates containing d ‐amino acid residue, which act as acyl acceptors as well as acyl donors for proteases in organic media, is reported. Dermorphin is a heptapeptide (H‐Tyr‐d ‐Ala‐Phe‐Gly‐Tyr‐Pro‐Ser‐NH₂) with potent analgesic activity. The N‐terminal tetrapeptide is the minimum sequence that retains dermorphin activity, and is selected as the model compound in our study. Two dermorphin‐(1–4) derivatives, Boc‐Tyr‐d ‐Ala‐Phe‐Gly‐N₂H₂Ph and Boc‐Tyr‐d ‐Ala‐Phe‐Gly‐NH₂, which contained a d ‐amino acid residue, were synthesized by proteases in organic media for the first time. The synthesis of these two dermorphin‐(1–4) derivatives could be catalyzed by subtilisin with Boc‐Tyr‐d ‐Ala‐OCH₂CF₃ as an acyl donor substrate in AcOEt. The synthesis of dermorphin‐(1–2) derivative Boc‐Tyr‐d ‐Ala‐N₂H₂Ph was catalyzed by α‐chymotrypsin in different organic solvents and d ‐Ala‐N₂H₂Ph was used as an acyl acceptor substrate. Factors influencing the above enzymatic reactions were systematically studied.     

Potent Opioid Peptide Agonists Containing 4′‐[N‐((4′‐phenyl)‐phenethyl)carboxamido]phenylalanine (Bcp) in Place of Tyr

Analogues of the opioid peptides H‐Tyr‐c[d ‐Cys‐Gly‐Phe(pNO₂)‐d ‐Cys]NH₂ (non‐selective), H‐Tyr‐d ‐Arg‐Phe‐Lys‐NH₂ (μ‐selective) and dynorphin A(1‐11)‐NH₂ (κ‐selective) containing 4′‐[N‐((4′‐phenyl)‐phenethyl)carboxamido]phenylanine (Bcp) in place of Tyr¹ were synthesized. All three Bcp¹‐opioid peptides retained high μ opioid receptor binding affinity, but showed very significant differences in the opioid receptor selectivity profiles as compared with the corresponding Tyr¹‐containing parent peptides. The cyclic peptide H‐Bcp‐c[d ‐Cys‐Gly‐Phe(pNO₂)‐d ‐Cys]NH₂ turned out to be an extraordinarily potent, μ‐selective opioid agonist, whereas the Bcp¹‐analogue of dynorphin A(1‐11)‐NH₂ displayed partial agonism at the μ receptor. The obtained results suggest that the large biphenylethyl substituent contained in these compounds may engage in a hydrophobic interaction with a receptor subsite and thereby may play a role in the ligand’s ability to induce a specific receptor conformation or to bind to a distinct receptor conformation in a situation of conformational receptor heterogeneity.     

Cyclic enkephalin analogs containing various para‐substituted phenylalanine derivatives in place of Tyr1 are potent opioid agonists*

Abstract: The cyclic enkephalin analog H‐Tyr‐c[d ‐Cys‐Gly‐Phe(pNO₂)‐d ‐Cys]NH₂ is a highly potent opioid agonist with IC₅₀s of 35 pm and 19 pm in the guinea‐pig ileum (GPI) and mouse vas deferens (MVD) assays, respectively. The Phe¹‐analog of this peptide showed 370‐fold and 6790‐fold lower agonist potency in the GPI and MVD assays, respectively, indicating the importance of the Tyr¹ hydroxyl‐group in the interaction with μ and δ opioid receptors. In the present study, the effect of various substituents (‐NH₂, ‐NO₂, ‐CN, ‐CH₃, ‐COOH, ‐COCH₃, ‐CONH₂) introduced in the para‐position of the Phe¹‐residue of H‐Phe‐c[d ‐Cys‐Gly‐Phe(pNO₂)‐d ‐Cys]NH₂ on the in vitro opioid activity profile was examined. Most analogs showed enhanced μ and δ agonist potencies in the two bioassays, except for the Phe(pCOOH)¹‐analog, which was weakly active, probably as a consequence of the negative charge. The most potent compounds were the Phe(pCOH₃)¹‐ and the Phe(pCONH₂)¹‐analogs. The latter compound showed subnanomolar μ and δ agonist potencies and represents the most potent enkephalin analog lacking the Tyr¹ hydroxyl‐group reported to date. Taken together, these results indicate that various substituents introduced in the para‐position of Phe¹ enhance opioid activity via hydrogen bonding or hydrophobic interactions with the receptor. Comparison with existing structure‐activity relationship on phenolic hydroxyl replacements in morphinans indicates that these nonpeptide opiates and some of the cyclic enkephalin analogs described here may have different modes of binding to the receptor.     

Synthesis and activity of the melanocortin Xaa‐d‐Phe‐Arg‐Trp‐NH2 tetrapeptides with amide bond modifications

Abstract: The melanocortin receptor (MCR) pathway has been identified as participating in several physiologically important pathways including pigmentation, energy homeostasis, inflammation, obesity, hypertension, and sexual function. All the endogenous MCR agonists contain a core His‐Phe‐Arg‐Trp sequence identified as important for receptor molecular recognition and stimulation. Several structure–activity studies using the Ac‐His‐d ‐Phe‐Arg‐Trp‐NH₂ tetrapeptide template have been performed in the context of modifying N‐terminal ‘capping’ groups and amino acid constituents. Herein, we report the synthesis and pharmacologic characterization of modified Xaa‐d ‐Phe‐Arg‐Trp‐NH₂ (Xaa = His or Phe) melanocortin tetrapeptides (N‐site selective methylation, permethylation, or amide bond reduction) at the mouse MC1, MC3, MC4 and MC5 receptors. The modified peptides generated in this study resulted in equipotent or reduced MCR potency when compared with control ligands. The reduced amide bond analog of the Phe‐d ‐Phe‐Arg‐Trp‐NH₂ peptide converted its agonist activity into an antagonistic at the central mMC3 and mMC4 receptors involved in the regulation of energy homeostasis, while retaining full agonist activity at the peripheral MC1 and MC5 receptors.     

Synthesis of Met‐enkephalin by solution‐phase peptide synthesis methodology utilizing para‐toluene sulfonic acid as N‐terminal masking of l‐methionine amino acid

The Met‐enkephalin, Tyr‐Gly‐Gly‐Phe‐Met, was synthesized by the solution‐phase synthesis (SPS) methodology employing ‐OBzl group as carboxyls' protection, while the t‐Boc groups were employed for the N‐terminal α‐amines' protection for the majority of the amino acids of the pentapeptide sequence. The l ‐methionine (l ‐Met) amino acid was used as PTSA.Met‐OBzl obtained from the simultaneous protection of the α‐amino, and carboxyl group with para‐toluene sulfonic acid (PTSA) and as‐OBzl ester, respectively in a C‐terminal start of the 2 + 2 + 1 fragments condensation convergent synthetic approach. The protection strategy provided a short, single‐step, simultaneous, orthogonal, nearly quantitative, robust, and stable process to carry through the protected l ‐methionine and l ‐phenylalanine coupling without any structural deformities during coupling and workups. The structurally confirmed final pentapeptide product was feasibly obtained in good yields through the current approach.     

An affinity label for δ‐opioid receptors derived from [d‐Ala2]deltorphin I*

Abstract: A series of potential affinity label derivatives of the amphibian opioid peptide [d ‐Ala²]deltorphin I were prepared by incorporation at the para position of Phe³ (in the ‘message’ sequence) or Phe⁵ (in the ‘address’ sequence) of an electrophilic group (i.e. isothiocyanate or bromoacetamide). The introduction of the electrophile was accomplished by incorporating Fmoc‐Phe(p‐NHAlloc) into the peptide, followed later in the synthesis by selective deprotection of the Alloc group and modification of the resulting amine. While para substitution decreased the δ‐opioid receptor affinity, selected analogs retained nanomolar affinity for δ receptors. [d ‐Ala²,Phe(p‐NCS)³]deltorphin I exhibited moderate affinity (IC₅₀ = 83 nm ) and high selectivity for δ receptors, while the corresponding amine and bromoacetamide derivatives showed pronounced decreases in δ‐receptor affinity (80‐ and >1200‐fold, respectively, compared with [d ‐Ala²]deltorphin I). In the ‘address’ sequence, the Phe(p‐NH₂)⁵ derivative showed the highest δ‐receptor affinity (IC₅₀ = 32 nm ), while the Phe(p‐NHCOCH₂Br)⁵ and Phe(p‐NCS)⁵ peptides displayed four‐ and tenfold lower δ‐receptor affinities, respectively. [d ‐Ala²,Phe(p‐NCS)³]deltorphin I exhibited wash‐resistant inhibition of [³H][d ‐Pen²,D‐Pen⁵]enkephalin (DPDPE) binding to δ receptors at a concentration of 80 nm . [d ‐Ala², Phe(p‐NCS)³]deltorphin I represents the first affinity label derivative of one of the potent and selective amphibian opioid peptides, and the first electrophilic affinity label derivative of an agonist containing the reactive functionality in the ‘message’ sequence of the peptide.     

Novel endomorphin‐2 analogs with μ‐opioid receptor antagonist activity

Abstract: A series of position 4‐substituted endomorphin‐2 (Tyr‐Pro‐Phe‐Phe‐NH₂) analogs containing 3‐(1‐naphthyl)‐alanine (1‐Nal) or 3‐(2‐naphthyl)‐alanine (2‐Nal) in l ‐ or d ‐configuration, was synthesized. The opioid activity profiles of these peptides were determined in the μ‐opioid receptor representative binding assay and in the Guinea‐Pig Ileum assay/Mouse Vas Deferens assay (GPI/MVD) bioassays in vitro, as well as in the mouse hot‐plate test of analgesia in vivo. In the binding assay the affinity of all new analogs for the μ‐opioid receptor was reduced compared with endomorphin‐2. The two most potent analogs were [d ‐1‐Nal⁴]‐ and [d ‐2‐Nal⁴]endomorphin‐2, with IC₅₀ values 14 ± 1.25 and 19 ± 2.1 nm , respectively, compared with 1.9 ± 0.21 nm for endomorphin‐2. In the GPI assay these analogs were found to be weak antagonists and they were inactive in the MVD assay. The in vitro GPI assay results were in agreement with those obtained in the in vivo hot‐plate test. Antinociception induced by endomorphin‐2 was reversed by concomitant intracerebroventricula (i.c.v.) administration of [d ‐1‐Nal⁴]‐ and [d ‐2‐Nal⁴]‐endomorphin‐2, indicating that these analogs were μ‐opioid antagonists. Their antagonist activity was compared with that of naloxone. At a dose 5 μg per animal naloxone almost completely inhibited antinociceptive action of endomorphin‐2, while [d ‐1‐Nal⁴]endomorphin‐2 in about 46%.     

Potent bradykinin antagonists containing N‐benzylglycine or N‐benzyl‐l‐alanine in position 8

Abstract: Two new analogues of a previously designed bradykinin (BK) antagonist, d ‐Arg‐Arg‐Pro‐Hyp‐Gly‐Thi‐Ser‐d ‐Phe‐Thi‐Arg, substituted in position 8 by N‐benzylglycine and N‐benzyl‐l ‐alanine were designed, synthesized and bioassayed. The results show an impressive enhancement of B₂ antagonistic potencies of both peptides in comparison with the model. In two further analogues these modifications were combined with acylation of the N‐terminus with 1‐adamantanacarboxylic acid. Acylated analogues exhibited higher antagonistic potency in comparison with the parent compounds, however, the range of effect was not as high as in previously described cases. The activity of analogues was assessed by their ability to inhibit vasodepressor response to exogenous BK (rat blood pressure test). Our results may be of value in the design of more potent BK antagonists.     

The chemical syntheses and bioactivities of novel peptide‐based endothelin antagonists

Abstract: Endothelin antagonists, novel tripeptides containing a series of unnatural amino acids, were synthesized and characterized. A linear peptide BQ‐485, perhydroazepin‐1‐yl‐l ‐leucyl (1)‐d ‐tryptophanyl (2)‐d ‐tryptophan (3), was selected as the parent compound. The introduction of d ‐Phe derivatives into these peptidic ET antagonists resulted in potent activity against the contraction of rat aortic smooth muscles induced by ET‐1 (10 nm ) which activated the ET receptors. Among these compounds, 15 tripeptides had high enough antagonistic activity at the level of 10^?7 mol/L (IC₅₀). The activity of three compounds was 10^?6 mol/L (IC₅₀). These HIM‐CO‐Leu‐d ‐Trp‐d ‐Phe(‐R)‐OH compounds as ET_A antagonists may provide a tool for the development of therapeutic agents in the treatment of putative ET‐1‐related disorders.     

Synthesis and biological evaluation of constrained analogues of the opioid peptide H‐Tyr‐d‐Ala‐Phe‐Gly‐NH2 using the 4‐amino‐2‐benzazepin‐3‐one scaffold

Abstract: The synthesis of conformationally restricted dipeptidic moieties 4‐amino‐1,2,4,5‐tetrahydro‐2‐benzazepin‐3‐one (Aba)‐Gly ([(4S)‐amino‐3‐oxo‐1,2,4,5‐tetrahydro‐1H‐2‐benzazepin‐2‐yl]‐acetic acid) and 8‐hydroxy‐4‐amino‐1,2,4,5‐tetrahydro‐2‐benzazepin‐3‐one (Hba)‐d ‐Ala ([(4S)‐amino‐8‐hydroxy‐3‐oxo‐1,2,4,5‐tetrahydro‐benzo[c]azepin‐2‐yl]‐propionic acid) was based on a synthetic strategy that uses an oxazolidinone as an N‐acyliminium precursor. Introducing these Aba scaffolds into the N‐terminal tetrapeptide of dermorphin (H‐Tyr‐d ‐Ala‐Phe‐Gly‐Tyr‐Pro‐Ser‐NH₂)‐induced remarkable shifts in affinity and selectivity towards the opioid μ‐ and δ‐receptors. This paper provides the synthesis and biological in vitro and in vivo evaluation of constricted analogues of the N‐terminal tetrapeptide H‐Tyr‐d ‐Ala‐Phe‐Gly‐NH₂, which is the minimal subunit of dermorphin needed for dermorphin‐like opiate activity.     

Cyclic Opioid Peptide Agonists and Antagonists Obtained Via Ring‐Closing Metathesis

The opioid peptide H‐Tyr‐c[D‐Cys‐Phe‐Phe‐Cys]NH₂ cyclized via a methylene dithiother is a potent and selective μ opioid agonist (Przydial M.J. et al., J Peptide Res, 66, 2005, 255). Dicarba analogues of this peptide with Tyr, 2′,6′‐dimethyltyrosine (Dmt), 3‐[2,6‐dimethyl‐4‐hydroxyphenyl)propanoic acid (Dhp) or (2S)‐2‐methyl‐3‐(2,6‐dimethyl‐4‐hydroxyphenyl)propanoic acid [(2S)‐Mdp] in the 1‐position were prepared. The peptides were synthesized on solid‐phase by substituting d ‐allylglycine and (2S)‐2‐amino‐5‐hexenoic acid in position 2 and 5, respectively, followed by ring‐closing metathesis. Mixtures of cis and trans isomers of the resulting olefinic peptides were obtained, and catalytic hydrogenation yielded the saturated –CH₂–CH₂– bridged peptides. All six Tyr¹‐ and Dmt¹‐dicarba analogues retained high μ and δ opioid agonist potency and showed only slight or no preference for μ over δ receptors. As expected, the six Dhp¹‐ and (2S)‐Mdp¹‐dicarba analogues turned out to be μ opioid antagonists but, surprisingly, displayed a range of different efficacies (agonism, partial agonism or antagonism) at the δ receptor. The obtained results indicate that the μ versus δ receptor selectivity and the efficacy at the δ receptor of these cyclic peptides depend on distinct conformational characteristics of the 15‐membered peptide ring structure, which may affect the spatial positioning of the exocyclic residue and of the Phe³ and Phe⁴ side chains.     

Synthesis and evaluation of potential affinity labels derived from endomorphin‐2

Abstract: In an attempt to identify potential peptide‐based affinity labels for opioid receptors, endomorphin‐2 (Tyr‐Pro‐Phe‐PheNH₂), a potent and selective endogenous ligand for µ‐opioid receptors, was chosen as the parent peptide for modification. The tetrapeptide analogs were prepared using standard Fmoc‐solid phase peptide synthesis in conjunction with incorporation of Fmoc‐Phe(p‐NHAlloc) and modification of the p‐amino group. The electrophilic groups isothiocyanate and bromoacetamide were introduced into the para position on either Phe³ or Phe⁴; the corresponding free amine‐containing peptides were also prepared for comparison. The peptides bearing an affinity label group and their free amine analogs were evaluated in a radioligand‐binding assay using Chinese hamster ovary (CHO) cells expressing µ‐ and δ‐opioid receptors. Modification on Phe⁴ was better tolerated than on Phe³ for µ‐receptor binding. Among the analogs tested, [Phe(p‐NH₂)⁴]endomorphin‐2 showed the highest affinity (IC₅₀ = 37 nm ) for µ‐receptors. The Phe(p‐NHCOCH₂Br)⁴ analog displayed the highest µ‐receptor affinity (IC₅₀ = 158 nm ) among the peptides containing an affinity label group. Most of the compounds exhibited negligible binding affinity for δ‐receptors, similar to the parent peptide.     

Dermorphin‐based potential affinity labels for µ‐opioid receptors*

Abstract: Dermorphin and [Lys⁷]dermorphin, selective µ‐opioid receptor ligands originating from amphibian skin, have been modified with various electrophiles in either the ‘message’ or ‘address’ sequences as potential peptide‐based affinity labels for µ‐receptors. Introduction of the electrophilic isothiocyanate and bromoacetamide groups on the para position of Phe³ and Phe⁵ was accomplished by incorporating Fmoc‐Phe(p‐NHAlloc) into the peptide followed by selective deprotection and modification. The corresponding amine‐containing peptides were also prepared. The pure peptides were evaluated in radioligand binding experiments using Chinese hamster ovary (CHO) cells expressing µ‐ and δ‐opioid receptors. In dermorphin, introduction of the electrophilic groups in the ‘message’ domain lowered the binding affinity by > 1000‐fold; only [Phe(p‐NH₂)³]dermorphin retained nanomolar affinity for µ‐receptors. Modifications in the ‘address’ region of both dermorphin and [Lys⁷]dermorphin were relatively well tolerated. In particular, [Phe(p‐NH₂)⁵,Lys⁷]dermorphin showed similar affinity to dermorphin, with almost 2‐fold higher selectivity for µ‐receptors. [Phe(p‐NHCOCH₂Br)⁵]‐ and [Phe(p‐NHCOCH₂Br)⁵,Lys⁷]dermorphin exhibited relatively high affinity (IC₅₀ = 27.7 and 15.1 nm , respectively) for µ‐receptors. However, neither of these peptides inhibited [³H]DAMGO binding in a wash‐resistant manner.     

Synthesis and evaluation of a new series of peptide‐based endothelin receptor antagonists

Abstract: Novel peptide‐based endothelin (ET) receptor antagonists were designed and synthesized in our laboratory. BQ‐485, HIM‐CO‐Leu‐d ‐Trp‐d ‐Trp‐OH, was selected as the leading compound. The primary structures of these new tripeptides were ABO‐CO‐Leu‐d ‐Trp‐d ‐AA(X)‐OH. The introduction of unnatural aromatic amino acids into these tripeptides was useful in the structure–activity relationship studies. Among the 20 tripeptides, 16 of them showed high activities against the contraction of rat aortic smooth muscles induced by ET‐1.     

Pseudopeptides containing the 2‐hydrazonoacyl fragment: analogs of the chemotactic agent HCO‐Met‐Leu‐Phe‐OMe

Abstract: In order to further examine the properties of pseudopeptides containing the 2‐hydrazonoacyl fragment, two new series of analogs of the prototypical chemotactic N‐formyl‐tripeptide HCO‐Met‐Leu‐Phe‐OMe were designed and synthesized. The first group contains the new fragment as the N‐terminal residue and is represented by the N‐aryl derivatives p‐Cl‐C₆H₄‐NH‐N=C(R)‐CO‐Leu‐Phe‐OMe ( 2 and 3 ) and by the corresponding N‐aroyl analogs p‐CH₃‐C₆H₄‐CO‐NH‐N=C(R)‐CO‐Leu‐Phe‐OMe ( 4 ). The second group contains the new fragment in place of the central Leu residue and is represented by compounds HCO‐Xaa‐NH‐N=C(R)‐CO‐Phe‐OMe ( 7a and 7b ) where Xaa is Nle and Met, respectively. The conformational and biochemical properties of the new products were examined.     

Design of high affinity cyclic pentapeptide ligands for κ‐opioid receptors

Abstract: Using results from our previously reported cyclic opioid peptide series and reliable models for μ‐, δ‐, and κ‐opioid receptors (MOR, DOR, and KOR, respectively) and their complexes with peptide ligands, we have designed and synthesized a series of cyclic pentapeptides of structure Tyr‐c[d ‐Cys‐Phe‐Phe‐X]‐NH₂, cyclized via disulfide, methylene, or ethylene dithioethers, and where X = d ‐ or l ‐Cys; or d ‐ or l ‐penicillamine (Pen; β,β‐dimethylcysteine). Determination of binding affinities to MOR, DOR, and KOR revealed that members of this series with X = d ‐ or l ‐Cys display KOR affinities in the low nanomolar range, demonstrating that a ‘DPDPE‐like’ tetrapeptide scaffold is suitable not only for DOR and MOR ligands, but also for KOR ligands. The cyclic pentapeptides reported here are not, however, selective for KOR, rather they display significant selectivity and high affinity for MOR. Indeed, peptide 8 , Tyr‐c[d ‐Cys‐Phe‐Phe‐Cys]‐NH₂‐cyclized via a methylene dithioether, shows picomolar binding affinity for MOR ( = 16 pm ) with more than 100‐fold selectivity for MOR vs. DOR or KOR, and may be of interest as a high affinity, high selectivity MOR ligand. Nonetheless, the high affinity KOR peptides in this series represent excellent leads for the development of structurally related, selective KOR ligands designed to exploit structurally specific features of KOR, MOR, and DOR.     

Novel Side‐Chain Glucosylated and Adamantylated [Asp2/Glu2]Enkephalin Analogs: Synthesis and In Vitro Growth Inhibition of Human Tumor Cells

A series of new backbone‐modified Leu‐ and Met‐enkephalin analogs ( 13 – 20 a and b ) were synthesized. Backbone manipulations involved the replacement of the Gly² residue in Tyr‐Gly‐Gly‐Phe‐Leu/Met with side‐chain glucosylated or adamantylated D /L ‐aspartic or ‐glutamic acids. The in vitro antiproliferative activity of these compounds was evaluated for several cell lines and the results were compared with the effect of Met‐enkephalin, the native opioid growth factor. The tested compounds modestly inhibited the growth of the tumor cells (20–50% inhibition at millimolar concentrations). Among the tested compounds, Tyr‐D ‐Glu(AdNH)‐Gly‐Phe‐Met ( 20b ) showed significant antiproliferative activity, somewhat more pronounced on MCF‐7 (breast carcinoma) and MOLT‐4 (lymphoblastic leukemia) cells.     

Carbopeptides: carbohydrates as potential templates for de novo design of protein models

Abstract: De novo design of proteins has evolved into a powerful approach for studying the factors governing protein folding and stability. Among the families of structures frequently studied is the ‘four‐helix bundle’ in which four α‐helical peptide strands, linked by loops, form a hydrophobic core. Assembly of protein models on a template has been suggested as a way to reduce the entropy of folding. Here we describe the potential use of a carbohydrate as such a template. The monosaccharide d ‐galactose was per‐O‐acylated with (N^β‐Fmoc‐βAla)₂O to give a penta‐substituted derivative, which was converted to the corresponding glycosyl bromide and used for the glycosylation of 4‐hydroxymethylbenzoic acid pentafluorophenyl ester (HMBA‐OPfp). The β‐glycosidic carbohydrate template (N^β‐Fmoc‐βAla)₄‐β‐d ‐Galp‐(1‐O)‐MBA‐OPfp thus obtained was coupled to a PAL‐PEG‐PS resin and simultaneously extended at the four arms to yield, after cleavage from the solid support, a carbopeptide with four identical peptide strands. Extension of this concept to, for example, synthesis of novel multiple antigenic peptides (MAPs) and synthesis of carbohydrate clusters can be easily envisioned. The ability to efficiently synthesize such structures sets the stage for further studies to test whether the carbohydrate templates do indeed nucleate folding.     

Synthesis and biological evaluation on hMC3, hMC4 and hMC5 receptors of γ‐MSH analogs substituted with l‐alanine

Abstract: To elucidate the molecular basis of the interaction of the native dodecapeptide γ‐MSH with the melanocortin receptors, we performed a structure?activity study in which we systematically replaced l ‐Ala in each position of this peptide. Here we report the binding affinity and agonist potency on human MC3R, MC4R and MC5R. Intracellular cAMP concentration was measured on CHO cells, and binding assays were carried out using membranes prepared from these cell lines which stably express hMC3R, hMC4R and hMC5R. Our results indicate that the last four amino acids in the C‐terminal region of γ‐MSH are not important determinants of biological activity and selectivity at human melanocortin receptors. Interesting results were obtained when l ‐Ala was substituted for His⁶, Phe⁷, Arg⁸ and Trp⁹. For these peptides, the affinity and activity at all three human receptors (MC3R, MC4R and MC5R) decreased significantly, demonstrating that the His‐Phe‐Arg‐Trp sequence in γ‐MSH is important for activity at these three melanocortin receptors. Similar results were obtained when Met³ was replaced with l ‐Ala, suggesting the importance of this position in the interaction with all three receptors. This study highlights the role played by the His‐Phe‐Arg‐Trp sequence in receptor binding and in agonist activity of γ‐MSH.     

Synthesis and biologic activity of conformationally constrained analogs of L‐363,301

Abstract: We report the synthesis, biological activity and conformational analysis of analogs of the cyclic hexapeptide L‐363,301, c[Pro⁶‐Phe⁷‐d ‐Trp⁸‐Lys⁹‐Thr¹⁰‐Phe¹¹] (numbering as in the native hormone somatostatin‐14). The d ‐Trp in position 8 was replaced with (2R,3S)‐ and (2R,3R)‐β‐MeTrp respectively, with an added methyl group in the beta position of Trp. The objective of our study was to determine the potency and selectivity generated by the added constraint in the beta position of the d ‐Trp upon binding to human somatostatin receptors hsst1‐5. We synthesized the building blocks enantioselectively and incorporated them into the peptides by SPPS. Competition binding assays revealed that both compounds 2 and 3 were selective for hsst2 over hsst5. The (2R,3S) analog 2 was approximately 30 times more potent at hsst2 than the (2R,3R) analog 3 . Interestingly, the (2R,3R) compound showed no binding affinity at hsst5.