RESUMEN
Macrocyclic peptides are an emerging class of therapeutics that can modulate protein-protein interactions. In contrast to the heavily automated high-throughput screening systems traditionally used for the identification of chemically synthesized small-molecule drugs, peptide-based macrocycles can be synthesized by ribosomal translation and identified using in vitro selection techniques, allowing for extremely rapid (hours to days) screening of compound libraries comprising more than 10(13) different species. Furthermore, chemical modification of translated peptides and engineering of the genetic code have greatly expanded the structural diversity of the available peptide libraries. In this review, we discuss the use of these technologies for the identification of bioactive macrocyclic peptides, emphasizing recent developments.
Asunto(s)
Química Farmacéutica/métodos , Diseño de Fármacos , Biblioteca de Péptidos , Péptidos/química , Membrana Celular/metabolismo , Código Genético , Ensayos Analíticos de Alto Rendimiento , Biosíntesis de Proteínas , Ingeniería de Proteínas/métodos , Mapeo de Interacción de Proteínas/métodos , ARN Mensajero/metabolismo , Ribosomas/químicaRESUMEN
Bromodomains (BDs) regulate gene expression by recognizing protein motifs containing acetyllysine. Although originally characterized as histone-binding proteins, it has since become clear that these domains interact with other acetylated proteins, perhaps most prominently transcription factors. The likely transient nature and low stoichiometry of such modifications, however, has made it challenging to fully define the interactome of any given BD. To begin to address this knowledge gap in an unbiased manner, we carried out mRNA display screens against a BD-the N-terminal BD of BRD3-using peptide libraries that contained either one or two acetyllysine residues. We discovered peptides with very strong consensus sequences and with affinities that are significantly higher than typical BD-peptide interactions. X-ray crystal structures also revealed modes of binding that have not been seen with natural ligands. Intriguingly, however, our sequences are not found in the human proteome, perhaps suggesting that strong binders to BDs might have been selected against during evolution.
Asunto(s)
Proteoma , Factores de Transcripción , Humanos , Proteoma/metabolismo , Factores de Transcripción/metabolismo , Dominios Proteicos , Secuencias de Aminoácidos , Péptidos/metabolismo , Unión Proteica , AcetilaciónRESUMEN
Two-chain hepatocyte growth factor (tcHGF), the mature form of HGF, is associated with malignancy and anticancer drug resistance; therefore, its quantification is an important indicator for cancer diagnosis. In tumors, activated tcHGF hardly discharges into the systemic circulation, indicating that tcHGF is an excellent target for molecular imaging using positron emission tomography (PET). We recently discovered HGF-inhibitory peptide-8 (HiP-8) that binds specifically to human tcHGF with nanomolar affinity. The purpose of this study was to investigate the usefulness of HiP-8-based PET probes in human HGF knock-in humanized mice. 64Cu-labeled HiP-8 molecules were synthesized using a cross-bridged cyclam chelator, CB-TE1K1P. Radio-high-performance liquid chromatography-based metabolic stability analyses showed that more than 90% of the probes existed in intact form in blood at least for 15 min. In PET studies, significantly selective visualization of hHGF-overexpressing tumors versus hHGF-negative tumors was observed in double-tumor-bearing mice. The accumulation of labeled HiP-8 into the hHGF-overexpressing tumors was significantly reduced by competitive inhibition. In addition, the radioactivity and distribution of phosphorylated MET/HGF receptor were colocalized in tissues. These results demonstrate that the 64Cu-labeled HiP-8 probes are suitable for tcHGF imaging in vivo, and secretory proteins like tcHGF can be a target for PET imaging.
Asunto(s)
Factor de Crecimiento de Hepatocito , Neoplasias , Ratones , Humanos , Animales , Factor de Crecimiento de Hepatocito/metabolismo , Péptidos/química , Neoplasias/diagnóstico por imagen , Tomografía de Emisión de Positrones/métodos , Quelantes/química , Radioisótopos de Cobre/química , Línea Celular TumoralRESUMEN
Cyclic peptide library screening technologies show immense promise for identifying drug leads and chemical probes for challenging targets. However, the structural and functional diversity encoded within such libraries is largely undefined. We have systematically profiled the affinity, selectivity, and structural features of library-derived cyclic peptides selected to recognize three closely related targets: the acetyllysine-binding bromodomain proteins BRD2, -3, and -4. We report affinities as low as 100 pM and specificities of up to 106-fold. Crystal structures of 13 peptide-bromodomain complexes reveal remarkable diversity in both structure and binding mode, including both α-helical and ß-sheet structures as well as bivalent binding modes. The peptides can also exhibit a high degree of structural preorganization. Our data demonstrate the enormous potential within these libraries to provide diverse binding modes against a single target, which underpins their capacity to yield highly potent and selective ligands.
Asunto(s)
Biblioteca de Péptidos , Péptidos Cíclicos , Sitios de Unión , Descubrimiento de Drogas , Humanos , Péptidos Cíclicos/química , Péptidos Cíclicos/metabolismo , Unión Proteica , Dominios Proteicos , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
Cyclotides are plant-derived peptides with complex structures shaped by their head-to-tail cyclic backbone and cystine knot core. These structural features underpin the native bioactivities of cyclotides, as well as their beneficial properties as pharmaceutical leads, including high proteolytic stability and cell permeability. However, their inherent structural complexity presents a challenge for cyclotide engineering, particularly for accessing libraries of sufficient chemical diversity to design potent and selective cyclotide variants. Here, we report a strategy using mRNA display enabling us to select potent cyclotide-based FXIIa inhibitors from a library comprising more than 1012 members based on the cyclotide scaffold of Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II). The most potent and selective inhibitor, cMCoFx1, has a pM inhibitory constant toward FXIIa with greater than three orders of magnitude selectivity over related serine proteases, realizing specific inhibition of the intrinsic coagulation pathway. The cocrystal structure of cMCoFx1 and FXIIa revealed interactions at several positions across the contact interface that conveyed high affinity binding, highlighting that such cyclotides are attractive cystine knot scaffolds for therapeutic development.
Asunto(s)
Proteínas Sanguíneas/farmacología , Ciclotidas/farmacología , Factor XIIa/metabolismo , Proteínas Sanguíneas/química , Ciclotidas/química , Factor XIIa/genética , Regulación de la Expresión Génica/efectos de los fármacos , HumanosRESUMEN
Activation of hepatocyte growth factor (HGF) by proteolytic processing is triggered in cancer microenvironments, and subsequent signaling through the MET receptor is involved in cancer progression. However, the structure of HGF remains elusive, and few small/medium-sized molecules can modulate HGF. Here, we identified HiP-8, a macrocyclic peptide consisting of 12 amino acids, which selectively recognizes active HGF. Biochemical analysis and real-time single-molecule imaging by high-speed atomic force microscopy demonstrated that HiP-8 restricted the dynamic domains of HGF into static closed conformations, resulting in allosteric inhibition. Positron emission tomography using HiP-8 as a radiotracer enabled noninvasive visualization and simultaneous inhibition of HGF-MET activation status in tumors in a mouse model. Our results illustrate the conformational change in proteolytic activation of HGF and its detection and inhibition by a macrocyclic peptide, which may be useful for diagnosis and treatment of cancers.
Asunto(s)
Factor de Crecimiento de Hepatocito/análisis , Compuestos Macrocíclicos/química , Neoplasias Experimentales/diagnóstico por imagen , Imagen Óptica , Péptidos/química , Animales , Factor de Crecimiento de Hepatocito/antagonistas & inhibidores , Factor de Crecimiento de Hepatocito/metabolismo , Compuestos Macrocíclicos/farmacología , Ratones , Neoplasias Experimentales/tratamiento farmacológico , Péptidos/farmacología , Tomografía de Emisión de PositronesRESUMEN
High-resolution structure-activity analysis of polypeptides requires amino acid structures that are not present in the universal genetic code. Examination of peptide and protein interactions with this resolution has been limited by the need to individually synthesize and test peptides containing nonproteinogenic amino acids. We describe a method to scan entire peptide sequences with multiple nonproteinogenic amino acids and, in parallel, determine the thermodynamics of binding to a partner protein. By coupling genetic code reprogramming to deep mutational scanning, any number of amino acids can be exhaustively substituted into peptides, and single experiments can return all free energy changes of binding. We validate this approach by scanning two model protein-binding peptides with 21 diverse nonproteinogenic amino acids. Dense structure-activity maps were produced at the resolution of single aliphatic atom insertions and deletions. This permits rapid interrogation of interaction interfaces, as well as optimization of affinity, fine-tuning of physical properties, and systematic assessment of nonproteinogenic amino acids in binding and folding.
Asunto(s)
Péptidos Cíclicos/genética , Secuencia de Aminoácidos , Aminoácidos/genética , Código Genético/genética , Unión Proteica/genética , Proteínas/genética , Relación Estructura-Actividad , TermodinámicaRESUMEN
Macrocyclic peptides make up an emerging class of candidate therapeutics and chemical probes, with properties that make them potentially applicable to a wide range of targets that are intractable using current pharmacological agents. Additionally, a number of biochemical screening strategies have been developed, particularly over the past decade, that allow for the massively parallel screening of cyclic peptide libraries of up to 1 trillion compounds or more, leading to the isolation of molecules with exceptional target affinity, selectivity, and bioactivity. Clinical development of compounds derived from such screens is already underway, but the nature of these molecules means that such development is likely to follow pathways different from those of traditional small molecule drugs or well-established biologics such as monoclonal antibodies. In addition, recent work has shown that the biochemical techniques used to identify macrocyclic peptides can also be used to rapidly characterize and optimize them. These findings are likely to facilitate the development of these compounds as chemical probes and as therapeutics for areas of unmet medical need.
Asunto(s)
Descubrimiento de Drogas , Péptidos Cíclicos/química , Animales , Humanos , Ligandos , Estructura Molecular , Biblioteca de Péptidos , Relación Estructura-ActividadRESUMEN
Targeting chemokine signaling is an attractive avenue for the treatment of inflammatory disorders. Tyrosine sulfation is an important post-translational modification (PTM) that enhances chemokine-receptor binding and is also utilized by a number of pathogenic organisms to improve the binding affinity of immune-suppressive chemokine binding proteins (CKBPs). Here we report the display selection of tyrosine-sulfated cyclic peptides using a reprogrammed genetic code to discover high-affinity ligands for the chemokine CCL11 (eotaxin-1). The selected cyclic sulfopeptides possess high affinity for the target chemokine (as well as one or more of the related family members CCL2, CCL7 and CCL24) and inhibit CCL11 activation of CC chemokine receptor 3 (CCR3). This work demonstrates the utility of exploiting native PTMs as binding motifs for the generation of new leads for medicinal chemistry.
Asunto(s)
Quimiocina CCL11/antagonistas & inhibidores , Descubrimiento de Drogas , Péptidos/farmacología , ARN Mensajero/efectos de los fármacos , Quimiocina CCL11/genética , Quimiocina CCL11/metabolismo , Humanos , Estructura Molecular , Péptidos/química , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Backbone-cyclic peptides are an attractive class for therapeutic development. However, in vitro display technologies coupled with ribosomal synthesis are intrinsically inapplicable to such "phenotypes" because of loss of the C-terminal peptide region linking to "genotype". Here, we report a methodology enabling the display of backbone-cyclic peptides. To achieve this, genetic code reprogramming was utilized to implement a rearrangement strategy involving the ribosomal incorporation of a designer initiator containing a thiazolidine-protected cysteine and 2-chloroacetoamide (ClAc) side chain, followed by an α-thio acid and cysteine at downstream positions. Upon expression of the linear peptide, spontaneous thioester rearrangement occurs between the α-thioester and the thiol group of the cysteine, liberating the α-thio group and resulting in cross-linking to the upstream ClAc side-chain group. Then selective deprotection of the thiazolidine-protected cysteine immediately promotes intramolecular native chemical ligation, as demonstrated for various sequences and ring sizes. In this approach, the backbone-cyclic peptides retain their C-terminal peptide regions via the side-chain thioether covalent linkage, making them compatible with in vitro display.
Asunto(s)
Péptidos Cíclicos/biosíntesis , Péptidos Cíclicos/química , Ribosomas/metabolismo , Secuencia de AminoácidosRESUMEN
The site-specific introduction of non-canonical amino acids into polypeptides through genetic code reprogramming has become a powerful tool for biochemical studies and bioorganic synthesis. Although a variety of such techniques have been developed, all are based on the 'mis-acylation' of tRNA molecules with non-canonical amino acids. Multiple strategies have been devised to synthesize such non-canonical aminoacyl-tRNAs; for example, those based on protein or ribozyme aminoacyl-tRNA synthetase enzymes are particularly useful. Such techniques have enabled the incorporation of hundreds of different non-canonical amino acids into polypeptides in vitro. This review discusses the development and application of in vitro genetic code reprogramming techniques, especially enzymatic mis-acylation, and examines recent efforts to engineer the translational machinery to increase the range of translatable non-canonical amino acids.
Asunto(s)
Código Genético , Ingeniería Genética , Biosíntesis de Péptidos , Animales , Humanos , Técnicas In Vitro , ARN Catalítico/genéticaRESUMEN
Bioactive naturally occurring macrocyclic peptides often exhibit a strong bias for hydrophobic residues. Recent advances in in vitro display technologies have made possible the identification of potent macrocyclic peptide ligands to protein targets of interest. However, such approaches have so far been restricted to using libraries composed of peptides containing mixtures of hydrophobic and hydrophilic/charged amino acids encoded by the standard genetic code. In the present study, we have demonstrated ribosomal expression of exotic macrocyclic peptides under a radically reprogrammed, relatively hydrophobic, genetic code, comprising 12 proteinogenic and 11 nonproteinogenic amino acids. Screening of this library for affinity to the interleukin-6 receptor (IL6R) as a case study successfully identified exotic macrocyclic peptide ligands with high affinity, validating the feasibility of this approach for the discovery of relatively hydrophobic exotic macrocyclic peptide ligands.
Asunto(s)
Aminoácidos/genética , Aminoácidos/metabolismo , Código Genético , Ingeniería Genética , Compuestos Macrocíclicos/metabolismo , Péptidos/genética , Péptidos/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Ligandos , Compuestos Macrocíclicos/química , Estructura Molecular , Péptidos/químicaRESUMEN
The combination of genetic code reprogramming and mRNA display is a powerful approach for the identification of macrocyclic peptides with high affinities to a target of interest. We have previously used such an approach to identify a potent inhibitor (CP2) of the human KDM4A and KDM4C lysine demethylases; important regulators of gene expression. In the present study, we have used genetic code reprogramming to synthesise very high diversity focused libraries (>1012 compounds) based on CP2 and, through affinity screening, used these to delineate the structure activity relationship of CP2 binding to KDM4A. In the course of these experiments we identified a CP2 analogue (CP2f-7) with â¼4-fold greater activity than CP2 in in vitro inhibition assays. This work will facilitate the development of more potent, selective inhibitors of lysine demethylases.
Asunto(s)
Inhibidores Enzimáticos/química , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Péptidos Cíclicos/química , Secuencia de Aminoácidos , Inhibidores Enzimáticos/metabolismo , Humanos , Concentración 50 Inhibidora , Histona Demetilasas con Dominio de Jumonji/metabolismo , Células MCF-7 , Microscopía Confocal , Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/metabolismo , Relación Estructura-ActividadRESUMEN
Ebola virus infection leads to severe hemorrhagic fever in human and non-human primates with an average case fatality rate of 50%. To date, numerous potential therapies are in development, but FDA-approved drugs or vaccines are yet unavailable. Ebola viral protein 24 (VP24) is a multifunctional protein that plays critical roles in the pathogenesis of Ebola virus infection, e.g. innate immune suppression by blocking the interaction between KPNA and PY-STAT1. Here we report macrocyclic peptide inhibitors of the VP24-KPNA5 protein-protein interaction (PPI) by means of the RaPID (Random non-standard Peptides Integrated Discovery) system. These macrocyclic peptides showed remarkably high affinity to recombinant Zaire Ebola virus VP24 (eVP24), with a dissociation constant in the single digit nanomolar range, and could also successfully disrupt the eVP24-KPNA interaction. This work provides for the first time a chemical probe capable of modulating this PPI interaction and is the starting point for the development of unique anti-viral drugs against the Ebola virus.
Asunto(s)
Compuestos Macrocíclicos/farmacología , Péptidos/farmacología , Proteínas Virales/metabolismo , alfa Carioferinas/metabolismo , Relación Dosis-Respuesta a Droga , Polarización de Fluorescencia , Humanos , Compuestos Macrocíclicos/química , Péptidos/química , Unión Proteica/efectos de los fármacos , Relación Estructura-Actividad , Proteínas Virales/química , alfa Carioferinas/químicaRESUMEN
Techniques facilitating the synthesis and screening of very high diversity nonstandard macrocyclic peptide libraries have led to such compounds receiving increasing attention as potential drug candidates. Specifically, approaches which allow the use of non-proteinogenic amino acids are proving to be particularly effective, since they expand the accessible chemical space of the starting library and thus allow the identification of compounds with structural similarity to known drugs. This review focuses on mRNA display screening platforms for drug discovery and their combined use with genetic code reprogramming to identify novel macrocyclic peptides with high affinities for disease-related targets of interest.
Asunto(s)
Péptidos/genética , Ligandos , Compuestos Macrocíclicos , ARN Mensajero/genéticaRESUMEN
In contemporary organisms the aminoacylation of tRNAs is performed exclusively by protein aminoacyl-tRNA synthetases. However, in vitro selection experiments have identified RNA enzymes that exhibit the necessary characteristics to charge tRNA molecules with acyl groups in a way that is compatible with ribosomal translation, suggesting that such ribozymes may have fulfilled this function prior to the evolution of proteinaceous life. The current chapter provides a review of the history, structure, and function of these RNA aminoacyl synthetases, and discusses their practical application to "genetic reprogramming" and other biotechnologies.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Evolución Molecular , Aminoacil-ARNt Sintetasas/química , Aminoacilación , Secuencia de Bases , Biosíntesis de Proteínas , Procesamiento Proteico-Postraduccional , ARN Catalítico/metabolismoRESUMEN
INTRODUCTION: Cyclic peptides are an established class of pharmaceuticals, with the ability to bind to a broader range of protein targets than traditional small molecules while also being capable of oral availability and cell penetration. Historically, cyclic peptide drugs have been discovered almost exclusively through natural product mining approaches; however, the last two decades have seen the development of display screening approaches capable of rapidly identifying de novo (i.e. not natural product derived) cyclic peptide ligands to targets of interest. AREAS COVERED: In this review, the authors describe the current clinical landscape for cyclic peptide pharmaceuticals. This article focuses on the discovery approaches that have led to the development of different classes of molecules and how the development of newer technologies, particularly phage and mRNA display, has broadened the clinical applicability of such molecules. EXPERT OPINION: The field of de novo cyclic peptide drug discovery is reaching maturity, with the first drugs identified through display screening approaches reaching the market in recent years. Many more are in clinical trials; however, significant technical challenges remain. Technological improvements will be required over the coming years to facilitate the identification of membrane permeable cyclic peptides capable of oral availability and targeting intracellular proteins.
Asunto(s)
Descubrimiento de Drogas , Péptidos Cíclicos , Humanos , Descubrimiento de Drogas/métodos , Péptidos Cíclicos/farmacología , Animales , Ligandos , Desarrollo de Medicamentos/métodos , Biblioteca de Péptidos , Administración OralRESUMEN
The regenerative functions associated with cytokines and growth factors have immense therapeutic potential; however, their poor pharmacokinetics, resulting from structural features, hinder their effectiveness. In this study, we aimed to enhance the pharmacokinetics of growth factors by designing receptor-binding macrocyclic peptides through in vitro mRNA display and grafting them into loops of immunoglobulin's crystallizable region (Fc). As a model, we developed peptide-grafted Fc proteins with hepatocyte growth factor (HGF) functionality that exhibited a prolonged circulation half-life and could be administered subcutaneously. The Fc-based HGF mimetic alleviated liver fibrosis in a mouse model fed a choline-deficient high-fat diet, which induces hepatic features of non-alcoholic steatohepatitis, including fibrosis, showcasing its potential as a therapeutic intervention. This study provides a basis for developing growth factor and cytokine mimetics with improved pharmacokinetics, expanding their therapeutic applications.