RESUMEN
Peptides are valuable for therapeutic development, with multicyclic peptides showing promise in mimicking antigen-binding potency of antibodies. However, our capability to engineer multicyclic peptide scaffolds, particularly for the construction of large combinatorial libraries, is still limited. Here, we study the interplay of disulfide pairing between three biscysteine motifs, and designed a range of triscysteine motifs with unique disulfide-directing capability for regulating the oxidative folding of multicyclic peptides. We demonstrate that incorporating these motifs into random sequences allows the design of disulfide-directed multicyclic peptide (DDMP) libraries with up to four disulfide bonds, which have been applied for the successful discovery of peptide binders with nanomolar affinity to several challenging targets. This study encourages the use of more diverse disulfide-directing motifs for creating multicyclic peptide libraries and opens an avenue for discovering functional peptides in sequence and structural space beyond existing peptide scaffolds, potentially advancing the field of peptide drug discovery.
Asunto(s)
Cisteína , Disulfuros , Biblioteca de Péptidos , Disulfuros/química , Cisteína/química , Secuencias de Aminoácidos , Descubrimiento de Drogas/métodos , Secuencia de Aminoácidos , Péptidos/química , Péptidos/metabolismo , Péptidos Cíclicos/química , Péptidos Cíclicos/metabolismo , Unión Proteica , Humanos , Oxidación-Reducción , Pliegue de ProteínaRESUMEN
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.
Asunto(s)
Permeabilidad de la Membrana Celular , Péptidos Cíclicos , Péptidos Cíclicos/química , Péptidos Cíclicos/metabolismo , Aprendizaje Automático , Aprendizaje Profundo , Biología Computacional/métodosRESUMEN
Cyclization provides a general strategy for improving the proteolytic stability, cell membrane permeability and target binding affinity of peptides. Insertion of a stable, non-reducible linker into a disulphide bond is a commonly used approach for cyclizing phage-displayed peptides. However, among the vast collection of cysteine reactive linkers available, few provide the selectivity required to target specific cysteine residues within the peptide in the phage display system, whilst sparing those on the phage capsid. Here, we report the development of a cyclopropenone-based proximity-driven chemical linker that can efficiently cyclize synthetic peptides and peptides fused to a phage-coat protein, and cyclize phage-displayed peptides in a site-specific manner, with no disruption to phage infectivity. Our cyclization strategy enables the construction of stable, highly diverse phage display libraries. These libraries can be used for the selection of high-affinity cyclic peptide binders, as exemplified through model selections on streptavidin and the therapeutic target αvß3.
Asunto(s)
Biblioteca de Péptidos , Péptidos Cíclicos , Ciclización , Péptidos Cíclicos/química , Péptidos Cíclicos/metabolismo , Estreptavidina/química , Estreptavidina/metabolismo , Humanos , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Proteínas de la Cápside/genética , Cisteína/química , Cisteína/metabolismo , Ciclopropanos/química , Péptidos/química , Péptidos/metabolismoRESUMEN
We designed and synthesized an N-ortho-nitrobenzylated benzanilide-based amino acid having a cis-amide structure that facilitates cyclization of peptides containing it. Photo-induced removal of the nitrobenzyl group from this residue in the resulting cyclized peptides dramatically alters their conformation and passive membrane permeability via complete cis-amide to trans-amide conversion.
Asunto(s)
Aminoácidos , Permeabilidad de la Membrana Celular , Péptidos Cíclicos , Péptidos Cíclicos/química , Péptidos Cíclicos/metabolismo , Aminoácidos/química , Anilidas/química , Nitrobencenos/química , Ciclización , Conformación ProteicaRESUMEN
Sickle cell disease (SCD) is a monogenic blood disorder associated with a mutation in the hemoglobin subunit ß gene encoding for the ß-globin of normal adult hemoglobin (HbA). This mutation transcribes into a Glu-ß6 â Val-ß6 substitution in the ß-globins, inducing the polymerization of this hemoglobin form (HbS) when in the T-state. Despite advances in stem cell and gene therapy, and the recent approval of a new antisickling drug, therapeutic limitations persist. Herein, we demonstrate through molecular dynamics and umbrella sampling, that (unrestrained) blockage of the hydrophobic pocket involved in the lateral contact of the HbS fibers by 5-mer cyclic peptides, recently proposed as SCD aggregation inhibitors (Neto, V.; J. Med. Chem. 2023, 66, 16062-16074), is enough to turn the dimerization of HbS thermodynamically unfavorable. Among these potential drugs, some exhibit an estimated pocket abandonment probability of around 15-20% within the simulations' time frame, and an impressive specificity toward the mutated Val-ß6. Additionally, we show that the dimerization can be thermodynamically unfavored by blocking a nearby region while the pocket remains vacant. These results are compared with curcumin, an antisickling molecule and a pan-assay interference compound, with a good binding affinity for different proteins and protein domains. Our results confirm the potential of some of these cyclic peptides as antisickling drug candidates to reduce the concentration of aggregation-competent HbS.
Asunto(s)
Hemoglobina Falciforme , Simulación de Dinámica Molecular , Péptidos Cíclicos , Hemoglobina Falciforme/química , Hemoglobina Falciforme/metabolismo , Humanos , Péptidos Cíclicos/química , Péptidos Cíclicos/metabolismo , Péptidos Cíclicos/farmacología , Agregado de Proteínas/efectos de los fármacos , Anemia de Células Falciformes/tratamiento farmacológico , Anemia de Células Falciformes/metabolismo , TermodinámicaRESUMEN
Microbial plant pathogens deploy amphipathic cyclic lipopeptides to reduce surface tension in their environment. While plants can detect these molecules to activate cellular stress responses, the role of these lipopeptides or associated host responses in pathogenesis are not fully clear. The gramillin cyclic lipopeptide is produced by the Fusarium graminearum fungus and is a virulence factor and toxin in maize. Here, we show that gramillin promotes virulence and necrosis in both monocots and dicots by disrupting ion balance across membranes. Gramillin is a cation-conducting ionophore and causes plasma membrane depolarization. This disruption triggers cellular signaling, including a burst of reactive oxygen species (ROS), transcriptional reprogramming, and callose production. Gramillin-induced ROS depends on expression of host ILK1 and RBOHD genes, which promote fungal induction of virulence genes during infection and host susceptibility. We conclude that gramillin's ionophore activity targets plant membranes to coordinate attack by the F. graminearum fungus.
Asunto(s)
Membrana Celular , Fusarium , Lipopéptidos , Enfermedades de las Plantas , Fusarium/patogenicidad , Fusarium/metabolismo , Lipopéptidos/farmacología , Lipopéptidos/metabolismo , Virulencia , Membrana Celular/metabolismo , Enfermedades de las Plantas/microbiología , Péptidos Cíclicos/farmacología , Péptidos Cíclicos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Zea mays/microbiologíaRESUMEN
Peptide-based therapeutics hold immense promise for the treatment of various diseases. However, their effectiveness is often hampered by poor cell membrane permeability, hindering targeted intracellular delivery and oral drug development. This study addressed this challenge by introducing a novel graph neural network (GNN) framework and advanced machine learning algorithms to build predictive models for peptide permeability. Our models offer systematic evaluation across diverse peptides (natural, modified, linear and cyclic) and cell lines [Caco-2, Ralph Russ canine kidney (RRCK) and parallel artificial membrane permeability assay (PAMPA)]. The predictive models for linear and cyclic peptides in Caco-2 and RRCK cell lines were constructed for the first time, with an impressive coefficient of determination (R2) of 0.708, 0.484, 0.553, and 0.528 in the test set, respectively. Notably, the GNN framework behaved better in permeability prediction with larger data sets and improved the accuracy of cyclic peptide prediction in the PAMPA cell line. The R2 increased by about 0.32 compared with the reported models. Furthermore, the important molecular structural features that contribute to good permeability were interpreted; the influence of cell lines, peptide modification, and cyclization on permeability were successfully revealed. To facilitate broader use, we deployed these models on the user-friendly KNIME platform (https://github.com/ifyoungnet/PharmPapp). This work provides a rapid and reliable strategy for systematically assessing peptide permeability, aiding researchers in drug delivery optimization, peptide preselection during drug discovery, and potentially the design of targeted peptide-based materials.
Asunto(s)
Permeabilidad de la Membrana Celular , Células CACO-2 , Perros , Humanos , Animales , Péptidos Cíclicos/metabolismo , Péptidos Cíclicos/química , Aprendizaje Automático , Redes Neurales de la Computación , Péptidos/química , Péptidos/metabolismo , Permeabilidad , Línea Celular , Membranas Artificiales , AlgoritmosRESUMEN
Viscosin is a membrane-permeabilizing, cyclic lipopeptide (CLiP) produced by Pseudomonas species. Here, we have studied four synthetic analogs (L1W, V4W, L5W, and L7W), each with one leucine (Leu; L) or valine residue exchanged for tryptophan (Trp; W) by means of time-resolved fluorescence spectroscopy of Trp. To this end, we recorded the average fluorescence lifetime, rotational correlation time and limiting anisotropy, dipolar relaxation time and limiting extent of relaxation, rate constant of acrylamide quenching, effect of H2O-D2O exchange, and time-resolved half-width of the spectrum in the absence and presence of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) liposomes. Structure, localization, and hydration of the peptides were described by molecular dynamics simulations. The combination of the parameters provides a good description of the molecular environments of the Trp positions and the behavior of viscosin as a whole. Of particular value for characterizing the impact of viscosin on the membrane is the dipolar relaxation of Trp4 in V4W, which is deeply embedded in the hydrophobic core. The limiting relaxation level represents the membrane perturbation-unlike typical membrane probes-at the site of the perturbant. Fractions of Trp4 relax at different rates; the one not in contact with water upon excitation relaxes via recruitment of a water molecule on the 10-ns timescale. This rate is sensitive to the concerted membrane perturbation by more than one lipopeptide, which appears at high lipopeptide concentration and is assumed a prerequisite for the final formation of a membrane-permeabilizing defect. Trp7 relaxes primarily with respect to neighboring Ser residues. Trp5 flips between a membrane-inserted and surface-exposed orientation.
Asunto(s)
Simulación de Dinámica Molecular , Espectrometría de Fluorescencia , Triptófano , Triptófano/química , Péptidos Cíclicos/química , Péptidos Cíclicos/metabolismo , Fosfatidilcolinas/química , Lipopéptidos/química , Lipopéptidos/metabolismo , Membrana Celular/metabolismo , Membrana Celular/química , Factores de Tiempo , Liposomas/química , Liposomas/metabolismoRESUMEN
Cyclotides and lanthipeptides are cyclic peptide natural products with promising bioengineering potential. No peptides have been isolated that contain both structural motifs defining these two families, an N-to-C cyclised backbone and lanthionine linkages. We combined their biosynthetic machineries to produce hybrid structures that possess improved activity or stability, demonstrate how the AEP-1 plant cyclase can be utilised to complete the maturation of the sactipeptide subtilosin A, and present head-to-tail cyclisation of the glycocin sublancin. These studies show the plasticity of AEP-1 and its utilisation alongside other post-translational modifications.
Asunto(s)
Ciclotidas , Ciclotidas/química , Ciclotidas/metabolismo , Péptidos Cíclicos/química , Péptidos Cíclicos/metabolismo , CiclizaciónRESUMEN
Nonproteinogenic amino acids, including d-α-, ß-, and γ-amino acids, present in bioactive peptides play pivotal roles in their biochemical activities and proteolytic stabilities. d-α-Amino acids (dαAA) are widely used building blocks that can enhance the proteolytic stability. Cyclic ß2,3-amino acids (cßAA), for instance, can fold peptides into rigid secondary structures, improving the binding affinity and proteolytic stability. Cyclic γ2,4-amino acids (cγAA) are recently highlighted as rigid residues capable of preventing the proteolysis of flanking residues. Simultaneous incorporation of all dαAA, cßAA, and cγAA into a peptide is expected to yield l-α/d-α/ß/γ-hybrid peptides with improved stability and potency. Despite challenges in the ribosomal incorporation of multiple nonproteinogenic amino acids, our engineered tRNAPro1E2 successfully reaches such a difficulty. Here, we report the ribosomal synthesis of macrocyclic l-α/d-α/ß/γ-hybrid peptide libraries and their application to in vitro selection against interferon gamma receptor 1 (IFNGR1). One of the resulting l-α/d-α/ß/γ-hybrid peptides, IB1, exhibited remarkable inhibitory activity against the IFN-γ/IFNGR1 protein-protein interaction (PPI) (IC50 = 12 nM), primarily attributed to the presence of a cßAA in the sequence. Additionally, cγAAs and dαAAs in the resulting peptides contributed to their serum stability. Furthermore, our peptides effectively inhibit IFN-γ/IFNGR1 PPI at the cellular level (best IC50 = 0.75 µM). Altogether, our platform expands the chemical space available for exploring peptides with high activity and stability, thereby enhancing their potential for drug discovery.
Asunto(s)
Receptor de Interferón gamma , Interferón gamma , Receptores de Interferón , Interferón gamma/metabolismo , Receptores de Interferón/metabolismo , Receptores de Interferón/química , Humanos , Unión Proteica , Compuestos Macrocíclicos/química , Compuestos Macrocíclicos/farmacología , Péptidos/química , Péptidos/farmacología , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Péptidos Cíclicos/metabolismoRESUMEN
Prenylation of peptides is widely observed in the secondary metabolites of diverse organisms, granting peptides unique chemical properties distinct from proteinogenic amino acids. Discovery of prenylated peptide agents has largely relied on isolation or genome mining of naturally occurring molecules. To devise a platform technology for de novo discovery of artificial prenylated peptides targeting a protein of choice, here we have integrated the thioether-macrocyclic peptide (teMP) library construction/selection technology, so-called RaPID (Random nonstandard Peptides Integrated Discovery) system, with a Trp-C3-prenyltransferase KgpF involved in the biosynthesis of a prenylated natural product. This unique enzyme exhibited remarkably broad substrate tolerance, capable of modifying various Trp-containing teMPs to install a prenylated residue with tricyclic constrained structure. We constructed a vast library of prenylated teMPs and subjected it to in vitro selection against a phosphoglycerate mutase. This selection platform has led to the identification of a pseudo-natural prenylated teMP inhibiting the target enzyme with an IC50 of 30â nM. Importantly, the prenylation was essential for the inhibitory activity, enhanced serum stability, and cellular uptake of the peptide, highlighting the benefits of peptide prenylation. This work showcases the de novo discovery platform for pseudo-natural prenylated peptides, which is readily applicable to other drug targets.
Asunto(s)
Prenilación , Ligandos , Humanos , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Péptidos Cíclicos/metabolismo , Péptidos/química , Péptidos/metabolismo , Péptidos/farmacología , Compuestos Macrocíclicos/química , Compuestos Macrocíclicos/farmacología , Compuestos Macrocíclicos/metabolismo , Productos Biológicos/química , Productos Biológicos/farmacología , Productos Biológicos/metabolismo , Dimetilaliltranstransferasa/metabolismo , Dimetilaliltranstransferasa/química , Dimetilaliltranstransferasa/antagonistas & inhibidores , Prenilación de ProteínaRESUMEN
Peptide-bile acid hybrids offer promising drug candidates due to enhanced pharmacological properties, such as improved protease resistance and oral bioavailability. However, it remains unknown whether bile acids can be incorporated into peptide chains by the ribosome to produce a peptide-bile acid hybrid macrocyclic peptide library for target-based de novo screening. In this study, we achieved the ribosomal incorporation of lithocholic acid (LCA)-d-tyrosine into peptide chains. This led to the construction of a peptide-LCA hybrid macrocyclic peptide library, which enabled the identification of peptides TP-2C-4L3 (targeting Trop2) and EP-2C-4L5 (targeting EphA2) with strong binding affinities. Notably, LCA was found to directly participate in binding to EphA2 and confer on the peptides improved stability and resistance to proteases. Cell staining experiments confirmed the high specificity of the peptides for targeting Trop2 and EphA2. This study highlights the benefits of LCA in peptides and paves the way for de novo discovery of stable peptide-LCA hybrid drugs.
Asunto(s)
Ácido Litocólico , Biblioteca de Péptidos , Péptidos , Ribosomas , Ácido Litocólico/química , Ácido Litocólico/análogos & derivados , Ácido Litocólico/metabolismo , Ribosomas/metabolismo , Humanos , Péptidos/química , Péptidos/metabolismo , Receptor EphA2/metabolismo , Receptor EphA2/química , Descubrimiento de Drogas , Péptidos Cíclicos/química , Péptidos Cíclicos/metabolismoRESUMEN
Cyclic peptides, such as most ribosomally synthesized and post-translationally modified peptides (RiPPs), represent a burgeoning area of interest in therapeutic and biotechnological research because of their conformational constraints and reduced susceptibility to proteolytic degradation compared to their linear counterparts. Herein, an expression system is reported that enables the production of structurally diverse lanthipeptides and derivatives in mammalian cells. Successful targeting of lanthipeptides to the nucleus, the endoplasmic reticulum, and the plasma membrane is demonstrated. In vivo expression and targeting of such peptides in mammalian cells may allow for screening of lanthipeptide-based cyclic peptide inhibitors of native, organelle-specific protein-protein interactions in mammalian systems.
Asunto(s)
Retículo Endoplásmico , Humanos , Retículo Endoplásmico/metabolismo , Péptidos Cíclicos/metabolismo , Núcleo Celular/metabolismo , Membrana Celular/metabolismo , Células HEK293 , Procesamiento Proteico-PostraduccionalRESUMEN
Microorganisms interact with plant roots through colonization of the root surface, i.e., the rhizoplane or the surrounding soil, i.e., the rhizosphere. Beneficial rhizosphere bacteria such as Pseudomonas spp. can promote plant growth and protect against pathogens by producing a range of bioactive compounds, including specialized metabolites like cyclic lipopeptides (CLPs) known for their biosurfactant and antimicrobial activities. However, the role of CLPs in natural soil systems during bacteria-plant interactions is underexplored. Here, Pseudomonas fluorescens SBW25, producing the CLP viscosin, was used to study the impact of viscosin on bacterial root colonization and microbiome assembly in two cultivars of winter wheat (Heerup and Sheriff). We inoculated germinated wheat seeds with SBW25 wild type or a viscosin-deficient mutant and grew the plants in agricultural soil. After 2 weeks, enhanced root colonization of SBW25 wild type compared to the viscosin-deficient mutant was observed, while no differences were observed between wheat cultivars. In contrast, the impact on root-associated microbial community structure was plant-genotype-specific, and SBW25 wild type specifically reduced the relative abundance of an unclassified oomycete and Phytophthora in Sheriff and Heerup, respectively. This study provides new insights into the natural role of viscosin and specifically highlights the importance of viscosin in wheat root colonization under natural soil conditions and in shaping the root microbial communities associated with different wheat cultivars. Furthermore, it pinpoints the significance of microbial microdiversity, plant genotype, and microbe-microbe interactions when studying colonization of plant roots. IMPORTANCE: Understanding parameters governing microbiome assembly on plant roots is critical for successfully exploiting beneficial plant-microbe interactions for improved plant growth under low-input conditions. While it is well-known from in vitro studies that specialized metabolites are important for plant-microbe interactions, e.g., root colonization, studies on the ecological role under natural soil conditions are limited. This might explain the often-low translational power from laboratory testing to field performance of microbial inoculants. Here, we showed that viscosin synthesis potential results in a differential impact on the microbiome assembly dependent on wheat cultivar, unlinked to colonization potential. Overall, our study provides novel insights into factors governing microbial assembly on plant roots, and how this has a derived but differential effect on the bacterial and protist communities.
Asunto(s)
Genotipo , Microbiota , Raíces de Plantas , Pseudomonas fluorescens , Rizosfera , Microbiología del Suelo , Triticum , Triticum/microbiología , Pseudomonas fluorescens/genética , Pseudomonas fluorescens/metabolismo , Raíces de Plantas/microbiología , Microbiota/genética , Suelo/química , Lipopéptidos/metabolismo , Lipopéptidos/genética , Lipopéptidos/farmacología , Péptidos Cíclicos/genética , Péptidos Cíclicos/metabolismoRESUMEN
Somatostatin receptor 5 (SSTR5) is an important G protein-coupled receptor and drug target for neuroendocrine tumors and pituitary disorders. This study presents two high-resolution cryogenicelectron microscope structures of the SSTR5-Gi complexes bound to the cyclic neuropeptide agonists, cortistatin-17 (CST17) and octreotide, with resolutions of 2.7 Å and 2.9 Å, respectively. The structures reveal that binding of these peptides causes rearrangement of a "hydrophobic lock", consisting of residues from transmembrane helices TM3 and TM6. This rearrangement triggers outward movement of TM6, enabling Gαi protein engagement and receptor activation. In addition to hydrophobic interactions, CST17 forms conserved polar contacts similar to somatostatin-14 binding to SSTR2, while further structural and functional analysis shows that extracellular loops differently recognize CST17 and octreotide. These insights elucidate agonist selectivity and activation mechanisms of SSTR5, providing valuable guidance for structure-based drug development targeting this therapeutically relevant receptor.
Asunto(s)
Octreótido , Receptores de Somatostatina , Receptores de Somatostatina/metabolismo , Receptores de Somatostatina/agonistas , Receptores de Somatostatina/química , Humanos , Octreótido/química , Octreótido/farmacología , Octreótido/metabolismo , Neuropéptidos/metabolismo , Neuropéptidos/química , Microscopía por Crioelectrón , Unión Proteica , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Péptidos Cíclicos/metabolismo , Somatostatina/metabolismo , Somatostatina/química , Somatostatina/análogos & derivados , Modelos Moleculares , Células HEK293RESUMEN
Despite recent success in the computational approaches of cyclic peptide design, current studies face challenges in modeling noncanonical amino acids and nonstandard cyclizations due to limited data. To address this challenge, we developed an integrated framework for the tailored design of stapled peptides (SPs) targeting the bromodomain of CREBBP (CREBBP-BrD). We introduce a powerful combination of anchored stapling and hierarchical molecular dynamics to design and optimize SPs by employing the MultiScale integrative conformational dynamics assessment (MSICDA) strategy, which involves an initial virtual screening of over 1.5 million SPs, followed by comprehensive simulations amounting to 154.54 µs across 5418 of instances. The MSICDA method provides a detailed and holistic stability view of peptide-protein interactions, systematically isolated optimized peptides and identified two leading candidates, DA#430 and DA#99409, characterized by their enhanced stability, optimized binding, and high affinity toward the CREBBP-BrD. In cell-free assays, DA#430 and DA#99409 exhibited 2- to 12-fold greater potency than inhibitor SGC-CBP30. Cell studies revealed higher peptide selectivity for cancerous versus normal cells over small molecules. DA#430 combined with (+)-JQ-1 showed promising synergistic effects. Our approach enables the identification of peptides with optimized binding, high affinity, and enhanced stability, leading to more precise and effective cyclic peptide design, thereby establishing MSICDA as a generalizable and transformative tool for uncovering novel targeted drug development in various therapeutic areas.
Asunto(s)
Proteína de Unión a CREB , Simulación de Dinámica Molecular , Proteína de Unión a CREB/química , Proteína de Unión a CREB/metabolismo , Proteína de Unión a CREB/antagonistas & inhibidores , Humanos , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Péptidos Cíclicos/metabolismo , Dominios Proteicos , Conformación Proteica , Péptidos/química , Péptidos/metabolismo , Péptidos/farmacología , Línea Celular Tumoral , Unión ProteicaRESUMEN
The cyclic peptide OS1 (amino acid sequence: CTERMALHNLC), which has a disulfide bond between both termini cysteine residues, inhibits complex formation between the platelet glycoprotein Ibα (GPIbα) and the von Willebrand factor (vWF) by forming a complex with GPIbα. To study the binding mechanism between GPIbα and OS1 and, therefore, the inhibition mechanism of the protein-protein GPIbα-vWF complex, we have applied our multicanonical molecular dynamics (McMD)-based dynamic docking protocol starting from the unbound state of the peptide. Our simulations have reproduced the experimental complex structure, although the top-ranking structure was an intermediary one, where the peptide was bound in the same location as in the experimental structure; however, the ß-switch of GPIbα attained a different conformation. Our analysis showed that subsequent refolding of the ß-switch results in a more stable binding configuration, although the transition to the native configuration appears to take some time, during which OS1 could dissociate. Our results show that conformational changes in the ß-switch are crucial for successful binding of OS1. Furthermore, we identified several allosteric binding sites of GPIbα that might also interfere with vWF binding, and optimization of the peptide to target these allosteric sites might lead to a more effective inhibitor, as these are not dependent on the ß-switch conformation.
Asunto(s)
Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Péptidos Cíclicos , Complejo GPIb-IX de Glicoproteína Plaquetaria , Unión Proteica , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Péptidos Cíclicos/metabolismo , Complejo GPIb-IX de Glicoproteína Plaquetaria/química , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Conformación Proteica , Factor de von Willebrand/química , Factor de von Willebrand/metabolismo , Humanos , Sitios de UniónRESUMEN
Triceptides are a class of ribosomally synthesized and post-translationally modified peptides defined by an aromatic C(sp2) to Cß(sp3) bond. The Gly-rich repeat family of triceptide maturases (TIGR04261) are paired with precursor peptides (TIGR04260) containing a Gly-rich core peptide. These maturases are prevalent in cyanobacteria and catalyze cyclophane formation on multiple Ω1-X2-X3 motifs (Ω1 = Trp and Phe) of the Gly-rich precursor peptide. The topology of the individual rings has not been completely elucidated, and the promiscuity of these enzymes is not known. In this study, we characterized all the cyclophane rings formed by the triceptide maturase OscB and show the ring topology is uniform with respect to the substitution at Trp-C7 and the atropisomerism (planar chirality). Additionally, the enzyme OscB demonstrated substrate promiscuity on Gly-rich precursors and can accommodate a diverse array of engineered sequences. These findings highlight the versatility and implications for using OscB as a biocatalyst for producing polycyclophane-containing peptides for biotechnological applications.
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Glicina , Especificidad por Sustrato , Glicina/química , Glicina/metabolismo , Péptidos/química , Péptidos/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Cianobacterias/enzimología , Cianobacterias/metabolismo , Péptidos Cíclicos/química , Péptidos Cíclicos/metabolismo , Biocatálisis , CiclofanosRESUMEN
Lipopeptides produced by beneficial bacilli present promising alternatives to chemical pesticides for plant biocontrol purposes. Our research explores the distinct plant biocontrol activities of lipopeptides surfactin (SRF) and fengycin (FGC) by examining their interactions with lipid membranes. Our study shows that FGC exhibits a direct antagonistic activity against Botrytis cinerea and no marked immune-eliciting activity in Arabidopsis thaliana while SRF only demonstrates an ability to stimulate plant immunity. It also reveals that SRF and FGC exhibit diverse effects on membrane integrity and lipid packing. SRF primarily influences membrane physical state without significant membrane permeabilization, while FGC permeabilizes membranes without significantly affecting lipid packing. From our results, we can suggest that the direct antagonistic activity of lipopeptides is linked to their capacity to permeabilize lipid membrane while the stimulation of plant immunity is more likely the result of their ability to alter the mechanical properties of the membrane. Our work also explores how membrane lipid composition modulates the activities of SRF and FGC. Sterols negatively impact both lipopeptides' activities while sphingolipids mitigate the effects on membrane lipid packing but enhance membrane leakage. In conclusion, our findings emphasize the importance of considering both membrane lipid packing and leakage mechanisms in predicting the biological effects of lipopeptides. It also sheds light on the intricate interplay between the membrane composition and the effectiveness of the lipopeptides, providing insights for targeted biocontrol agent design.
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Botrytis , Lipopéptidos , Lípidos de la Membrana , Péptidos Cíclicos , Lipopéptidos/farmacología , Lipopéptidos/química , Péptidos Cíclicos/farmacología , Péptidos Cíclicos/química , Péptidos Cíclicos/metabolismo , Lípidos de la Membrana/metabolismo , Lípidos de la Membrana/química , Botrytis/efectos de los fármacos , Arabidopsis/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Antifúngicos/farmacología , Antifúngicos/químicaRESUMEN
Iturin A biosynthesis has garnered considerable interest, yet bottlenecks persist in its low productivity in wild strains and the ability to engineer Bacillus amyloliquefaciens producers. This study reveals that deleting the endogenous plasmid, plas1, from the wild-type B. amyloliquefaciens HM618 notably enhances iturin A synthesis, likely related to the effect of the Rap phosphatase gene within plas1. Furthermore, inactivating Rap phosphatase-related genes (rapC, rapF, and rapH) in the genome of the strain also improved the iturin A level and specific productivity while reducing cell growth. Strategic rap genes and plasmid elimination achieved a synergistic balance between cell growth and iturin A production. Engineered strain HM-DR13 exhibited an increase in iturin A level to 849.9 mg/L within 48 h, significantly shortening the production period. These insights underscore the critical roles of endogenous plasmids and Rap phosphatases in iturin A biosynthesis, presenting a novel engineering strategy to optimize iturin A production in B. amyloliquefaciens.