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The G protein-coupled receptor cascade leading to production of the second messenger cAMP is replete with pharmacologically targetable proteins, with the exception of the Gα subunit, Gαs. GTPases remain largely undruggable given the difficulty of displacing high-affinity guanine nucleotides and the lack of other drug binding sites. We explored a chemical library of 1012 cyclic peptides to expand the chemical search for inhibitors of this enzyme class. We identified two macrocyclic peptides, GN13 and GD20, that antagonize the active and inactive states of Gαs, respectively. Both macrocyclic peptides fine-tune Gαs activity with high nucleotide-binding-state selectivity and G protein class-specificity. Co-crystal structures reveal that GN13 and GD20 distinguish the conformational differences within the switch II/α3 pocket. Cell-permeable analogs of GN13 and GD20 modulate Gαs/Gßγ signaling in cells through binding to crystallographically defined pockets. The discovery of cyclic peptide inhibitors targeting Gαs provides a path for further development of state-dependent GTPase inhibitors.
Assuntos
Peptídeos , Receptores Acoplados a Proteínas G , GTP Fosfo-Hidrolases , Nucleotídeos de Guanina , Nucleotídeos , Peptídeos/química , Peptídeos Cíclicos/farmacologiaRESUMO
Sponges (Porifera) contain many peptide-specialized metabolites with potent biological activities and significant roles in shaping marine ecology. It is well established that symbiotic bacteria produce bioactive "sponge" peptides, both on the ribosome (RiPPs) and nonribosomally. Here, we demonstrate that sponges themselves also produce many bioactive macrocyclic peptides, such as phakellistatins and related proline-rich macrocyclic peptides (PRMPs). Using the Stylissa carteri sponge transcriptome, methods were developed to find sequences encoding 46 distinct RiPP-type core peptides, of which ten encoded previously identified PRMP sequences. With this basis set, the genome and transcriptome of the sponge Axinella corrugata was interrogated to find 35 PRMP precursor peptides encoding 31 unique core peptide sequences. At least 11 of these produced cyclic peptides that were present in the sponge and could be characterized by mass spectrometry, including stylissamides A-D and seven previously undescribed compounds. Precursor peptides were encoded in the A. corrugata genome, confirming their animal origin. The peptides contained signal peptide sequences and highly repetitive recognition sequence-core peptide elements with up to 25 PRMP copies in a single precursor. In comparison to sponges without PRMPs, PRMP sponges are incredibly enriched in potentially secreted polypeptides, with >23,000 individual signal peptide encoding genes found in a single transcriptome. The similarities between PRMP biosynthetic genes and neuropeptides in terms of their biosynthetic logic suggest a fundamental biology linked to circular peptides, possibly indicating a widespread and underappreciated diversity of signaling peptide post-translational modifications across the animal kingdom.
Assuntos
Peptídeos Cíclicos , Peptídeos , Animais , Peptídeos/genética , Peptídeos Cíclicos/genética , Sequência de Aminoácidos , Bandagens , Sinais Direcionadores de ProteínasRESUMO
Cyclic peptides offer a range of notable advantages, including potent antibacterial properties, high binding affinity and specificity to target molecules, and minimal toxicity, making them highly promising candidates for drug development. However, a comprehensive database that consolidates both synthetically derived and naturally occurring cyclic peptides is conspicuously absent. To address this void, we introduce CyclicPepedia (https://www.biosino.org/iMAC/cyclicpepedia/), a pioneering database that encompasses 8744 known cyclic peptides. This repository, structured as a composite knowledge network, offers a wealth of information encompassing various aspects of cyclic peptides, such as cyclic peptides' sources, categorizations, structural characteristics, pharmacokinetic profiles, physicochemical properties, patented drug applications, and a collection of crucial publications. Supported by a user-friendly knowledge retrieval system and calculation tools specifically designed for cyclic peptides, CyclicPepedia will be able to facilitate advancements in cyclic peptide drug development.
Assuntos
Bases de Conhecimento , Peptídeos Cíclicos , Peptídeos Cíclicos/química , Bases de Dados de ProteínasRESUMO
Cyclic peptides are versatile therapeutic agents that boast high binding affinity, minimal toxicity, and the potential to engage challenging protein targets. However, the pharmaceutical utility of cyclic peptides is limited by their low membrane permeability-an essential indicator of oral bioavailability and intracellular targeting. Current machine learning-based models of cyclic peptide permeability show variable performance owing to the limitations of experimental data. Furthermore, these methods use features derived from the whole molecule that have traditionally been used to predict small molecules and ignore the unique structural properties of cyclic peptides. This study presents CycPeptMP: an accurate and efficient method to predict cyclic peptide membrane permeability. We designed features for cyclic peptides at the atom-, monomer-, and peptide-levels and seamlessly integrated these into a fusion model using deep learning technology. Additionally, we applied various data augmentation techniques to enhance model training efficiency using the latest data. The fusion model exhibited excellent prediction performance for the logarithm of permeability, with a mean absolute error of $0.355$ and correlation coefficient of $0.883$. Ablation studies demonstrated that all feature levels contributed and were relatively essential to predicting membrane permeability, confirming the effectiveness of augmentation to improve prediction accuracy. A comparison with a molecular dynamics-based method showed that CycPeptMP accurately predicted peptide permeability, which is otherwise difficult to predict using simulations.
Assuntos
Permeabilidade da Membrana Celular , Peptídeos Cíclicos , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Aprendizado de Máquina , Aprendizado Profundo , Biologia Computacional/métodosRESUMO
Adenosine triphosphate-binding cassette (ABC) transporters, such as multidrug resistance protein 1 (MRP1), protect against cellular toxicity by exporting xenobiotic compounds across the plasma membrane. However, constitutive MRP1 function hinders drug delivery across the blood-brain barrier, and MRP1 overexpression in certain cancers leads to acquired multidrug resistance and chemotherapy failure. Small-molecule inhibitors have the potential to block substrate transport, but few show specificity for MRP1. Here we identify a macrocyclic peptide, named CPI1, which inhibits MRP1 with nanomolar potency but shows minimal inhibition of a related multidrug transporter P-glycoprotein. A cryoelectron microscopy (cryo-EM) structure at 3.27 Å resolution shows that CPI1 binds MRP1 at the same location as the physiological substrate leukotriene C4 (LTC4). Residues that interact with both ligands contain large, flexible sidechains that can form a variety of interactions, revealing how MRP1 recognizes multiple structurally unrelated molecules. CPI1 binding prevents the conformational changes necessary for adenosine triphosphate (ATP) hydrolysis and substrate transport, suggesting it may have potential as a therapeutic candidate.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Trifosfato de Adenosina/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Transporte Biológico , Microscopia Crioeletrônica , Leucotrieno C4/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Peptídeos/metabolismo , Peptídeos Cíclicos/farmacologiaRESUMO
Cyclotides are plant-derived disulfide-rich cyclic peptides that have a natural function in plant defense and potential for use as agricultural pesticides. Because of their highly constrained topology, they are highly resistant to thermal, chemical, or enzymatic degradation. However, the stability of cyclotides at alkaline pH for incubation times of longer than a few days is poorly studied but important since these conditions could be encountered in the environment, during storage or field application as insecticides. In this study, kalata B1 (kB1), the prototypical cyclotide, was engineered to improve its long-term stability and retain its insecticidal activity via point mutations. We found that substituting either Asn29 or Gly1 to lysine or leucine increased the stability of kB1 by twofold when incubated in an alkaline buffer (pH = 9.0) for 7 days, while retaining its insecticidal activity. In addition, when Gly1 was replaced with lysine or leucine, the mutants could be cyclized using an asparaginyl endopeptidase, in vitro with a yield of â¼90% within 5 min. These results demonstrate the potential to manufacture kB1 mutants with increased stability and insecticidal activity recombinantly or in planta. Overall, the discovery of mutants of kB1 that have enhanced stability could be useful in leading to longer term activity in the field as bioinsecticides.
Assuntos
Ciclotídeos , Inseticidas , Oldenlandia , Ciclotídeos/genética , Ciclotídeos/farmacologia , Ciclotídeos/química , Inseticidas/química , Inseticidas/farmacologia , Leucina , Lisina/genética , Mutagênese , Proteínas de Plantas/metabolismo , Oldenlandia/química , Estabilidade Proteica , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacosRESUMO
Tris-(2-carboxyethyl)phosphine (TCEP) linked to agarose beads is widely used for reducing disulfide bridges in proteins and peptides. The immobilization of TCEP on beads allows efficient removal after reduction to prevent its reaction with alkylating reagents and thus interference with conjugation reactions. However, a limitation of agarose TCEP is its relatively low reduction capacity per milliliter of wet beads (about 15â µmol/ml), making it unsuitable for the reduction of disulfides from molecules at millimolar concentrations. In this work, we tested the immobilization of TCEP to a range of different solid supports and found that conjugation to silica gel offers TCEP beads with about 8-fold higher reduction capacity (129±16â µmol/ml wet beads). We show that it allows reducing disulfide-cyclized peptides at millimolar concentrations for subsequent cyclization by bis-electrophile linker reagents. Given the substantially higher reduction capacity, the robust performance in different solvents, the low cost of the silica gel, and the ease of functionalization with TCEP, the silica gel-TCEP is suited for reducing disulfide bridges in essentially any peptide and is particularly useful for reducing peptides at higher concentrations.
Assuntos
Fosfinas , Dióxido de Silício , Compostos de Sulfidrila , Sefarose , Sílica Gel , Peptídeos/química , Indicadores e Reagentes , Alquilação , Dissulfetos/química , OxirreduçãoRESUMO
Peptide therapeutics have gained great interest due to their multiple advantages over small molecule and antibody-based drugs. Peptide drugs are easier to synthesize, have the potential for oral bioavailability, and are large enough to target protein-protein interactions that are undruggable by small molecules. However, two major limitations have made it difficult to develop novel peptide therapeutics not derived from natural products, including the metabolic instability of peptides and the difficulty of reaching antibody-like potencies and specificities. Compared to linear and disulfide-monocyclized peptides, multicyclic peptides can provide increased conformational rigidity, enhanced metabolic stability, and higher potency in inhibiting protein-protein interactions. The identification of novel multicyclic peptide binders can be difficult, however, recent advancements in the construction of multicyclic phage libraries have greatly advanced the process of identifying novel multicyclic peptide binders for therapeutically relevant protein targets. This review will describe the current approaches used to create multicyclic peptide libraries, highlighting the novel chemistries developed and the proof-of-concept work done on validating these libraries against different protein targets.
Assuntos
Biblioteca de Peptídeos , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Humanos , Peptídeos/química , Peptídeos/metabolismoRESUMO
BACKGROUND: The aim of the present study was to develop a novel 64Cu-labeled cyclic peptide ([64Cu]Cu-FAP-NOX) that targets fibroblast activation protein (FAP) and may offer advantages in terms of image contrast, imaging time window, and low uptake in normal tissues. METHODS: The novel cyclic peptide featuring with a N-oxalyl modified tail was constructed and conjugated to NOTA for 64Cu labeling. Biochemical and cellular assays were performed with A549.hFAP cells. The performance of [64Cu]Cu-FAP-NOX was compared to that of two established tracers ([64Cu]Cu-FAPI-04 and [68Ga]Ga-FAP-2286) and three different NOTA-conjugates in HEK-293T.hFAP xenograft mice using micro-PET imaging. Ex vivo biodistribution studies were performed to confirm the FAP specificity and to validate the PET data. Furthermore, a first-in-human study of this novel tracer was conducted on one patient with lung cancer. RESULTS: Compared to [64Cu]Cu-FAPI-04, [64Cu]Cu-FAP-NOX demonstrated faster and higher rates of cellular uptake and internalization in A549.hFAP cells, but lower rates of cellular efflux. All six radiotracers were rapidly taken up by the tumor within the first 4 h post-injection. However, [64Cu]Cu-FAP-NOX had more intense tumor accumulation and slower washout from the target. The ratios of the tumor to normal tissue (including kidneys and muscles) increased significantly over time, with [64Cu]Cu-FAP-NOX reaching the highest ratio among all tracers. In the patient, [64Cu]Cu-FAP-NOX PET showed a comparable result to FDG PET in the primary malignant lesion while exhibiting higher uptake in pleural metastases, consistent with elevated FAP expression as confirmed by immunohistochemistry. CONCLUSION: [64Cu]Cu-FAP-NOX is a promising FAP-targeted tracer with a highly flexible imaging time window, as evidenced by preclinical evaluation encompassing biodistribution and micro-PET studies, along with a successful patient application. Furthermore, [64Cu]Cu-FAP-NOX showed enhanced image contrast and favorable pharmacokinetic properties for FAP PET imaging, warranting translation into large cohort studies.
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Cyclisation of peptides by forming thioether (lanthionine), disulfide (cystine) or methylene thioacetal bridges between side chains is established as an important tool to stabilise a given structure, enhance metabolic stability and optimise both potency and selectivity. However, a systematic comparative study of the effects of differing bridging modalities on peptide conformation has not previously been carried out. In this paper, we have used the NMR deconvolution algorithm, NAMFIS, to determine the conformational ensembles, in aqueous solution, of three cyclic analogues of angiotensin(1-7), incorporating either disulfide, or non-reducible thioether or methylene thioacetal bridges. We demonstrate that the major solution conformations are conserved between the different bridged peptides, but the distribution of conformations differs appreciably. This suggests that subtle differences in ring size and bridging structure can be exploited to fine-tune the conformational properties of cyclic peptides, which may modulate their bioactivities.
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The mesenchymal-epithelial transition factor (c-Met) is a receptor tyrosine kinase linked to the proliferation, survival, invasion, and metastasis of several types of cancers, including colorectal cancer (CRC), particularly when aberrantly activated. Our study strategically designs peptides derived from interactions between c-Met and the antibody Onartuzumab. By utilizing a cyclic strategy, we achieved significantly enhanced peptide stability and affinity. Our in vitro assessments confirmed that the cyclic peptide HYNIC-cycOn exhibited a higher affinity (KD = 83.5 nM) and greater specificity compared with its linear counterpart. Through in vivo experiments, [99mTc]Tc-HYNIC-cycOn displayed exceptional tumor-targeting capabilities and minimal absorption in nontumor cells, as confirmed by single-photon emission computed tomography. Notably, the ratios of tumor to muscle and tumor to intestine, 1 h postinjection, were 4.78 ± 0.86 and 3.24 ± 0.47, respectively. Comparable ratios were observed in orthotopic CRC models, recording 4.94 ± 0.32 and 3.88 ± 0.41, respectively. In summary, [99mTc]Tc-HYNIC-cycOn shows substantial promise as a candidate for clinical applications. We show that [99mTc]Tc-HYNIC-cycOn can effectively target and visualize c-Met-expressing tumors in vivo, providing a promising approach for enhancing diagnostic accuracy when detecting c-Met in CRC.
Assuntos
Neoplasias Colorretais , Peptídeos Cíclicos , Proteínas Proto-Oncogênicas c-met , Neoplasias Colorretais/diagnóstico por imagem , Proteínas Proto-Oncogênicas c-met/metabolismo , Peptídeos Cíclicos/química , Humanos , Animais , Camundongos , Linhagem Celular Tumoral , Camundongos Nus , Tomografia Computadorizada de Emissão de Fóton Único/métodos , Camundongos Endogâmicos BALB C , Feminino , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Peptides are mid-size molecules (700-2000 g/mol) and have attracted particular interest as therapeutic modalities as they are superior in controlling protein-protein interactions, a process that is a typical drug target category, compared with small molecules (<500 g/mol). In 2020, we identified KS-58 (1333 g/mol) as a K-Ras(G12D)-inhibitory bicyclic peptide and suggested its cell membrane permeability. However, the membrane permeability mechanism had not been elucidated. In this study, we aim to clarify the mechanism by molecular dynamics (MD) simulations. Initially, we simulated the molecular conformations of KS-58 in water (a polar solvent) and in chloroform (a non-polar solvent). The identified stable conformations were significantly different in each solvent. KS-58 behaves as a chameleon-like molecule as it alters its polar surface area (PSA) depending on the solvent environment. It was also discovered that orientation of Asp's side chain is a critical energy barrier for KS-58 altering its conformation from hydrophilic to lipophilic. Taking these properties into consideration, we simulated its lipid bilayer membrane permeability. KS-58 shifted toward the inside of the lipid bilayer membrane with altering its conformations to lipophilic. When the simulation condition was set in deionized form of that carboxy group of Asp, KS-58 traveled deeper inside the cell membrane. PSA and the depth of the membrane penetration correlated. In vitro data suggested that cell membrane permeability of KS-58 is improved in weakly acidic conditions leading to partial deionization of the carboxy group. Our data provide an example of the molecular properties of mid-size peptides with membrane accessibility and propose an effective metadynamics approach to elucidate such molecular mechanisms by MD simulations.
Assuntos
Bicamadas Lipídicas , Simulação de Dinâmica Molecular , Peptídeos Cíclicos , Bicamadas Lipídicas/química , Peptídeos/química , Solventes/química , PermeabilidadeRESUMO
Near-infrared photoimmunotherapy (NIR-PIT) is a new cancer treatment that involves photoimmunotherapy drug injection and NIR light exposure. In NIR-PIT, antibodies are commonly used as target-directed molecules carrying IRDye700DX (IR700). However, antibodies have disadvantages, such as high cost, complex development strategies, and poor tumor penetration. In contrast, peptides have lower production costs, can be easy to chemically synthesize and modify, and can also be used for tumor-targeting like antibodies. In this study, we developed a novel PIT drug using a peptide as the target-directed molecule. Epidermal growth factor receptor (EGFR) was selected as the target, and monovalent and bivalent EGFR-binding peptides were synthesized. The bivalent peptide showed sufficient binding to EGFR-positive cells, and a bivalent peptide-IR700 conjugate with a long linker induced morphological changes in EGFR-positive cells. Additionally, the drug significantly reduced cell viability in vitro in an NIR light-dose- and drug-concentration-dependent manner. These results indicate the feasibility of NIR-PIT in treating cancer using peptide-based drugs.
Assuntos
Sobrevivência Celular , Receptores ErbB , Imunoterapia , Raios Infravermelhos , Peptídeos , Fototerapia , Receptores ErbB/metabolismo , Receptores ErbB/antagonistas & inibidores , Humanos , Peptídeos/química , Peptídeos/farmacologia , Peptídeos/síntese química , Sobrevivência Celular/efeitos dos fármacos , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Ensaios de Seleção de Medicamentos Antitumorais , Proliferação de Células/efeitos dos fármacos , Estrutura Molecular , Relação Dose-Resposta a Droga , Relação Estrutura-Atividade , Linhagem Celular Tumoral , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/síntese químicaRESUMO
Cyclic peptides are attracting attention as therapeutic agents due to their potential for oral absorption and easy access to tough intracellular targets. LUNA18, a clinical KRAS inhibitor, was transformed-without scaffold hopping-from the initial hit by using an mRNA display library that met our criteria for drug-likeness. In drug discovery using mRNA display libraries, hit compounds always possess a site linked to an mRNA tag. Here, we describe our examination of the Structure-Activity Relationship (SAR) using X-ray structures for chemical optimization near the site linked to the mRNA tag, equivalent to the C-terminus. Structural modifications near the C-terminus demonstrated a relatively wide range of tolerance for side chains. Furthermore, we show that a single atom modification is enough to change the pharmacokinetic (PK) profile. Since there are four positions where side chain modification is permissible in terms of activity, it is possible to flexibly adjust the pharmacokinetic profile by structurally optimizing the side chain. The side chain transformation findings demonstrated here may be generally applicable to hits obtained from mRNA display libraries.
Assuntos
Peptídeos Cíclicos , Proteínas Proto-Oncogênicas p21(ras) , RNA Mensageiro , Relação Estrutura-Atividade , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/farmacocinética , Humanos , RNA Mensageiro/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Estrutura Molecular , Animais , Relação Dose-Resposta a DrogaRESUMO
The non-POU domain-containing octamer-binding protein (NONO) is a nucleic acid-binding protein with diverse functions that has been identified as a potential cancer target in cell biology studies. Little is known about structural motifs that mediate binding to NONO apart from its ability to form homodimers, as well as heterodimers and oligomers with related homologues. We report a stapling approach to macrocyclise helical peptides derived from the insulin-like growth factor binding protein (IGFBP-3) that NONO interacts with, and also from the dimerisation domain of NONO itself. Using a range of chemistries including Pd-catalysed cross-coupling, cysteine arylation and cysteine alkylation, we successfully improved the helicity and observed modest peptide binding to the NONO dimer, although binding could not be saturated at micromolar concentrations. Unexpectedly, we observed cell permeability and preferential nuclear localisation of various dye-labelled peptides in live confocal microscopy, indicating the potential for developing peptide-based tools to study NONO in a cellular context.
Assuntos
Proteínas de Ligação a DNA , Proteínas de Ligação a RNA , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Cisteína , Peptídeos/metabolismo , PermeabilidadeRESUMO
Fungal biosynthetic gene clusters often include genes encoding transmembrane proteins, which have been mostly thought to be transporters exporting the products. However, there is little knowledge about subcellular compartmentalization of transmembrane proteins essential for biosynthesis. Fungal mycotoxin cyclochlorotine is synthesized by non-ribosomal peptide synthetase, which is followed by modifications with three transmembrane UstYa-family proteins. Heterologous expression in Aspergillus oryzae revealed that total biosynthesis of cyclochlorotine requires additional two transporter proteins. Here, we investigated subcellular localizations of the five transmembrane proteins under heterologous expression in A. oryzae. Enhanced green fluorescent protein (EGFP) fusions to the transmembrane proteins, which were confirmed to normally function in cyclochlorotine production, were expressed together with organellar markers. All the transmembrane proteins exhibited localizations commonly in line of the trans-Golgi, endosomes, and vacuoles. This study suggests that subcellular compartmentalization of UstYa family proteins and transporters allows corporative functions of delivering intermediates and subsequent modifications, completing cyclochlorotine biosynthesis.
RESUMO
The SPRY domain-containing SOCS box proteins SPSB1, SPSB2, and SPSB4 utilize their SPRY/B30.2 domain to interact with a short region in the N-terminus of inducible nitric oxide synthase (iNOS), and recruit an E3 ubiquitin ligase complex to polyubiquitinate iNOS, resulting in the proteasomal degradation of iNOS. Inhibitors that can disrupt the endogenous SPSB-iNOS interactions could be used to augment cellular NO production, and may have antimicrobial and anticancer activities. We previously reported the rational design of a cyclic peptide inhibitor, cR8, cyclo(RGDINNNV), which bound to SPSB2 with moderate affinity. We, therefore, sought to develop SPSB inhibitors with higher affinity. Here, we show that cyclic peptides cR7, cyclo(RGDINNN), and cR9, cyclo(RGDINNNVE), have ~6.5-fold and ~2-fold, respectively, higher SPSB2-bindng affinities than cR8. We determined high-resolution crystal structures of the SPSB2-cR7 and SPSB2-cR9 complexes, which enabled a good understanding of the structure-activity relationships for these cyclic peptide inhibitors. Moreover, we show that these cyclic peptides displace full-length iNOS from SPSB2, SPSB1, and SPSB4, and that their inhibitory potencies correlate well with their SPSB2-binding affinities. The strongest inhibition was observed for cR7 against all three iNOS-binding SPSB proteins.
Assuntos
Peptídeos Cíclicos , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Humanos , Proteínas Supressoras da Sinalização de Citocina/química , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo II/química , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
Kappa opioid receptor (KOR) antagonists have potential therapeutic applications in the treatment of stress-induced relapse to substance abuse and mood disorders. The dynorphin A analog arodyn (Ac[Phe1,2,3,Arg4,D-Ala8]dynorphin A-(1-11)-NH2) exhibits potent and selective kappa opioid receptor antagonism. Multiple cyclizations in longer peptides, such as dynorphin and its analogs, can extend the conformational constraint to additional regions of the peptide beyond what is typically constrained by a single cyclization. Here, we report the design, synthesis, and pharmacological evaluation of a bicyclic arodyn analog with two constraints in the opioid peptide sequence. The peptide, designed based on structure-activity relationships of monocyclic arodyn analogs, was synthesized by solid-phase peptide synthesis and cyclized by sequential ring-closing metathesis (RCM) in the C- and N-terminal sequences. Molecular modeling studies suggest similar interactions of key aromatic and basic residues in the bicyclic peptide with KOR as found in the cryoEM structure of KOR-bound dynorphin, despite substantial differences in the backbone conformations of the two peptides. The bicyclic peptide's affinities at KOR and mu opioid receptors (MOR) were determined in radioligand binding assays, and its KOR antagonism was determined in the [35S]GTPγS assay in KOR-expressing cells. The bicyclic analog retains KOR affinity and selectivity (Ki = 26 nM, 97-fold selectivity over MOR) similar to arodyn and exhibits potent KOR antagonism in the dynorphin-stimulated [35S]GTPγS assay. This bicyclic peptide represents a promising advance in preparing cyclic opioid peptide ligands and opens avenues for the rational design of additional bicyclic opioid peptide analogs.
Assuntos
Dinorfinas , Receptores Opioides kappa , Receptores Opioides kappa/antagonistas & inibidores , Receptores Opioides kappa/metabolismo , Dinorfinas/química , Dinorfinas/farmacologia , Humanos , Animais , Relação Estrutura-Atividade , Modelos Moleculares , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Peptídeos Cíclicos/síntese química , Sequência de AminoácidosRESUMO
We report the discovery of a novel cyclic nonribosomal peptide (NRP), acyl-surugamide A2, from a marine-derived Streptomyces albidoflavus RKJM-0023 (CP133227). The structure of acyl-surugamide A2 was elucidated using a combination of NMR spectroscopy, MS2 fragmentation analysis, and comparative analysis of the sur biosynthetic gene cluster. Acyl-surugamide A2 contains all eight core amino acids of surugamide A, with a modified N-ε-acetyl-L-lysine residue. Our study highlights the potential of marine Streptomyces strains to produce novel natural products with potential therapeutic applications. The structure of cyclic peptides can be solved using MS2 spectra and analysis of their biosynthetic gene clusters.
Assuntos
Lisina , Streptomyces , Aminoácidos , Peptídeos Cíclicos , Streptomyces/genéticaRESUMO
Cyclic peptides are fascinating molecules abundantly found in nature and exploited as molecular format for drug development as well as other applications, ranging from research tools to food additives. Advances in peptide technologies made over many years through improved methods for synthesis and drug development have resulted in a steady stream of new drugs, with an average of around one cyclic peptide drug approved per year. Powerful technologies for screening random peptide libraries, and de novo generating ligands, have enabled the development of cyclic peptide drugs independent of naturally derived molecules and now offer virtually unlimited development opportunities. In this review, we feature therapeutically relevant cyclic peptides derived from nature and discuss the unique properties of cyclic peptides, the enormous technological advances in peptide ligand development in recent years, and current challenges and opportunities for developing cyclic peptides that address unmet medical needs.