ABSTRACT
Cu(II)-mediated C-H sulphenylation or selenylation of Trp indole by a derivative of cysteine or selenocysteine enables access to the tryptathionine unit or its selenium congener. The mechanism of these protocols, which allow macrocyclization of Trp-containing peptides, has been studied.
Subject(s)
Copper/chemistry , Peptides, Cyclic/chemical synthesis , Selenium/chemistry , Tryptophan/chemistry , Amino Acid Sequence , Catalysis , Cyclization , Disulfides/chemistry , Indoles/chemistry , Lactams/chemistry , Oxidation-Reduction , Phenothiazines/chemistry , Pyrrolidinones/chemistry , Trypsin/chemistryABSTRACT
The rising opioid crisis has become a worldwide societal and public health burden, resulting from the abuse of prescription opioids. Targeting the κ-opioid receptor (KOR) in the periphery has emerged as a powerful approach to develop novel pain medications without central side effects. Inspired by the traditional use of sunflower (Helianthus annuus) preparations for analgesic purposes, we developed novel stabilized KOR ligands (termed as helianorphins) by incorporating different dynorphin A sequence fragments into a cyclic sunflower peptide scaffold. As a result, helianorphin-19 selectively bound to and fully activated the KOR with nanomolar potency. Importantly, helianorphin-19 exhibited strong KOR-specific peripheral analgesic activity in a mouse model of chronic visceral pain, without inducing unwanted central effects on motor coordination/sedation. Our study provides a proof of principle that cyclic peptides from plants may be used as templates to develop potent and stable peptide analgesics applicable via enteric administration by targeting the peripheral KOR for the treatment of chronic abdominal pain.
Subject(s)
Abdominal Pain/drug therapy , Analgesics/pharmacology , Peptides, Cyclic/pharmacology , Plant Extracts/pharmacology , Receptors, Opioid, kappa/antagonists & inhibitors , Analgesics/chemical synthesis , Analgesics/chemistry , Animals , Cells, Cultured , Chronic Disease , Dose-Response Relationship, Drug , Drug Design , HEK293 Cells , Helianthus/chemistry , Humans , Male , Mice , Mice, Inbred C57BL , Molecular Structure , Peptides, Cyclic/chemical synthesis , Peptides, Cyclic/chemistry , Plant Extracts/chemical synthesis , Plant Extracts/chemistry , Receptors, Opioid, kappa/metabolism , Seeds/chemistry , Structure-Activity RelationshipABSTRACT
Total synthesis-the ultimate proving ground for the invention and field-testing of new methods, exploration of disruptive strategies, final structure confirmation, and empowerment of medicinal chemistry on natural products-is one of the oldest and most enduring subfields of organic chemistry. In the early days of this field, its sole emphasis focused on debunking the concept of vitalism, that living organisms could create forms of matter accessible only to them. Emphasis then turned to the use of synthesis to degrade and reconstitute natural products to establish structure and answer questions about biosynthesis. It then evolved to not only an intricate science but also a celebrated form of art. As the field progressed, a more orderly and logical approach emerged that served to standardize the process. These developments even opened up the possibility of computer-aided design using retrosynthetic analysis. Finally, the elevation of this field to even higher levels of sophistication showed that it was feasible to synthesize any natural product, regardless of complexity, in a laboratory. During this remarkable evolution, as has been reviewed elsewhere, many of the principles and methods of organic synthesis were refined and galvanized. In the modern era, students and practitioners are still magnetically attracted to this field due to the excitement of the journey, the exhilaration of creation, and the opportunity to invent solutions to challenges that still persist. Contemporary total synthesis is less concerned with demonstrating a proof of concept or a feasible approach but rather aims for increased efficiency, scalability, and even "ideality." In general, the molecules of Nature are created biosynthetically with levels of practicality that are still unimaginable using the tools of modern synthesis. Thus, as the community strives to do more with less (i.e., innovation), total synthesis is now focused on a pursuit for simplicity rather than a competition for maximal complexity. In doing so, the practitioner must devise outside-the-box strategies supplemented with forgotten or newly invented methods to reduce step count and increase the overall economy of the approach. The downstream applications of this pursuit not only empower students who often go on to apply these skills in the private sector but also lead to new discoveries that can impact numerous disciplines of societal importance. This account traces some select case studies from our laboratory over the past five years that vividly demonstrate our own motivation for dedicating so much effort to this classic field. In aiming for simplicity, we focus on the elusive goal of achieving ideality, a term that, when taken in the proper context, can serve as a guiding light to point the way to furthering progress in organic synthesis.
Subject(s)
Biological Products/chemical synthesis , Alkaloids/chemical synthesis , Alkaloids/chemistry , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Biological Products/chemistry , Oligopeptides/chemical synthesis , Oligopeptides/chemistry , Peptides, Cyclic/chemical synthesis , Peptides, Cyclic/chemistry , Solid-Phase Synthesis Techniques , Thiazolidines/chemical synthesis , Thiazolidines/chemistry , Ubiquinone/analogs & derivatives , Ubiquinone/chemical synthesis , Ubiquinone/chemistryABSTRACT
The discovery of a pan-genotypic hepatitis C virus (HCV) NS3/4A protease inhibitor based on a P1-P3 macrocyclic tripeptide motif is described. The all-carbon tether linking the P1-P3 subsites of 21 is functionalized with alkyl substituents, which are shown to effectively modulate both potency and absorption, distribution, metabolism, and excretion (ADME) properties. The CF3Boc-group that caps the P3 amino moiety was discovered to be an essential contributor to metabolic stability, while positioning a methyl group at the C1 position of the P1' cyclopropyl ring enhanced plasma trough values following oral administration to rats. The C7-fluoro, C6-CD3O substitution pattern of the P2* isoquinoline heterocycle of 21 was essential to securing the targeted potency, pharmacokinetic (PK), and toxicological profiles. The C6-CD3O redirected metabolism away from a problematic pathway, thereby circumventing the time-dependent cytochrome P (CYP) 450 inhibition observed with the C6-CH3O prototype.
Subject(s)
Antiviral Agents/pharmacology , Peptides, Cyclic/pharmacology , Serine Proteinase Inhibitors/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Animals , Antiviral Agents/chemical synthesis , Antiviral Agents/metabolism , Antiviral Agents/pharmacokinetics , CHO Cells , Cricetulus , Drug Discovery , Drug Stability , Hepacivirus/drug effects , Hepacivirus/enzymology , Microbial Sensitivity Tests , Microsomes, Liver/metabolism , Molecular Structure , Peptides, Cyclic/chemical synthesis , Peptides, Cyclic/metabolism , Peptides, Cyclic/pharmacokinetics , Rats , Serine Proteinase Inhibitors/chemical synthesis , Serine Proteinase Inhibitors/metabolism , Serine Proteinase Inhibitors/pharmacokinetics , Structure-Activity RelationshipABSTRACT
Small, cyclic peptides are reported to have many bioactivities. In bacteria and fungi, they can be made by nonribosomal peptide synthetases, but in plants they are exclusively ribosomal. Cyclic peptides from the Annona genus possess cytotoxic and anti-inflammatory activities, but their biosynthesis is unknown. The medicinal soursop plant, Annona muricata, contains annomuricatins A (cyclo-PGFVSA) and B (cyclo-PNAWLGT). Here, using de novo transcriptomics and tandem mass spectrometry, we identify a suite of short transcripts for precursor proteins for 10 validated annomuricatins, 9 of which are novel. In their precursors, annomuricatins are preceded by an absolutely conserved Glu and each peptide sequence has a conserved proto-C-terminal Pro, revealing parallels with the segetalin orbitides from the seed of Vaccaria hispanica, which are processed through ligation by a prolyl oligopeptidase in a transpeptidation reaction.
Subject(s)
Annona/chemistry , Anti-Inflammatory Agents/chemistry , Peptides, Cyclic/chemical synthesis , Plant Extracts/chemistry , Amino Acid Sequence , Anti-Inflammatory Agents/analysis , Molecular Structure , Peptides, Cyclic/chemistry , Plant Leaves/chemistry , Plants, MedicinalABSTRACT
Multidrug exporters expressed in pathogens efflux substrate drugs such as antibiotics, and thus, the development of inhibitors against them has eagerly been anticipated. Furthermore, the crystal structures of multidrug exporters with their inhibitors provide novel insights into the inhibitory mechanism and the development of more specific and effective inhibitors. We previously reported the complex structures of the Multidrug And Toxic compound Extrusion (MATE)-type multidrug exporter with the macrocyclic peptides, which inhibit the efflux of substrates by the MATE-type multidrug exporter (Tanaka et al., Nature 496:247-251, 2013). In this chapter, we describe methodologies of the screening and synthesis of macrocyclic peptides as inhibitors, as well as the purification, crystallization, and structure determination of the complexes of the MATE-type multidrug exporter with its inhibitors.
Subject(s)
Organic Cation Transport Proteins/antagonists & inhibitors , Peptides, Cyclic/chemical synthesis , Pyrococcus furiosus/metabolism , Archaeal Proteins/antagonists & inhibitors , Archaeal Proteins/chemistry , Crystallography, X-Ray , Drug Evaluation, Preclinical , Models, Molecular , Organic Cation Transport Proteins/chemistry , Peptides, Cyclic/chemistry , Peptides, Cyclic/pharmacology , Protein ConformationABSTRACT
Naturally occurring peptides and proteins often use dynamic disulfide bonds to impart defined tertiary/quaternary structures for the formation of binding pockets with uniform size and function. Although peptide synthesis and modification are well established, controlling quaternary structure formation remains a significant challenge. Here, we report the facile incorporation of aryl aldehyde and acyl hydrazide functionalities into peptide oligomers via solid-phase copper-catalysed azide-alkyne cycloaddition (SP-CuAAC) click reactions. When mixed, these complementary functional groups rapidly react in aqueous media at neutral pH to form peptide-peptide intermolecular macrocycles with highly tunable ring sizes. Moreover, sequence-specific figure-of-eight, dumbbell-shaped, zipper-like and multi-loop quaternary structures were formed selectively. Controlling the proportions of reacting peptides with mismatched numbers of complementary reactive groups results in the formation of higher-molecular-weight sequence-defined ladder polymers. This also amplified antimicrobial effectiveness in select cases. This strategy represents a general approach to the creation of complex abiotic peptide quaternary structures.
Subject(s)
Anti-Bacterial Agents/pharmacology , Antimicrobial Cationic Peptides/pharmacology , Peptides, Cyclic/pharmacology , Quaternary Ammonium Compounds/pharmacology , Aldehydes/chemical synthesis , Alkynes/chemistry , Amino Acid Sequence , Anti-Bacterial Agents/chemical synthesis , Antimicrobial Cationic Peptides/chemical synthesis , Azides/chemistry , Click Chemistry , Cycloaddition Reaction , Hydrazines/chemical synthesis , Peptides, Cyclic/chemical synthesis , Pseudomonas aeruginosa/drug effects , Quaternary Ammonium Compounds/chemical synthesis , Staphylococcus aureus/drug effectsABSTRACT
The melanocortin receptor 4 (MC4R) subtype of the melanocortin receptor family is a target for therapeutics to ameliorate metabolic dysfunction. Endogenous MC4R agonists possess a critical pharmacophore (HFRW), and cyclization of peptide agonists often enhances potency. Thus, 17 cyclized peptides were synthesized by solid phase click chemistry to develop novel, potent, selective MC4R agonists. Using cAMP measurements and a transcriptional reporter assay, we observed that several constrained agonists generated by a cycloaddition reaction displayed high selectivity (223- to 467-fold) toward MC4R over MC3R and MC5R receptor subtypes without compromising agonist potency. Significant variation was also observed between the EC50 values for the two assays, with robust levels of reporter expression measured at lower concentrations than those effecting appreciable increases in cAMP levels for the majority of the compounds tested. Collectively, we characterized significant elements that modulate the activity of the core pharmacophore for MC4R and provide a rationale for careful assay selection for agonist screening.
Subject(s)
Click Chemistry/methods , Peptides, Cyclic/chemical synthesis , Receptor, Melanocortin, Type 4/agonists , Animals , Cyclic AMP/analysis , Drug Evaluation, Preclinical/methods , Humans , Peptides, Cyclic/pharmacology , Structure-Activity RelationshipABSTRACT
We have demonstrated that arginine-rich and poly-arginine peptides possess potent neuroprotective properties with arginine content and peptide positive charge being particularly critical for neuroprotective efficacy. In addition, the presence of other amino acids within arginine-rich peptides, as well as chemical modifications, peptide length and cell-penetrating properties also influence the level of neuroprotection. Against this background, we have examined the neuroprotective efficacy of arginine-rich protamine peptides, a cyclic (R12-c) poly-arginine peptide and a R22 poly-arginine peptide, as well as arginine peptides containing tryptophan or other amino acids (phenylalanine, tyrosine, glycine or leucine) in in vitro glutamic acid excitotoxicity and in vivo rat permanent middle cerebral artery occlusion models of stroke. In vitro studies demonstrated that protamine and poly-arginine peptides (R12-c, R22) were neuroprotective. Arginine-tryptophan-containing peptides were highly neuroprotective, with R12W8a being the most potent arginine-rich peptide identified in our laboratory. Peptides containing phenylalanine or tyrosine substituted in place of tryptophan in R12W8a were also highly neuroprotective, whereas leucine, and in particular glycine substitutions, decreased peptide efficacy. In vivo studies with protamine administered intravenously at 1000 nmol/kg 30 min after MCAO significantly reduced infarct volume and cerebral oedema by 22.5 and 38.6%, respectively. The R12W8a peptide was highly toxic when administered intravenously at 300 or 100 nmol/kg and ineffective at reducing infarct volume when administered at 30 nmol/kg 30 min after MCAO, unlike R18 (30 nmol/kg), which significantly reduced infarct volume by 20.4%. However, both R12W8a and R18 significantly reduced cerebral oedema by 19.8 and 42.2%, respectively. Protamine, R12W8a and R18 also reduced neuronal glutamic acid-induced calcium influx. These findings further highlight the neuroprotective properties of arginine-rich peptides and support the view that they represent a new class of neuroprotective agent.
Subject(s)
Glutamic Acid/toxicity , Infarction, Middle Cerebral Artery/drug therapy , Neurons/drug effects , Neuroprotective Agents/therapeutic use , Oligopeptides/therapeutic use , Amino Acids/pharmacology , Animals , Arginine/chemistry , Astrocytes/drug effects , Calcium Signaling/drug effects , Cells, Cultured , Cerebral Cortex/cytology , Cerebral Cortex/embryology , Drug Evaluation, Preclinical , In Vitro Techniques , Infarction, Middle Cerebral Artery/pathology , Male , Neuroprotective Agents/chemical synthesis , Neuroprotective Agents/pharmacology , Oligopeptides/chemical synthesis , Oligopeptides/pharmacology , Peptides, Cyclic/chemical synthesis , Peptides, Cyclic/pharmacology , Peptides, Cyclic/therapeutic use , Protamines/chemistry , Rats , Rats, Sprague-Dawley , Tryptophan/chemistryABSTRACT
The newly discovered short (9 amino acid) non-RGD S-S bridged cyclic peptide ALOS-4 (H-cycl(Cys-Ser-Ser-Ala-Gly-Ser-Leu-Phe-Cys)-OH), which binds to integrin αvß3 is investigated as peptide carrier for targeted drug delivery against human metastatic melanoma. ALOS4 binds specifically the αvß3 overexpressing human metastatic melanoma WM-266-4 cell line both in vitro and in ex vivo assays. Coupling ALOS4 to the topoisomerase I inhibitor Camptothecin (ALOS4-CPT) increases the cytotoxicity of CPT against human metastatic melanoma cells while reduces dramatically the cytotoxicity against non-cancerous cells as measured by the levels of γH2A.X, active caspase 3 and cell viability. Moreover, conjugating ALOS4 to CPT even increases the chemo-stability of CPT under physiological pH. Bioinformatic analysis using Rosetta platform revealed potential docking sites of ALOS4 on the αvß3 integrin which are distinct from the RGD binding sites. We propose to use this specific non-RGD cyclic peptide as the therapeutic carrier for conjugation of drugs in order to improve efficacy and reduce toxicity of currently available treatments of human malignant melanoma.
Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Drug Discovery , Peptides, Cyclic/chemistry , Peptides, Cyclic/pharmacology , Peptides, Cyclic/therapeutic use , Animals , Antineoplastic Agents/chemical synthesis , Caspase 3/metabolism , Cell Line, Tumor , Cell Survival/drug effects , DNA Damage/drug effects , Disease Models, Animal , Drug Evaluation, Preclinical , Drug Stability , Humans , Integrin alphaVbeta3/chemistry , Integrin alphaVbeta3/metabolism , Melanoma/drug therapy , Melanoma/metabolism , Melanoma/pathology , Mice , Models, Molecular , Molecular Conformation , Molecular Structure , Oligopeptides/chemical synthesis , Oligopeptides/chemistry , Oligopeptides/pharmacology , Peptides, Cyclic/chemical synthesis , Protein Binding , Xenograft Model Antitumor AssaysABSTRACT
The solution-phase synthesis of a proline and glycine-rich plant-derived cyclic heptapeptide, gypsophin E (8), is reported via coupling of a tetrapeptide unit Glycyl-L-leucyl-L-valyl-L-proline-OMe with a tripeptide unit Boc-L-isoleucyl-glycyl-L-proline-OH, followed by cyclization of the linear fragment having seven amino acid units. The structure of the newly synthesized cycloheptapeptide was confirmed by means of chemical and spectroscopic methods. The newly synthesized cyclopolypeptide displayed potent antifungal and anthelmintic activities against the pathogenic yeast Candida albicans, the dermatophytes Trichophyton mentagrophytes and Microsporum audouinii at the 6 µg/mL level, and the earthworms Megascoplex konkanensis, Pontoscotex corethruses and - - Eudrilus eugeniea at a concentration of 2 mg/mL.
Subject(s)
Biological Products/chemical synthesis , Peptides, Cyclic/chemical synthesis , Peptides, Cyclic/pharmacology , Animals , Anthelmintics/chemical synthesis , Anthelmintics/pharmacology , Antifungal Agents/chemical synthesis , Antifungal Agents/pharmacology , Biological Products/pharmacology , Fungi/drug effects , Molecular Structure , Oligochaeta/drug effectsABSTRACT
Cyclic peptides with two phosphoserines and two glutamic acids were developed to mimic high-affinity binding sites for uranyl found in proteins such as osteopontin, which is believed to be a privileged target of this ion in vivo. These peptides adopt a ß-sheet structure that allows the coordination of the latter amino acid side chains in the equatorial plane of the dioxo uranyl cation. Complementary spectroscopic and analytical methods revealed that these cyclic peptides are efficient uranyl chelating peptides with a large contribution from the phosphorylated residues. The conditional affinity constants were measured by following fluorescence tryptophan quenching and are larger than 10(10) at physiological pH. These compounds are therefore promising models for understanding uranyl chelation by proteins, which is relevant to this actinide ion toxicity.
Subject(s)
Chelating Agents/chemistry , Molecular Mimicry , Peptides, Cyclic/chemistry , Phosphopeptides/chemistry , Uranyl Nitrate/chemistry , Amino Acid Sequence , Binding Sites , Calcium/chemistry , Chelating Agents/chemical synthesis , Circular Dichroism , Glutamic Acid/chemistry , Imino Acids , Osteopontin/chemistry , Peptides, Cyclic/chemical synthesis , Phosphopeptides/chemical synthesis , Phosphoserine/chemistry , Protein Structure, Secondary , Spectrometry, Mass, Electrospray Ionization , Tryptophan/chemistryABSTRACT
Standard mechanism inhibitors are attractive design templates for engineering reversible serine protease inhibitors. When optimizing interactions between the inhibitor and target protease, many studies focus on the nonprimed segment of the inhibitor's binding loop (encompassing the contact ß-strand). However, there are currently few methods for screening residues on the primed segment. Here, we designed a synthetic inhibitor library (based on sunflower trypsin inhibitor-1) for characterizing the P2' specificity of various serine proteases. Screening the library against 13 different proteases revealed unique P2' preferences for trypsin, chymotrypsin, matriptase, plasmin, thrombin, four kallikrein-related peptidases, and several clotting factors. Using this information to modify existing engineered inhibitors yielded new variants that showed considerably improved selectivity, reaching up to 7000-fold selectivity over certain off-target proteases. Our study demonstrates the importance of the P2' residue in standard mechanism inhibition and unveils a new approach for screening P2' substitutions that will benefit future inhibitor engineering studies.
Subject(s)
Peptides, Cyclic/chemical synthesis , Peptides, Cyclic/pharmacology , Serine Proteinase Inhibitors/chemical synthesis , Serine Proteinase Inhibitors/pharmacology , Amino Acid Sequence , Blood Coagulation/drug effects , Drug Design , Drug Evaluation, Preclinical , Fibrinolysis/drug effects , Kallikreins/antagonists & inhibitors , Kallikreins/chemistry , Models, Molecular , Peptides, Cyclic/chemistry , Protein EngineeringABSTRACT
We describe a general synthetic strategy for developing high-affinity peptide binders against specific epitopes of challenging protein biomarkers. The epitope of interest is synthesized as a polypeptide, with a detection biotin tag and a strategically placed azide (or alkyne) presenting amino acid. This synthetic epitope (SynEp) is incubated with a library of complementary alkyne or azide presenting peptides. Library elements that bind the SynEp in the correct orientation undergo the Huisgen cycloaddition, and are covalently linked to the SynEp. Hit peptides are tested against the full-length protein to identify the best binder. We describe development of epitope-targeted linear or macrocycle peptide ligands against 12 different diagnostic or therapeutic analytes. The general epitope targeting capability for these low molecular weight synthetic ligands enables a range of therapeutic and diagnostic applications, similar to those of monoclonal antibodies.
Subject(s)
Drug Design , Epitopes/chemistry , Peptides, Cyclic/chemical synthesis , Peptides, Cyclic/pharmacology , Proteins/chemistry , Ligands , Molecular Weight , Peptides, Cyclic/chemistry , Proteins/antagonists & inhibitorsABSTRACT
The melanocortin-3 (MC3R) and melanocortin-4 (MC4R) receptors are expressed in the brain and are implicated in the regulation of food intake and energy homeostasis. The endogenous agonist ligands for these receptors (α-, ß-, γ-MSH and ACTH) are linear peptides with limited receptor subtype selectivity and metabolic stability, thus minimizing their use as probes to characterize the overlapping pharmacological and physiological functions of the melanocortin receptor subtypes. In the present study, an engineered template, in which the peptide backbone was modified by a heterocyclic reverse turn mimetic at the Trp(7) residue, was synthesized using solid phase peptide synthesis and characterized by a ß-galactosidase cAMP based reporter gene assay. The functional assay identified a â¼5 nM mouse MC4R agonist (AST3-88) with more than 50-fold selectivity over the mMC3R. Biophysical studies (2D (1)H NMR spectroscopy and molecular dynamics) of AST3-88 identified a type VIII ß-turn secondary structure spanning the pharmacophore domain stabilized by the intramolecular interactions between the side chains of the His and Trp residues. Enzymatic studies of AST3-88 revealed enhanced stability of AST3-88 over the α-MSH endogenous peptide in rat serum. Upon central administration of AST3-88 into rats, a decreased food intake response was observed. This is the first study to probe the in vivo physiological activity of this engineered peptide-heterocycle template. These findings advance the present knowledge of pharmacophore design for potent, selective, and metabolically stable melanocortin ligands.
Subject(s)
Neurotransmitter Agents/pharmacology , Peptides, Cyclic/pharmacology , Receptor, Melanocortin, Type 4/agonists , Animals , Chromatography, Liquid , Drug Evaluation, Preclinical , Eating/drug effects , HEK293 Cells , Humans , Male , Mass Spectrometry , Mice , Molecular Dynamics Simulation , Molecular Structure , Neurotransmitter Agents/chemical synthesis , Peptides, Cyclic/chemical synthesis , Proton Magnetic Resonance Spectroscopy , Rats , Rats, Sprague-Dawley , Receptor, Melanocortin, Type 3/agonists , Receptor, Melanocortin, Type 3/metabolism , Receptor, Melanocortin, Type 4/genetics , Receptor, Melanocortin, Type 4/metabolism , Transfection , alpha-MSH/metabolismABSTRACT
BACKGROUND: A detailed comprehension of central mechanisms underlying feeding behavior holds considerable promise for the treatment of alimentary disorders. METHODS: In order to elucidate the tight interrelationships occurring at the hypothalamic neuronal endings between aminergic neurotransmitters and co-localized appetite modulators, we initially studied the effects of two anorexigenic peptides structurally related to thyrotropin-releasing hormone (TRH, 1), namely cyclo(His-Pro) (CHP, 2) and pGlu-His-Gly-OH (3), on [(3)H]-norepinephrine and [(3)H]-dopamine release from perfused rat hypothalamic synaptosomes. Furthermore, a number of TRH and CHP analogues were synthesized and tested for their ability to influence neurotransmitter release in the selected neuronal model. RESULTS: Peptide 3 showed only a slight inhibitory activity on norepinephrine release, whereas no effect was observed for compound 2. TRH analogue 8, metabolically stabilized by the replacement of pyroglutamate with the pyrohomocysteic acid (pHcs), was found to be inactive. Conversely, a significant inhibitory effect on dopamine and norepinephrine release was observed for the CHP-related diketopiperazines cyclo(Leu-Pro) (11) and cyclo(His-Gly) (14). CONCLUSIONS: These results suggest a potential role for cyclo-dipeptides 11 and 14 in the hypothalamic modulation of appetite suppressant circuitry.
Subject(s)
Catecholamines/metabolism , Oligopeptides/pharmacology , Peptides, Cyclic/pharmacology , Peptides/chemical synthesis , Peptides/pharmacology , Piperazines/pharmacology , Pyrrolidonecarboxylic Acid/analogs & derivatives , Thyrotropin-Releasing Hormone/analogs & derivatives , Thyrotropin-Releasing Hormone/pharmacology , Animals , Hypothalamus/drug effects , Hypothalamus/metabolism , Male , Oligopeptides/chemical synthesis , Peptides, Cyclic/chemical synthesis , Piperazines/chemical synthesis , Pyrrolidonecarboxylic Acid/chemical synthesis , Pyrrolidonecarboxylic Acid/pharmacology , Rats , Synaptosomes/drug effects , Synaptosomes/metabolismSubject(s)
Disulfides/chemistry , Norepinephrine Plasma Membrane Transport Proteins/antagonists & inhibitors , Peptidomimetics , Adrenergic Uptake Inhibitors/blood , Adrenergic Uptake Inhibitors/chemical synthesis , Adrenergic Uptake Inhibitors/chemistry , Amino Acid Sequence , Animals , Drug Evaluation, Preclinical , Half-Life , Humans , Magnetic Resonance Spectroscopy , Norepinephrine Plasma Membrane Transport Proteins/metabolism , Peptides, Cyclic/blood , Peptides, Cyclic/chemical synthesis , Peptides, Cyclic/chemistry , Protein Structure, Tertiary , RatsABSTRACT
The interplay between cardiac sarcoplasmic Ca(2+)ATPase and phospholamban is a key regulating factor of contraction and relaxation in the cardiac muscle. In heart failure, aberrations in the inhibition of sarcoplasmic Ca(2+)ATPase by phospholamban are associated with anomalies in cardiac functions. In experimental heart failure models, modulation of the interaction between these two proteins has been shown to be a potential therapeutic approach. The aim of our research was to find molecules able to interfere with the inhibitory activity of phospholamban on sarcoplasmic Ca(2+)ATPase. For this purpose, a portion of phospholamban was synthesized and used as target for a phage-display peptide library screening. The cyclic peptide C-Y-W-E-L-E-W-L-P-C-A was found to bind to phospholamban (1-36) with high specificity. Its functional activity was tested in Ca(2+)uptake assays utilizing preparations from cardiac sarcoplasmic reticulum. By synthesizing and testing a series of alanine point-mutated cyclic peptides, we identified which amino acid was important for the inhibition of the phospholamban function. The structures of active and inactive alanine-mutated cyclic peptides, and of phospholamban (1-36), were determined by NMR. This structure-activity analysis allowed building a model of phospholamban -cyclic peptide complex. Thereafter, a simple pharmacophore was defined and used for the design of small molecules. Finally, examples of such molecules were synthesized and characterized as phospholamban inhibitors.
Subject(s)
Calcium-Binding Proteins/metabolism , Peptides, Cyclic/chemistry , Amino Acid Sequence , Animals , Calcium/metabolism , Calcium-Binding Proteins/antagonists & inhibitors , Calcium-Binding Proteins/chemical synthesis , Drug Design , Drug Evaluation, Preclinical , Guinea Pigs , Heart/drug effects , Humans , Models, Molecular , Myocardium/metabolism , Peptide Library , Peptides, Cyclic/chemical synthesis , Peptides, Cyclic/pharmacology , Protein Binding , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Structure-Activity RelationshipABSTRACT
This paper deals with the design, synthesis, and evaluation of a new series of receptors for protein surface recognition. The design of these agents is based around the attachment of four constrained dipeptide chains onto a central resorc[4]arene scaffold. By varying the sequence, nature, and stereochemistry of the chains we prepared anionically functionalized N-linked peptidoresorc[4]arenes 12, 13, and 17 by Pd/C-catalyzed hydrogenation of the corresponding benzyl esters 10, 11, and 16. From this family of receptors we have identified noncompetitive inhibitors of α-chymotrypsin (ChT), which function by binding to the surface of the enzyme in the neighborhood of the active site cleft (K(i) values ranging from 12.4 ± 5.1 µM for free carboxylic acid (+)-12b to 0.76 ± 0.14 µM for benzyl ester (-)-16a). For anionically functionalized receptors 12, 13, and 17 the ChT inhibition is based essentially on electrostatic interaction, and the bound enzyme can be released from the resorcarene surface by increasing the ionic strength, with its activity almost completely restored. For receptors with terminal benzyl ester groups (10 and 16) a hydrophobic network can be suggested.