Tryptophan Analogues with Fixed Side-Chain Orientation: Expanding the Scope

A generalized synthetic strategy is proposed here for the synthesis of asymmetric β-indoylated amino acids by 8-aminoquinoline (8AQ)-directed C(sp³)-H functionalization of suitably protected precursors. Peptides containing one of the four stereoisomers of (indol-3-yl)-3-phenylalanine at position 2 of the parent peptide KwFwLL-NH₂ (w=d -Trp) cover a wide range of activities as ghrelin receptor inverse agonists, among them the most active described until now. This application exemplarily shows how β-indoylated amino acids can be used for the systematic variation of the position of an indole group in a bioactive peptide.     

Structure-activity relations of β-adrenergic agents

A review of the structure-activity relations in aryloxypropanolamine β-adrenoceptor drugs is presented. The effect of substitution in various parts of the molecule on β₁/β₂ selectivity and degree of partial agonism is discussed. Cardiac (β₁) selectivity is achieved by hydrophilic compounds with either p-amide substitution in the aryl ring, or a hydrogen bonding group in the amine side chain. Lipophilic compounds with branched chains tend to be β₂ selective. Partial agonist activity may also be controlled by hydrogen bonding groups in the side chain and by aromatic substitution. Here 3,4-dihydroxyl groups give full agonists, mono-and des-hydroxy (OH) groups partial agonists. The size of any o-substituent is critical and a plot of Taft's steric factor versus partial agonism gives a linear relation. By manipulation of the above factors all combinations of selectivity and agonist activity may be achieved.     

Synthesis and Biological Evaluation of Hapten-Clicked Analogues of The Antigenic Peptide Melan-A/MART-126(27L)-35

A click-chemistry-based approach was implemented to prepare peptidomimetics designed in silico and made from aromatic azides and a propargylated GIGI-mimicking platform derived from the altered Melan-A/MART-1_26(27L)-35 antigenic peptide ELAGIGILTV. The Cu^I-catalyzed Huisgen cycloaddition was carried out on solid support to generate rapidly a first series of peptidomimetics, which were evaluated for their capacity to dock at the interface between the major histocompatibility complex class-I (MHC-I) human leucocyte antigen (HLA)-A2 and T-cell receptors (TCRs). Despite being a weak HLA-A2 ligand, one of these 11 first synthetic compounds bearing a p-nitrobenzyl-triazole side chain was recognized by the receptor proteins of Melan-A/MART-1-specific T-cells. After modification of the N and C termini of this agonist, which was intended to enhance HLA-A2 binding, one of the resulting seven additional compounds triggered significant T-cell responses. Thus, these results highlight the capacity of naturally circulating human TCRs that are specific for the native Melan-A/MART-1_26-35 peptide to cross-react with peptidomimetics bearing organic motifs structurally different from the native central amino acids.     

Structure–Activity Relationships of 1-Benzoylazulenes at the OX1 and OX2 Orexin Receptors

We previously demonstrated the potential of di- or trisubstituted azulenes as ligands (potentiators, weak agonists, and antagonists) of the orexin receptors. In this study we investigated 27 1-benzoylazulene derivatives, uncovering seven potentiators of the orexin response on OX₁ and two weak dual orexin receptor agonists. For potentiators, replacement of the azulene scaffold by indole retained the activity of four out of six compounds. The structure–activity relationships for agonism and potentiation can be summarized into a bicyclic aromatic ring system substituted with two hydrogen-bond acceptors (1-position, benzoyl; 6-position, carboxyl/ester) within 7–8 Å of each other; a third acceptor at the 3-position is also well tolerated. The same pharmacophoric signature is found in the preferred conformations of the orexin receptor agonist Nag26 from molecular dynamics simulations. Subtle changes switch the activity between weak agonism and potentiation, suggesting overlapping binding sites.     

6,5-Fused Ring,C2-Salvinorin Ester,Dual Kappa and Mu Opioid Receptor Agonists as Analgesics Devoid of Anxiogenic Effects**

Current common analgesics are mediated through the mu or kappa opioid receptor agonism. Unfortunately, selective mu or kappa receptor agonists often cause harmful side effects. However, ligands exhibiting dual agonism to the opioid receptors, such as to mu and kappa, or to mu and delta, have been suggested to temper undesirable adverse effects while retaining analgesic activity. Herein we report an introduction of various 6,5-fused rings to C2 of the salvinorin scaffold via an ester linker. In vitro studies showed that many of these compounds have dual agonism on kappa and mu opioid receptors. In vivo studies on the lead dual kappa and mu opioid receptor agonist demonstrated supraspinal thermal analgesic activity while avoiding anxiogenic effects in male mice, thus providing further strong evidence in support of the therapeutic advantages of dual opioid receptor agonists over selective opioid receptor agonists.     

Linoleic Acid Oxidation Catalysed by Various Amino Acids and Cupric Ions in Aqueous Media

Model systems were designed to study the linoleic acid oxidation in the presence and absence of various amino acids and with or without cupric ions. The tested amino acids have shown to possess potential pro-oxidant capacity in linoleic acid dispersed in aqueous media. The effectiveness of various amino acids on linoleic acid oxidation decreased in the following order: cysteine > serine > tryptophan > phenylalanine > histidine > alanine. The addition of alanine, serine, phenylalanine, histidine or tryptophan to linoleic acid have shown features of an autocatalytic oxidation chain reaction. With cysteine, there was a linear relation between concentration of hydroperoxides and time during the early stages of oxidation. The pro-oxidative activity of the tested amino acids in general could be attributed to the presence of the α-amino group in the form H₃-N-R. The apparent difference in the pro-oxidative activity is mainly due to the functional groups attached to β-carbon in the amino acid molecules. The addition of cupric ions at concentration of 10^–5 M to linoleic acid catalysed with various α-amino acids have shown that these amino acids had no significant effect. The increasing copper concentration from 10^–5 to 10^–1 M shortened the induction period of linoleic acid catalysed by amino acids having aromatic side chain, had no effect on the induction period but increased the oxidation rate during the propagation step in the model systems catalysed by alanine and serine and in the model system containing cysteine increased the linoleic acid oxidation linearly from the start of the reaction.     

Linoleic acid oxidation catalyzed by various amino acids and cupric ions in aqueous media

Model systems were designed to study linoleic acid oxidation in the presence and absence of various amino acids with or without cupric ions. The tested amino acids exhibited a potential prooxidant effect in linoleic acid dispersed in aqueous media. The effectiveness of various amino acids on linoleic acid oxidation decreased in the following order: cysteine > serine > tryptophan > phenylalanine > histidine > alanine. The addition of alanine, serine, phenylalanine, histidine, or tryptophan to linoleic acid showed an autocatalytic chain reaction. With cysteine, there was a linear relation between concentration of hydroperoxides and time during the early stages of oxidation. The prooxidative activity of the tested amino acids in general could be attributed to the presence of the a-amino group in the form H₃-N-R. The apparent difference in the prooxidative activity is mainly due to the functional groups attached in the β-carbon atom in the amino acid molecules. The addition of cupric ions at a concentration of 10^-5M to linoleic acid catalyzed with various a-amino acids showed that these amino acids had no significant effect. Increasing the copper concentration from 10^-5M to 10^-3M had the following effects: a shortening of the induction period of linoleic acid catalyzed by amino acids having an aromatic side chain, no effect on the induction period but an increase in the oxidation rate during the propagation step in the model systems catalyzed by alanine and serine, and in the model system containing cysteine a linear increase in the linoleic acid oxidation from the start of the reaction.     

Development of Potent and Metabolically Stable APJ Ligands with High Therapeutic Potential

The apelin ligand receptor system is an important target to develop treatment strategies for cardiovascular diseases. Although apelin exhibits strong inotropic effects, its pharmaceutical application is limited because no agonist with suitable properties is available. On the one hand, peptide ligands are too instable, and on the other hand, small‐molecule agonists show only low potency. This study describes the development of apelin (APJ) receptor agonists with not only high activity but also metabolic stability. Several strategies including capping of termini, insertion of unnatural amino acids, cyclization, and lipidation were analyzed. Peptide activity was tested using a Ca²⁺‐mobilization assay and the degradation of selected analogues was analyzed in rat plasma. The best results were obtained by N‐terminal lipidation of a 13‐mer apelin derivative. This analogue displayed a half‐life of 29 h in rat plasma, compared with 0.025 h for the wild‐type peptide. Furthermore, in vivo pharmacokinetics revealed a clearance of 0.049 L h^?1 kg^?1 and a half‐life of 0.36 h. In summary, amino acid substitution and fatty acid modification resulted in a potent and 1000‐fold more stable peptide that exhibits high pharmaceutical potential.     

Antagonizing S1P3 Receptor with Cell-Penetrating Pepducins in Skeletal Muscle Fibrosis

S1P is the final product of sphingolipid metabolism, which interacts with five widely expressed GPCRs (S1P_1-5). Increasing numbers of studies have indicated the importance of S1P₃ in various pathophysiological processes. Recently, we have identified a pepducin (compound KRX-725-II) acting as an S1P₃ receptor antagonist. Here, aiming to optimize the activity and selectivity profile of the described compound, we have synthesized a series of derivatives in which Tyr, in position 4, has been substituted with several natural aromatic and unnatural aromatic and non-aromatic amino acids. All the compounds were evaluated for their ability to inhibit vascular relaxation induced by KRX-725 (as S1P₃ selective pepducin agonist) and KRX-722 (an S1P₁-selective pepducin agonist). Those selective towards S1P₃ (compounds V and VII) were also evaluated for their ability to inhibit skeletal muscle fibrosis. Finally, molecular dynamics simulations were performed to derive information on the preferred conformations of selective and unselective antagonists.     

BRET-Based Biosensors to Measure Agonist Efficacies in Histamine H1 Receptor-Mediated G Protein Activation,Signaling and Interactions with GRKs and β-Arrestins

The histamine H₁ receptor (H₁R) is a G protein-coupled receptor (GPCR) and plays a key role in allergic reactions upon activation by histamine which is locally released from mast cells and basophils. Consequently, H₁R is a well-established therapeutic target for antihistamines that relieve allergy symptoms. H₁R signals via heterotrimeric G_q proteins and is phosphorylated by GPCR kinase (GRK) subtypes 2, 5, and 6, consequently facilitating the subsequent recruitment of β-arrestin1 and/or 2. Stimulation of a GPCR with structurally different agonists can result in preferential engagement of one or more of these intracellular signaling molecules. To evaluate this so-called biased agonism for H₁R, bioluminescence resonance energy transfer (BRET)-based biosensors were applied to measure H₁R signaling through heterotrimeric G_q proteins, second messengers (inositol 1,4,5-triphosphate and Ca²⁺), and receptor-protein interactions (GRKs and β-arrestins) in response to histamine, 2-phenylhistamines, and histaprodifens in a similar cellular background. Although differences in efficacy were observed for these agonists between some functional readouts as compared to reference agonist histamine, subsequent data analysis using an operational model of agonism revealed only signaling bias of the agonist Br-phHA-HA in recruiting β-arrestin2 to H₁R over G_q biosensor activation.     

Palmitoylation of Prolactin-Releasing Peptide Increased Affinity for and Activation of the GPR10, NPFF-R2 and NPFF-R1 Receptors: In Vitro Study

The anorexigenic neuropeptide prolactin-releasing peptide (PrRP) is involved in the regulation of food intake and energy expenditure. Lipidization of PrRP stabilizes the peptide, facilitates central effect after peripheral administration and increases its affinity for its receptor, GPR10, and for the neuropeptide FF (NPFF) receptor NPFF-R2. The two most potent palmitoylated analogs with anorectic effects in mice, palm¹¹-PrRP31 and palm-PrRP31, were studied in vitro to determine their agonist/antagonist properties and mechanism of action on GPR10, NPFF-R2 and other potential off-target receptors related to energy homeostasis. Palmitoylation of both PrRP31 analogs increased the binding properties of PrRP31 to anorexigenic receptors GPR10 and NPFF-R2 and resulted in a high affinity for another NPFF receptor, NPFF-R1. Moreover, in CHO-K1 cells expressing GPR10, NPFF-R2 or NPFF-R1, palm¹¹-PrRP and palm-PrRP significantly increased the phosphorylation of extracellular signal-regulated kinase (ERK), protein kinase B (Akt) and cAMP-responsive element-binding protein (CREB). Palm¹¹-PrRP31, unlike palm-PrRP31, did not activate either c-Jun N-terminal kinase (JNK), p38, c-Jun, c-Fos or CREB pathways in cells expressing NPFF-1R. Palm-PrRP31 also has higher binding affinities for off-target receptors, namely, the ghrelin, opioid (KOR, MOR, DOR and OPR-L1) and neuropeptide Y (Y₁, Y₂ and Y₅) receptors. Palm¹¹-PrRP31 exhibited fewer off-target activities; therefore, it has a higher potential to be used as an anti-obesity drug with anorectic effects.     

Synthese von anionischen N-acylierten L-Aminosäuren,Oligopeptiden und Proteinhydrolysaten als Eiweißtenside und deren Anwendung in der Wollveredlung

Synthesis of anionic N-acylated L-amino acids, oligopeptides and protein hydrolysates as protein-based surfactants and their application in wool finishing. As model compounds for protein-fatty acid condensation products, ten L-amino acids were N-acylated with fatty acid chlorides by Schotten-Baumann reaction. Structural variation in the non-polar part of the molecule was achieved by using saturated fatty acids of different chain length (C₈–C₁₈). The terminal carboxyl group was modified by reaction with different amino acids. Furthermore, N-octanoyl-, N-dodecanoyl-di, -tripeptides and protein-fatty acid condensation products were synthesized by Schotten-Baumann reaction. A dependence of the surface activity on both molecular weight and amino acid composition of the peptide moiety was found after determination of the surface tension-concentration isotherms: critical micelle concentration (CMC) of the surfactant solutions increase with increasing molecular weight and higher polarity of the peptide part. This coincides with a reduction of surface activity. With increasing alkyl chain length, however, the surface activity increases as can be seen from the decrease in CMC. For the synthesized anionic protein-based surfactants good biodegradabilities (68–78% BOD₅/COD) were determined. The sorption characteristics of protein derivatives on wool were studied in wool finishing processes. An increased bath exhaustion and a more intense coloration of wool were obtained during dyeing of wool by addition of N-acyl-L-amino acids or commercially available protein-fatty acid condensates.     

N‐Arylsulfonyl Indolines as Retinoic Acid Receptor‐Related Orphan Receptor γ (RORγ) Agonists

The nuclear retinoic acid receptor‐related orphan receptor γ (RORγ; NR1F3) is a key regulator of inflammatory gene programs involved in T helper 17 (T_H17) cell proliferation. As such, synthetic small‐molecule repressors (inverse agonists) targeting RORγ have been extensively studied for their potential as therapeutic agents for various autoimmune diseases. Alternatively, enhancing T_H17 cell proliferation through activation (agonism) of RORγ may boost an immune response, thereby offering a potentially new approach in cancer immunotherapy. Herein we describe the development of N‐arylsulfonyl indolines as RORγ agonists. Structure–activity studies reveal a critical linker region in these molecules as the major determinant for agonism. Hydrogen/deuterium exchange coupled to mass spectrometry (HDX‐MS) analysis of RORγ–ligand complexes help rationalize the observed results.     

Synthesis and Evaluation of a Series of Long‐Acting Glucagon‐Like Peptide‐1 (GLP‐1) Pentasaccharide Conjugates for the Treatment of Type 2 Diabetes

The present study details the development of a family of novel D ‐Ala⁸ glucagon‐like peptide‐1 (GLP‐1) peptide conjugates by site specific conjugation to an antithrombin III (ATIII) binding carrier pentasaccharide through tetraethylene glycol linkers. All conjugates were found to possess potent insulin‐releasing activity. Peptides with short linkers (<25 atoms) conjugated at Lys³⁴ and Lys³⁷ displayed strong GLP‐1 receptor (GLP‐1‐R) binding affinity. All D ‐Ala⁸GLP‐1 conjugates exhibited prominent glucose‐lowering action. Biological activity of the Lys³⁷ short‐linker peptide was evident up to 72 h post‐injection. In agreement, the pharmacokinetic profile of this conjugate (t_1/2, 11 h) was superior to that of the GLP‐1‐R agonist, exenatide. Once‐daily injection of the Lys³⁷ short‐linker peptide in ob/ob mice for 21 days significantly decreased food intake and improved HbA_1c and glucose tolerance. Islet size was decreased, with no discernible change in islet number. The beneficial effects of the Lys³⁷ short‐linker peptide were similar to or better than either exenatide or liraglutide, another GLP‐1‐R agonist. In conclusion, GLP‐1 peptides conjugated to an ATIII binding carrier pentasaccharide have a substantially prolonged bioactive profile compatible for possible once‐weekly treatment of type 2 diabetes in humans.     

Synthesis and Characterization of Octapeptide Somatostatin Analogues with Backbone Cyclization: Comparison of Different Strategies,Biological Activities and Enzymatic Stabilities

Somatostatin octapeptide analogues of the general sequence DPhe⁵‐Phe ⁶‐Tyr⁷‐DTrp⁸‐Lys⁹‐Val¹⁰‐Ph ¹¹‐Thr¹²‐NH₂ containing two types of backbone cyclization have been synthesized by the solid phase methodology. Backbone cyclization in these peptides was achieved via N‐modified phenylalanines in position 6 and 11. The N‐modified amino acids were incorporated as dipeptide building units which have been prepared in solution prior to the solid phase synthesis. Two dipeptide units of structure a) Fmoc‐aa ₁ ψ[CO—N((CH₂)_n‐X)]Phe—OH or b) Fmoc‐aa₁ ψ[CH₂—N(COlpar;CH₂)_n‐X)]Phe—OH have been introduced into the peptide sequence. Different resins and linkers were examined for an optimized peptide assembly and monitoring. The synthesized somatostatin analogues are highly resistant against enzymatic degradation as determined in vitro by incubation with rat liver homogenate. The biological activity was determined in binding experiments to the somatostatin receptors expressed in CHO‐ or BON‐1 cells. Most analogues show moderate activity without differentiation between the receptor subtypes.     

A Helix‐Stabilized Cell‐Penetrating Peptide as an Intracellular Delivery Tool

Two types of cationic cyclic α,α‐disubstituted α‐amino acids: Api (which possesses a lysine mimic side chain) and Api^C2Gu (which possesses an arginine mimic side chain), were developed. These amino acids were incorporated into an arginine‐based peptide sequence [(l ‐Arg‐l ‐Arg‐dAA)₃: dAA=Api or Api^C2Gu], and the relationship between the secondary structures of the resulting peptides and their ability to pass through cell membranes was investigated. The peptide containing Api^C2Gu formed a stable α‐helical structure and was more effective at penetrating cells than the nonhelical Arg nonapeptide (R₉). Furthermore, the peptide was able to deliver plasmid DNA into various types of cells in a highly efficient manner.     

Ligand-binding domain of farnesoid X receptor (FXR) had the highest sensitivity and activity among FXR variants in a fluorescence-based assay

The farnesoid X receptor (FXR, NR1H4) has been recognized as an attractive therapeutic target because it is a nuclear hormone receptor that controls the expression level of cholesterol-7α-hydroxylase, which in turn regulates bile acid production and cholesterol excretion. To compare receptor activity between each domain and the full-length protein, human FXR cDNA was cloned from a human liver cDNA library. Three human FXR cDNA, designated FXR₂₀, FXR₃₃, and FXR₅₃ cDNA, were subcloned and ligated into a pET28a expression vector. Each protein was expressed in Escherichia coli (BL21) and purified by nickel-nitrilotriacetic acid column chromatography. Approximately 5 mg of FXR₃₃ (1–182 amino acids deleted from FXR, 37 kDa) and 2 mg of FXR₅₃ (the full-length protein of FXR, 59 kDa) was purified from 1 L of Luria-Bertani culture, achieving at least 90% purity. The coactivator recruitment assay for FXR activation was carried out with the three variants of the FXR protein by using dissociation-enhanced lanthanide fluoroimmunoassay-europium-N¹-labeled anti-His antibody. From an optimized assay, a saturated hyperbolic fluorescence signal curve was produced when 250 nM of FXR₃₃ and 100 nM of steroid receptor coactivator-1 peptide, a coactivator of FXR consisting of 26 amino acids, were used with a concentration dependence on chenodeoxycholic acid (from 0 to 200 μM). The ligand-binding domain of FXR (FXR₃₃) was the most suitable protein for studying the activation of FXR with a fluorescence-based assay, because it showed better structural stability than either the full length of FXR (FXR₅₃) or the DNA-binding domain of FXR (FXR₂₀).     

Oligomeric poly(imide-amides)s containing side 1,8-naphthalimidyl groups: synthesis and characterization

Poly(imide-amide)s (PIAs) were synthesized from isophthalic acids, which have in position 5 a bulky group like 1,8-naphthalimidyl bonded to amino acids as flexible spacers, and the diamine bis(4-aminophenyl)diphenylsilane, which provides a polar group in the main chain. Glycine, l-alanine, l-phenylalanine, l-valine and p-aminobenzoic acid were used as amino acids. Polymers were obtained according to the Yamazaki method and characterized by elemental analysis, optical activity, IR and ¹H, ¹³C and ²⁹Si NMR spectroscopy. PIAs were soluble in aprotic polar solvents but not in common organic solvents, and obtained with low η _inh values, which was an indicative of low molecular weights species, probably of oligomeric nature. According to the polymeric structure, the only difference between PIAs is the structure of the amino acid residue, and in this sense it was possible to see an increase of the T _g values when the volume of the amino acid residue also increased, due to the lower possibility of internal mobility of the side chains. The higher Tg value was obtained with the PIA-e, which includes an aromatic ring as a side chain, derived from p-aminobenzoic acid. PIAs, in spite of the good TDT values obtained, were not thermally stable in the sense that the 10 % of weight lost was obtained at lower temperature than 400 °C, with the exception of PIA-e derived from p-aminobenzoic acid. However, there was an increase of the TDT values when the volume of the amino acid residue increases. The PIAs do not show good UV–vis transparence probably due to the low free volume of those including an aliphatic amino acid residue.     

Different Binding Modes of Small and Large Binders of GAT1

Well‐known inhibitors of the γ‐aminobutyric acid (GABA) transporter GAT1 share a common scaffold of a small cyclic amino acid linked by an alkyl chain to a moiety with two aromatic rings. Tiagabine, the only FDA‐approved GAT1 inhibitor, is a typical example. Some small amino acids such as (R)‐nipecotic acid are medium‐to‐strong binders of GAT1, but similar compounds, such as proline, are very weak binders. When substituted with 4,4‐diphenylbut‐3‐en‐1‐yl (DPB) or 4,4‐bis(3‐methylthiophen‐2‐yl)but‐3‐en‐1‐yl (BTB) groups, the resulting compounds have similar pK_i and pIC₅₀ values, even though the pure amino acids have very different values. To investigate if small amino acids and their substituted counterparts share a similar binding mode, we synthesized butyl‐, DPB‐, and BTB‐substituted derivatives of small amino acids. Supported by the results of docking studies, we propose different binding modes not only for unsubstituted und substituted, but also for strong‐ and weak‐binding amino acids. These data lead to the conclusion that following a fragment‐based approach, not pure but N‐butyl‐substituted amino acids should be used as starting points, giving a better estimate of the activity when a BTB or DPB substituent is added.     

Structural Characterization of EnpA D,L-Endopeptidase from Enterococcus faecalis Prophage Provides Insights into Substrate Specificity of M23 Peptidases

The best-characterized members of the M23 family are glycyl-glycine hydrolases, such as lysostaphin (Lss) from Staphylococcus simulans or LytM from Staphylococcus aureus. Recently, enzymes with broad specificities were reported, such as EnpA_CD from Enterococcus faecalis, that cleaves D,L peptide bond between the stem peptide and a cross-bridge. Previously, the activity of EnpA_CD was demonstrated only on isolated peptidoglycan fragments. Herein we report conditions in which EnpA_CD lyses bacterial cells live with very high efficiency demonstrating great bacteriolytic potential, though limited to a low ionic strength environment. We have solved the structure of the EnpA_CD H109A inactive variant and analyzed it in the context of related peptidoglycan hydrolases structures to reveal the bases for the specificity determination. All M23 structures share a very conserved β-sheet core which constitutes the rigid bottom of the substrate-binding groove and active site, while variable loops create the walls of the deep and narrow binding cleft. A detailed analysis of the binding groove architecture, specificity of M23 enzymes and D,L peptidases demonstrates that the substrate groove, which is particularly deep and narrow, is accessible preferably for peptides composed of amino acids with short side chains or subsequent L and D-isomers. As a result, the bottom of the groove is involved in interactions with the main chain of the substrate while the side chains are protruding in one plane towards the groove opening. We concluded that the selectivity of the substrates is based on their conformations allowed only for polyglycine chains and alternating chirality of the amino acids.