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Melanocortin 4 receptor (MC4-R) antagonists are actively sought for treating cancer cachexia. We determined the structures of complexes with PG-934and SBL-MC-31. These peptides differ from SHU9119by substituting His6with Pro6and inserting Gly10or Arg10. The structures revealed two subpockets at the TM7-TM1-TM2 domains, separated by N2857.36. Two peptide series based on the complexed peptides led to an antagonist activity and selectivity SAR study. Most ligands retained the SHU9119potency, but several SBL-MC-31-derived peptides significantly enhanced MC4-R selectivity over MC1-R by 60- to 132-fold. We also investigated MC4-R coupling to the K+channel, Kir7.1. Some peptides activated the channel, whereas others induced channel closure independently of G protein coupling. In cell culture studies, channel activation correlated with increased feeding, while a peptide with Kir7.1 inhibitory activity reduced eating. These results highlight the potential for targeting the MC4-R:Kir7.1 complex for treating positive and restrictive eating disorders.

The melanocortin receptors (MC1R–MC5R) belong to class A G-protein-coupled receptors (GPCRs) and are known to have receptor-specific roles in normal and diseased states. Selectivity for MC4R is of particular interest due to its involvement in various metabolic disorders, including obesity, feeding regulation, and sexual dysfunctions. To further improve the potency and selectivity of MC4R (ant)agonist peptide ligands, we designed and synthesized a series of cyclic peptides based on the recent crystal structure of MC4R in complex with the well-characterized antagonist SHU-9119(Ac-Nle4-c[Asp5-His6-DNal(2′)7-Arg8-Trp9-Lys10]-NH2). These analogues were pharmacologically characterized in vitro, giving key insights into exploiting binding site subpockets to deliver more selective ligands. More specifically, the side chains of the Nle4, DNal(2′)7, and Trp9residues in SHU-9119, as well as the amide linkage between the Asp5and Lys10side chains, were found to represent structural features engaging a hMC4R/hMC3R selectivity switch.

One of the main challenges in contemporary medicinal chemistry is the development of safer analgesics, used in the treatment of pain. Currently, moderate to severe pain is still treated with the “gold standard” opioids whose long-term often leads to severe side effects. With the discovery of biased agonism, the importance of this area of pharmacology has grown exponentially over the past decade. Of these side effects, tolerance, opioid misuse, physical dependence and substance use disorder (SUD) stand out, since these have led to many deaths over the past decades in both USA and Europe. New therapeutic molecules that induce a biased response at the opioid receptors (MOR, DOR, KOR and NOP receptor) are able to circumvent these side effects and, consequently, serve as more advantageous therapies with great promise. The concept of biased signaling extends far beyond the already sizeable field of GPCR pharmacology and covering everything would be vastly outside the scope of this review which consequently covers the biased ligands acting at the opioid family of receptors. The limitation of quantifying bias, however, makes this a controversial subject, where it is dependent on the reference ligand, the equation or the assay used for the quantification. Hence, the major issue in the field of biased ligands remains the translation of the in vitroprofiles of biased signaling, with corresponding bias factors to in vivoprofiles showing the presence or the lack of specific side effects. This review comprises a comprehensive overview of biased ligands in addition to their bias factors at individual members of the opioid family of receptors, as well as bifunctional ligands.

The neuromedin U peptide sequence is highly conserved between various species. Neuromedin U is involved in a variety of physiological processes. It exerts its effects via two neuromedin U receptors, NMUR1 and NMUR2. These receptors are characterized by a distinct, yet complementary, tissue distribution with NMUR1 mostly found in the periphery, while NMUR2 is most abundant in the central nervous system. The capability of the neuropeptide to reduce food intake in rodents triggered the design and synthesis of a broad range of modified peptide ligands. The purpose of these ligands is to develop novel therapeutics which could be beneficial in the treatment of obesity and diabetes. Most compounds are derived either from the full-length neuromedin U sequence or are based on the truncated orthologs of this neuropeptide. Only a few non-peptidic ligands were developed. This review provides an overview on various neuromedin U analogs and mimetics that have been reported to date.

Fusion of nonopioid pharmacophores, such as neurotensin, with opioid ligands represents an attractive approach for pain treatment. Herein, the μ-/δ-opioid agonist tetrapeptide H-Dmt-d-Arg-Aba-β-Ala-NH2(KGOP01) was fused to NT(8-13)analogues. Since the NTS1 receptor has been linked to adverse effects, selective MOR-NTS2 ligands are preferred. Modifications were introduced within the native NT sequence, particularly a β3-homo amino acid in position 8 and Tyr11substitutions. Combination of β3hArg and Dmt led to peptide 7, a MOR agonist, showing the highest NTS2 affinity described to date (Ki= 3 pM) and good NTS1 affinity (Ki= 4 nM), providing a >1300-fold NTS2 selectivity. The (6-OH)Tic-containing analogue 9also exhibited high NTS2 affinity (Ki= 1.7 nM), with low NTS1 affinity (Ki= 4.7 μM), resulting in an excellent NTS2 selectivity (>2700). In mice, hybrid 7produced significant and prolonged antinociception (up to 8 h), as compared to the KGOP01opioid parent compound.

Recently, we presented a novel compound (PK20, Dmt-D-Lys-Phe-Phe-Lys-Lys-Pro-Phe-Tle-Leu-OH) that targets single entity opioid and neurotensin pharmacophores. This endomorphin-2-like opioid peptide was introduced as a highly active analgesic because it elicited a strong dose- and time-dependent antinociceptive response when administered centrally and peripherally. Its pain-relieving activity was observed as rapidly as 5min after drug injection. Such promising results led us to perform further studies, such as determining the resistance to enzymatic degradation, which resulted in obtaining a very stable opioid pharmacore PK20 metabolite.

Background: Recently, we presented a novel compound (PK20, Dmt-D-Lys-Phe-Phe-Lys-Lys-Pro-Phe-Tle-Leu-OH) that targets single entity opioid and neurotensin pharmacophores. This endomorphin-2-like opioid peptide was introduced as a highly active analgesic because it elicited a strong dose- and time-dependent antinociceptive response when administered centrally and peripherally. Its pain-relieving activity was observed as rapidly as 5 min after drug injection. Such promising results led us to perform further studies, such as determining the resistance to enzymatic degradation, which resulted in obtaining a very stable opioid pharmacore PK20 metabolite. Methods: The synthesis of PK20 and its N-terminal tetrapeptide fragment has been accomplished using solid phase peptide chemistry. The biological stability of peptides has been measured in human serum and analyzed by HPLC/MS. Peptides were pharmacologically characterized in in vitroMOP and DOP receptor binding as well as [35S]GTPyS receptor binding assays. Antinociceptive properties of compounds were measured by in vivoassays in C57Bl6 mice after intravenous or intrathecal applications. Results: Dmt-D-Lys-Phe-Phe-OH (PK20M), an N-terminal tetrapeptide metabolite of the opioid-neurotensin hybrid peptide PK20, is characterized by a long duration of action, as demonstrated by a preserved, long-lasting analgesic effect even 2 h post-injection (average % MPE = 69.33). In rat brain membranes, PK20M efficiently displaced both the MOP and DOP receptor selective radio-probes [3H]DAMGO and [3H]DIDI (pKi of 9.52 and 7.86, respectively) and potently stimulated [35S]GTPyS binding, proving full agonism at both receptor types. In the [35S]GTPyS assay, which measured the agonist-mediated G protein activation, PK20M together with PK20 and Met-enkephalin were potent stimulators of the regulatory G proteins. The relative affinities of PK20M for the µ and d receptor subtypes revealed µ-receptor selectivity. Conclusion: The novel MOP receptor selective metabolite has been shown to possess opioid subtype receptor selectivity, high potency, and effective analgesic activities as measured in various bioassays.

We report on the characteristics of the radical‐ion‐driven dissociation of a diverse array of β‐amino acids incorporated into α‐peptides, as probed by tandem electron‐capture and electron‐transfer dissociation (ECD/ETD) mass spectrometry. The reported results demonstrate a stronger ECD/ETD dependence on the nature of the amino acid side chain for β‐amino acids than for their α‐form counterparts. In particular, only aromatic (e.g., β‐Phe), and to a substantially lower extent, carbonyl‐containing (e.g., β‐Glu and β‐Gln) amino acid side chains, lead to NCβbond cleavage in the corresponding β‐amino acids. We conclude that radical stabilization must be provided by the side chain to enable the radical‐driven fragmentation from the nearby backbone carbonyl carbon to proceed. In contrast with the cleavage of backbones derived from α‐amino acids, ECD of peptides composed mainly of β‐amino acids reveals a shift in cleavage priority from the NCβto the CαC bond. The incorporation of CH2groups into the peptide backbone may thus drastically influence the backbone charge solvation preference. The characteristics of radical‐driven β‐amino acid dissociation described herein are of particular importance to methods development, applications in peptide sequencing, and peptide and protein modification (e.g., deamidation and isomerization) analysis in life science research.

The stereoisomers of N‐phthaloyl‐protected amino acids and dipeptidomimetics were separated on macrocyclic glycopeptide and cellulose‐based chiral stationary phases (CSPs) in the RP and polar‐ionic modes. The effects of the organic modifier, the mobile phase composition, and the pH on the separations were investigated. Optimization of these separations was achieved through variation of the mobile‐phase additive combinations. The elution sequence was determined for some of the samples. A practical application for the monitoring of the reaction conditions for N‐phthaloylation of (S)‐Phe was demonstrated.

The retro-Nα-carboxymethyl histidine moiety, short (NαHis)Ac, functions as an efficient chelator for the 99mTc(CO)3core which allows the labeling of the peptides with a very high specific activity. However as a general consequence of the neutral [99mTc(CO)3] (NαHis)Ac-complex, undesired accumulation in kidney and liver may be high. In order to improve the pharmacokinetic properties of the radiolabeled peptides containing this chelate, attempts have been made to conjugate a carbohydrate using the Maillard reaction. Since the (NαHis)Ac moiety has an unusually reactive Nα, various chemical strategies have been explored to perform the Maillard reaction followed by the Amadori rearrangement on peptides containing this chelator. This indicated that a selective protection of the secondary nitrogen in the chelator is necessary.The retro-Nα-carboxymethyl histidine moiety, short (NαHis)Ac, functions as an efficient chelator for the 99mTc(CO)3core which allows the labeling of the peptides with a very high specific activity. However as a general consequence of the neutral [99mTc(CO)3] (NαHis)Ac-complex, undesired accumulation in kidney and liver may be high. In order to improve the pharmacokinetic properties of the radiolabeled peptides containing this chelate, attempts have been made to conjugate a carbohydrate using the Maillard reaction. Since the (NαHis)Ac moiety has an unusually reactive Nα, various chemical strategies have been explored to perform the Maillard reaction followed by the Amadori rearrangement on peptides containing this chelator. This indicated that a selective protection of the secondary nitrogen in the chelator is necessary.

A series of Gly-neurotensin(8−13) analogues modified at the N-terminus by acyclic tetraamines (Demotensin 1−4) were obtained by solid-phase peptide synthesis techniques. Strategic replacement of amino acids and/or reduction of sensitive peptide bonds were performed to enhance conjugate resistance against proteolytic enzymes. During 99mTc-labeling, single species radiopeptides, 99mTcDemotensin 1−4, were easily obtained in high yields and typical specific activities of 1 Ci/mol. Peptide conjugates displayed a high affinity binding to the human neurotensin subtype 1 receptor (NTS1-R) expressed in colon adenocarcinoma HT-29 or WiDr cells and/or in human tumor sections. 99mTcDemotensin 1−4 internalized very rapidly in HT-29 or WiDr cells by a NTS1-R-mediated process. 99mTcDemotensin 3 and 4, which remained stable during 1 h incubation in murine plasma, were selectively studied in nude mice bearing human HT-29 and WiDr xenografts. After injection, 99mTcDemotensin 3 and 4 effectively and specifically localized in the experimental tumors and were rapidly excreted via the kidneys into the urine, exhibiting overall biodistribution patterns favorable for NTS1-R-targeted tumor imaging in man.

A series of 4-amino-tetrahydro-2-benzazepin-3-one derivatives (Ac–Aba–Xxx–NHMe) were prepared as tetrapeptide mimetics. Considering the structural resemblance with the so-called Freidinger lactams, their propensity to adopt a β-turn conformation was investigated by NMR spectroscopy (solvent and temperature dependence) and molecular modeling. Interestingly, most of these lactams adopt extended conformations, only the spiro-benzazepinone 9 has a strong preference for the formation of a β-turn. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)

The Aba-Gly scaffold, incorporated into Dmt-Tic ligands (H-Dmt-Tic-Gly-NH-CH2-Ph, H-Dmt-Tic-Gly-NH-Ph, H-Dmt-Tic-NH-CH2-Bid), exhibited mixed μ/δ or δ opioid receptor activities with μ agonism. Substitution of Tic by Aba-Gly coupled to −NH−CH2−Ph (1), −NH−Ph (2), or −Bid (Bid = 1H-benzimidazole-2-yl) (3) shifted affinity (Ki(μ) = 0.46, 1.48, and 19.9 nM, respectively), selectivity, and bioactivity to μ-opioid receptors. These compounds represent templates for a new class of lead opioid agonists that are easily synthesized and suitable for therapeutic pain relief.

The side chain orientation of the tyrosine residue included in a peptide, which is an excellent substrate of Syk tyrosine kinase, was fixed in different conformations by either incorporating the tyrosine in cyclic structures (6-OH-Tic, 5-OH-Aic, and Hat derivatives) or adding a sterically bulky substituent in the tyrosine side chain moiety (β-MeTyr). Synthetic peptides containing tyrosine analogues displaying different side chain orientations were analyzed by NMR techniques and tested as potential substrates of the nonreceptor tyrosine kinases Syk, Csk, Lyn, and Fyn. The “rotamer scan” of the phosphorylatable residue generated optimal substrates in terms of both phosphorylation efficiency and selectivity for Syk tyrosine kinase, while the peptidomimetics were not recognized by the other tyrosine kinases. In particular, l-β-MeTyr and d-Hat containing peptides resulted to be both suitable substrates for the specific monitoring of Syk and consensus sequence scaffolds for the design of potential inhibitors highly selective for this tyrosine kinase.

Two new 99mTc-labeled neurotensin(8−13) analogues containing the retro-Nα-carboxymethyl-histidine ((NαHis)Ac) chelator were synthesized as potential radiopharmaceuticals for visualization of pancreatic carcinoma. To improve the pharmacokinetic properties, (NαHis)Ac-Arg-NMeArg-Pro-Tyr-Tle-Leu (NT-XII), which is metabolically stabilized at two positions, was further modified. Shikimic acid (3,4,5-trihydroxy-1-cyclohexene-1-carboxylic acid) was introduced to obtain a more hydrophilic peptide (NT-XVIII), or Tyr11 was replaced by 2,6-dimethyltyrosine (Dmt) resulting in a triple-stabilized NT(8−13) analogue (NT-XIX). The latter has the best biodistribution profile.

The application of a chiral ligand‐exchange column (CLEC) for the direct high‐performance liquid chromatographic enantioseparation of unusual secondary amino acids using D‐penicillamine‐Cu(II) complex as chiral selector is reported. The amino acids investigated were pyrrolidine‐2‐carboxylic acid, piperidine‐2‐carboxylic acid, piperazine‐2‐carboxylic acid, morpholine‐3‐carboxylic acid, and thiomorpholine‐3‐carboxylic acid analogs. Chromatographic results are given as the retention, separation, and resolution factors. The chromatographic conditions were varied to achieve optimal separation. The elution sequence of the enantiomers was determined and in most cases the Sisomer eluted before R. Chirality 18:539–543, 2006. © 2006 Wiley‐Liss, Inc.

This paper focuses on the use of liver-derived in vitrosystems for biotransformation studies during early drug development, as exemplified by the two molecules recently studied in our laboratory: Trichostatin A (TSA) and its structural analogue 5-(4-dimethylaminobenzoyl)aminovaleric acid hydroxamide (4-Me2N-BAVAH). Phase I biotransformation of TSA, a histone deacetylase inhibitor with promising antifibrotic and antitumoural properties, was investigated in liver microsomal (rat and human) and in hepatocyte (rat) suspensions. Within 40 minutes, 50µM of TSA was completely metabolised by 2 × 106hepatocytes/ml. Reduction of the hydroxamic acid function to its corresponding amide and N-demethylation were the two major phase I biotransformation pathways, while hydrolysis products of TSA were minor metabolites. Lower concentrations of TSA (5µM and 25µM) were N-demethylated faster. Liver microsomes, however, metabolised TSA incompletely with the formation of two major metabolites, N-mono- and N-didemethylated TSA. Unlike TSA, 4-Me2N-BAVAH (50µM) could still be detected after 3 hours of incubation with 2 × 106rat hepatocytes/ml suspension. Hydrolysis and reduction of the hydroxamic acid function to its corresponding acid and amide, respectively, were shown to be the major phase I biotransformation pathways. Lower concentrations of 4-Me2N-BAVAH were hydrolysed more readily. 4-Me2N-BAVAH and its metabolites were less subjected to N-demethylation than TSA.

N-Glycyl-(1R,3S)-1-(m-hydroxybenzyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) was prepared as a Tyr-Tic dipeptide mimetic for exploration of its potential as a delta opioid receptor selective ligand. The compound was successfully obtained by a stereoselective synthesis starting from L-Tic. In the course of the reactions, unusual peptide bond cleavages were observed under mild conditions, and reaction mechanisms have been proposed. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)

Use of a quinine‐derived chiral anion‐exchanger stationary phase for the direct high‐performance liquid chromatographic enantiomer separation of N‐protected unusual α‐substituted proline analogues is reported. The chromatographic conditions (eluent composition, pH, and buffer concentration) were varied to achieve optimal separation. The readily prepared 2,4‐dinitrophenyl derivatives were well separable, with good efficiency and high resolution. The elution sequences of the enantiomers of the derivatives were determined. Knowledge of the elution sequence allows identification of the configuration of the α‐substituted proline analogues in peptide epimers resulting from use of the racemates in the peptide synthesis. The method also proved suitable for determination of the enantiomeric purity of proline analogues obtained by asymmetric synthesis.

Use of a quinine-derived chiral anion-exchanger stationary phase for the direct high-performance liquid chromatographic enantiomer separation of N-protected unusual α-substituted proline analogues is reported. The chromatographic conditions (eluent composition, pH, and buffer concentration) were varied to achieve optimal separation. The readily prepared 2,4-dinitrophenyl derivatives were well separable, with good efficiency and high resolution. The elution sequences of the enantiomers of the derivatives were determined. Knowledge of the elution sequence allows identification of the configuration of the α-substituted proline analogues in peptide epimers resulting from use of the racemates in the peptide synthesis. The method also proved suitable for determination of the enantiomeric purity of proline analogues obtained by asymmetric synthesis.

The potential utility of neurotensin (NT) in cancer diagnosis and therapy is limited by its rapid degradation. New stabilized analogues were synthesized, labeled with [99mTc] and screened in vitroand in vivo.High affinity and rapid internalization were obtained in binding assays. Despite their longer human plasma half-lives, a rapid degradation was observed with low concentrations as used in biodistribution tests. The tumor uptake rates were rather low but tumor/blood ratios increased according to the stability raise.

Novel endomorphin-2 analogs containing the unusual amphiphilic amino acid (R)- and (S)-α-hydroxymethyltyrosine in position 1 and (R)- and (S)-α-hydroxymethylphenylalanine in the positions 3 and 4 were synthesized via the solid-phase method. The binding characteristics of the synthetic analogs may suggest that α-hydroxymethyl substitution of amino acid residues influences the conformation of a peptide much more than simply increasing the local amphiphilic character of the peptide.

Novel endomorphin-2 analogs containing the unusual amphiphilic amino acid (R)- and (S)-α-hydroxymethyltyrosine in position 1 and (R)- and (S)-α-hydroxymethylphenylalanine in the positions 3 and 4 were synthesized via the solid-phase method. The binding characteristics of the synthetic analogs may suggest that α-hydroxymethyl substitution of aminoacid residues influences the conformation of a peptide much more than simply increasing the local amphiphilic character of the peptide.

The direct cyclization of 2-amino-4-chloro-3,3-dimethylbutanenitrile with potassium tert-butoxide in THF afforded 1-amino-2,2-dimethylcyclopropane-1-carbonitrile and a dimerization product. Various new cis- and trans-1-(tert-butylamino)-2-benzyl-2-methylcyclopropane-carbonitriles and the corresponding cyclopropanecarboxamides have been synthesized, with focus on the isolation of the pure stereoisomeric cyclopropanecarboxamides. The relative configuration of the stereoisomers was established by X-ray crystallographic analysis of one of the model compounds. A new route to the latter functionalized cyclopropanes was developed by reaction of 1-methoxycyclopropylamines with potassium cyanide. Some remarkable rearrangements of 1-aminocyclopropane-1-carbonitriles into azetidine and oxazine derivatives via Favorskii-derived intermediates are reported. Various aspects of the chemistry of geminally functionalized cyclopropanes are discussed.

Different olefination methods on Boc-phenylalanine methyl ketone for the synthesis of γ-methyl-E-olefin Phe-Gly isosteres are investigated. The Horner phosphonate reagent gave the highest yield of E-isomer. The conformational behaviour of four tetrapeptides containing the E-olefin isostere with and without a vinyl methyl group is studied by 1H NMR. No evidence for turn structures is found.

A new and stereoselective method to synthesize hydroxyethylamine and hydroxymethylamide peptide bond isosteres is developed. The key step is the addition of 2-trimethylsilylthiazole to a-aminoaldehydes, followed by transformation to a-hydroxy-ß-aminoaldehydes. The stereochemistry of the addition can be manipulated by the choice of the nitrogen substitution. The isosteres are easily synthesized via Solid Phase Peptide Synthesis, which rapidly gives the desired pseudopeptides.

The design process and the synthetic problems for the construction of conformationally constrained cyclic somatostatins are described. Highly potent analogs containing o-aminomethylphenylacetic acid or o-aminomethylphenylalanine are obtained.

A new cis-peptide bond mimetic, a-benzyl-o-aminomethylphenylacetic acid, was synthesized and incorporated in a homodetic somatostatin analogue. Biological binding tests and 2D NMR conformational analysis indicate that the configuration of the bridge-unit asymmetric center and the orientation of the benzyl side chain play a key role in the biological activity of this type of somatostatin analogues.

In a previous communication we reported the racemic synthesis of the cis peptide bond mimic α-benzyl-o-aminomethylphenylacetic acid and its incorporation in the cyclic somatostatin analogues c[α(R and S)Bn-o-AMPA-Phe7-d-Trp8-Lys9-Thr10]. Since the epimeric peptides exhibit different binding affinities, we completed the structure–activity study with an asymmetric synthesis. A model for the solution conformation of c[α(R and S)Bn-o-AMPA-Phe7-d-Trp8-Lys9-Thr10] is proposed on the basis of a 2D NMR study in CD3OH and restrained molecular dynamics.

In a previous communication we reported the racemic synthesis of the cis peptide bond mimic α-benzyl-o-aminomethylphenylacetic acid and its incorporation in the cyclic somatostatin analoguesc[α(RandS)Bn-o-AMPA-Phe7-d-Trp8-Lys9-Thr10]. Since the epimeric peptides exhibit different binding affinities, we completed the structure-activity study with an asymmetric synthesis. A model for the solution conformation ofc[α(RandS)Bn-o-AMPA-Phe7-d-Trp8-Lys9-Thr10] is proposed on the basis of a 2D NMR study in CD3OH and restrained molecular dynamics.