RESUMO
Drug safety and efficacy due to premature release into the bloodstream and poor biodistribution remains a problem despite seminal advances in this area. To circumvent these limitations, we report drug cyclization based on dynamic covalent linkages to devise a dual lock for the small-molecule anticancer drug, camptothecin (CPT). Drug activity is "locked" within the cyclic structure by the redox responsive disulfide and pH-responsive boronic acid-salicylhydroxamate and turns on only in the presence of acidic pH, reactive oxygen species and glutathione through traceless release. Notably, the dual-responsive CPT is more active (100-fold) than the non-cleavable (permanently closed) analogue. We further include a bioorthogonal handle in the backbone for functionalization to generate cyclic-locked, cell-targeting peptide- and protein-CPTs, for targeted delivery of the drug and traceless release in triple negative metastatic breast cancer cells to inhibit cell growth at low nanomolar concentrations.
Assuntos
Antineoplásicos , Nanopartículas , Neoplasias , Camptotecina/química , Distribuição Tecidual , Antineoplásicos/química , Micelas , Proteínas , Sistemas de Liberação de Medicamentos , Nanopartículas/química , Liberação Controlada de Fármacos , Linhagem Celular TumoralRESUMO
Viral infections pose a significant threat to human health, and effective antiviral strategies are urgently needed. Antiviral peptides have emerged as a promising class of therapeutic agents due to their unique properties and mechanisms of action. While effective on their own, combining antiviral peptides may allow us to enhance their potency and to prevent viral resistance. Here, we developed an orthogonal chemical strategy to prepare a heterodimeric peptide conjugate assembled on a protein-based nanoplatform. Specifically, we combined the optimized version of two peptides inhibiting HIV-1 by distinct mechanisms. Virus-inhibitory peptide (VIRIP) is a 20 amino acid fragment of α1-antitrypsin that inhibits HIV-1 by targeting the gp41 fusion peptide. Endogenous peptide inhibitor of CXCR4 (EPI-X4) is a 16-residue fragment of human serum albumin that prevents HIV-1 entry by binding to the viral CXCR4 co-receptor. Optimized forms of both peptides are assembled on supramolecular nanoplatforms through the streptavidin-biotin interaction. We show that the construct consisting of the two different peptides (SAv-VIR-102C9-EPI-X4 JM#173-C) shows increased activity against CCR5- and CXCR4-tropic HIV-1 variants. Our results are a proof of concept that peptides with different modes of action can be assembled on nanoplatforms to enhance their antiviral activity.
Assuntos
Infecções por HIV , HIV-1 , Humanos , Infecções por HIV/tratamento farmacológico , Infecções por HIV/prevenção & controle , Peptídeos/farmacologia , Albumina Sérica Humana , AntiviraisRESUMO
Advanced derivatives of the Endogenous Peptide Inhibitor of CXCR4 (EPI-X4) have shown therapeutic efficacy upon topical administration in animal models of asthma and dermatitis. Here, we studied the plasma stability of the EPI-X4 lead compounds WSC02 and JM#21, using mass spectrometry to monitor the chemical integrity of the peptides and a functional fluorescence-based assay to determine peptide function in a CXCR4-antibody competition assay. Although mass spectrometry revealed very rapid disappearance of both peptides in human plasma within seconds, the functional assay revealed a significantly higher half-life of 9 min for EPI-X4 WSC02 and 6 min for EPI-X4 JM#21. Further analyses demonstrated that EPI-X4 WSC02 and EPI-X4 JM#21 interact with low molecular weight plasma components and serum albumin. Albumin binding is mediated by the formation of a disulfide bridge between Cys10 in the EPI-X4 peptides and Cys34 in albumin. These covalently linked albumin-peptide complexes have a higher stability in plasma as compared with the non-bound peptides and retain the ability to bind and antagonize CXCR4. Remarkably, chemically synthesized albumin-EPI-X4 conjugates coupled by non-breakable bonds have a drastically increased plasma stability of over 2 h. Thus, covalent coupling of EPI-X4 to albumin in vitro before administration or in vivo post administration may significantly increase the pharmacokinetic properties of this new class of CXCR4 antagonists.
Assuntos
Receptores CXCR4 , Albumina Sérica Humana , Animais , Humanos , Receptores CXCR4/metabolismo , Peptídeos/química , Meia-Vida , Albumina Sérica/metabolismoRESUMO
Dynamic covalent chemistry (DCvC) has emerged as a versatile synthetic tool for devising stable, stimuli-responsive linkers or conjugates. The interplay of binding affinity, association and dissociation constants exhibits a strong influence on the selectivity of the reaction, the conversion rate, as well as the stability in aqueous solutions. Nevertheless, dynamic covalent interactions often exhibit fast binding and fast dissociation events or vice versa, affecting their conversion rates or stabilities. To overcome the limitation in linker design, we reported herein dual responsive dynamic covalent peptide tags combining a pH responsive boronate ester with fast association and dissociation rates, and a redox-active disulfide with slow formation and dissociation rate. Precoordination by boronic acid-catechol interaction improves self-sorting and selectivity in disulfide formation into heterodimers. The resulting bis-peptide conjugate exhibited improved complex stability in aqueous solution and acidic tumor-like extracellular microenvironment. Furthermore, the conjugate responds to pH changes within the physiological range as well as to redox conditions found inside cancer cells. Such tags hold great promise, through cooperative effects, for controlling the stability of bioconjugates under dilution in aqueous media, as well as designing intelligent pharmaceutics that react to distinct biological stimuli in cells.
Assuntos
Ácidos BorônicosRESUMO
Site-selective protein functionalization serves as an invaluable tool for investigating protein structures and functions in complicated cellular environments and accomplishing semi-synthetic protein conjugates such as traceable therapeutics with improved features. Dual functionalization of proteins allows the incorporation of two different types of functionalities at distinct location(s), which greatly expands the features of native proteins. The attachment and crosstalk of a fluorescence donor and an acceptor dye provides fundamental insights into the folding and structural changes of proteins upon ligand binding in their native cellular environments. Moreover, the combination of drug molecules with different modes of action, imaging agents or stabilizing polymers provides new avenues to design precision protein therapeutics in a reproducible and well-characterizable fashion. This review aims to give a timely overview of the recent advancements and a future perspective of this relatively new research area. First, the chemical toolbox for dual functionalization of proteins is discussed and compared. The strengths and limitations of each strategy are summarized in order to enable readers to select the most appropriate method for their envisaged applications. Thereafter, representative applications of these dual-modified protein bioconjugates benefiting from the synergistic/additive properties of the two synthetic moieties are highlighted.
Assuntos
Proteínas/metabolismo , Aminoácidos/química , Aminoácidos/metabolismo , Transferência Ressonante de Energia de Fluorescência , Modelos Moleculares , Estrutura Molecular , Proteínas/químicaRESUMO
An inverse electron demand Diels-Alder reaction between tetrazine and trans-cyclooctene (TCO) holds great promise for protein modification and manipulation. Herein, we report the design and synthesis of a tetrazine-based disulfide rebridging reagent, which allows the site-selective installation of a tetrazine group into disulfide-containing peptides and proteins such as the hormone somatostatin (SST) and the antigen binding fragment (Fab) of human immunoglobulin G (IgG). The fast and efficient conjugation of the tetrazine modified proteins with three different TCO-containing substrates to form a set of bioconjugates in a site-selective manner was successfully demonstrated for the first time. Homogeneous, well-defined bioconjugates were obtained underlining the great potential of our method for fast bioconjugation in emerging protein therapeutics. The formed bioconjugates were stable against glutathione and in serum, and they maintained their secondary structure. With this work, we broaden the scope of tetrazine chemistry for site-selective protein modification to prepare well-defined SST and Fab conjugates with preserved structures and good stability under biologically relevant conditions.
Assuntos
Ciclo-Octanos/metabolismo , Dissulfetos/metabolismo , Compostos Heterocíclicos/química , Imunoglobulina G/metabolismo , Ciclo-Octanos/química , Dissulfetos/síntese química , Dissulfetos/química , Humanos , Imunoglobulina G/química , Modelos Moleculares , Estrutura Molecular , Processamento de Proteína Pós-TraducionalRESUMO
Nature has evolved an optimal synthetic factory in the form of translational and posttranslational processes by which millions of proteins with defined primary sequences and 3D structures can be built. Nature's toolkit gives rise to protein building blocks, which dictates their spatial arrangement to form functional protein nanostructures that serve a myriad of functions in cells, ranging from biocatalysis, formation of structural networks, and regulation of biochemical processes, to sensing. With the advent of chemical tools for site-selective protein modifications and recombinant engineering, there is a rapid development to develop and apply synthetic methods for creating structurally defined, functional protein nanostructures for a broad range of applications in the fields of catalysis, materials and biomedical sciences. In this review, design principles and structural features for achieving and characterizing functional protein nanostructures by synthetic approaches are summarized. The synthetic customization of protein building blocks, the design and introduction of recognition units and linkers and subsequent assembly into structurally defined protein architectures are discussed herein. Key examples of these supramolecular protein nanostructures, their unique functions and resultant impact for biomedical applications are highlighted.
Assuntos
Nanoestruturas/química , Nanotecnologia/métodos , Proteínas/química , Animais , Catálise , Técnicas de Química Sintética/métodos , Humanos , Modelos Moleculares , Nanoestruturas/ultraestrutura , Conformação Proteica , Engenharia de Proteínas/métodos , Proteínas/síntese química , Proteínas/genética , Proteínas/ultraestruturaRESUMO
The development of small protein tags that exhibit bioorthogonality, bond stability, and reversibility, as well as biocompatibility, holds great promise for applications in cellular environments enabling controlled drug delivery or for the construction of dynamic protein complexes in biological environments. Herein, we report the first application of dynamic covalent chemistry both for purification and for reversible assembly of protein conjugates using interactions of boronic acid with diols and salicylhydroxamates. Incorporation of the boronic acid (BA) tag was performed in a site-selective fashion by applying disulfide rebridging strategy. As an example, a model protein enzyme (lysozyme) was modified with the BA tag and purified using carbohydrate-based column chromatography. Subsequent dynamic covalent "click-like" bioconjugation with a salicylhydroxamate modified fluorescent dye (BODIPY FL) was accomplished while retaining its original enzymatic activity.
Assuntos
Ácidos Borônicos/química , Química Click , Proteínas/química , Compostos de Boro/química , Cromatografia Líquida/métodos , Dissulfetos/química , Corantes Fluorescentes/química , Muramidase/química , Muramidase/metabolismo , Proteínas/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectrometria de Massas em Tandem/métodosRESUMO
The attachment of two different functionalities in a site-selective fashion represents a great challenge in protein chemistry. We report site specific dual functionalizations of peptides and proteins capitalizing on reactivity differences of cysteines in their free (thiol) and protected, oxidized (disulfide) forms. The dual functionalization of interleukin 2 and EYFP proceeded with no loss of bioactivity in a stepwise fashion applying maleimide and disulfide rebridging allyl-sulfone groups. In order to ensure broader applicability of the functionalization strategy, a novel, short peptide sequence that introduces a disulfide bridge was designed and site-selective dual labeling in the presence of biogenic groups was successfully demonstrated.
Assuntos
Compostos Alílicos/química , Cisteína/química , Maleimidas/química , Peptídeos/química , Proteínas/química , Compostos de Sulfidrila/química , Sulfonas/química , Compostos Alílicos/síntese química , Animais , Proteínas de Bactérias/síntese química , Proteínas de Bactérias/química , Linhagem Celular , Cisteína/síntese química , Humanos , Interleucina-2/síntese química , Interleucina-2/química , Proteínas Luminescentes/síntese química , Proteínas Luminescentes/química , Maleimidas/síntese química , Camundongos , Modelos Moleculares , Peptídeos/síntese química , Proteínas/síntese química , Proteínas Recombinantes/síntese química , Proteínas Recombinantes/química , Coloração e Rotulagem/métodos , Compostos de Sulfidrila/síntese química , Sulfonas/síntese químicaRESUMO
Once materials come into contact with a biological fluid containing proteins, proteins are generally-whether desired or not-attracted by the material's surface and adsorb onto it. The aim of this Review is to give an overview of the most commonly used characterization methods employed to gain a better understanding of the adsorption processes on either planar or curved surfaces. We continue to illustrate the benefit of combining different methods to different surface geometries of the material. The thus obtained insight ideally paves the way for engineering functional materials that interact with proteins in a predetermined manner.
Assuntos
Nanoestruturas/química , Proteínas/química , Portadores de Fármacos/química , Ligação Proteica , Coroa de Proteína/química , Dobramento de Proteína , Proteínas/metabolismo , Propriedades de Superfície , Nanomedicina TeranósticaRESUMO
The synthetic transformation of polypeptides with molecular accuracy holds great promise for providing functional and structural diversity beyond the proteome. Consequently, the last decade has seen an exponential growth of site-directed chemistry to install additional features into peptides and proteins even inside living cells. The disulfide rebridging strategy has emerged as a powerful tool for site-selective modifications since most proteins contain disulfide bonds. In this Review, we present the chemical design, advantages and limitations of the disulfide rebridging reagents, while summarizing their relevance for synthetic customization of functional protein bioconjugates, as well as the resultant impact and advancement for biomedical applications.
Assuntos
Dissulfetos/química , Peptídeos/química , Proteínas/química , Humanos , ProteomaRESUMO
CONSPECTUS: Chemical programming of macromolecular structures to instill a set of defined chemical properties designed to behave in a sequential and precise manner is a characteristic vision for creating next generation nanomaterials. In this context, biopolymers such as proteins and nucleic acids provide an attractive platform for the integration of complex chemical design due to their sequence specificity and geometric definition, which allows accurate translation of chemical functionalities to biological activity. Coupled with the advent of amino acid specific modification techniques, "programmable" areas of a protein chain become exclusively available for any synthetic customization. We envision that chemically reprogrammed hybrid proteins will bridge the vital link to overcome the limitations of synthetic and biological materials, providing a unique strategy for tailoring precision therapeutics. In this Account, we present our work toward the chemical design of protein- derived hybrid polymers and their supramolecular responsiveness, while summarizing their impact and the advancement in biomedicine. Proteins, in their native form, represent the central framework of all biological processes and are an unrivaled class of macromolecular drugs with immense specificity. Nonetheless, the route of administration of protein therapeutics is often vastly different from Nature's biosynthesis. Therefore, it is imperative to chemically reprogram these biopolymers to direct their entry and activity toward the designated target. As a consequence of the innate structural regularity of proteins, we show that supramolecular interactions facilitated by stimulus responsive chemistry can be intricately designed as a powerful tool to customize their functions, stability, activity profiles, and transportation capabilities. From another perspective, a protein in its denatured, unfolded form serves as a monodispersed, biodegradable polymer scaffold decorated with functional side chains available for grafting with molecules of interest. Additionally, we are equipped with analytical tools to map the fingerprint of the protein chain, directly elucidating the structure at the molecular level. Contrary to conventional polymers, these biopolymers facilitate a more systematic avenue to investigate engineered macromolecules, with greater detail and accuracy. In this regard, we focus on denaturing serum albumin, an abundant blood protein, and exploit its peptidic array of functionalities to program supramolecular architectures for bioimaging, drug and gene delivery. Ultimately, we seek to assimilate the evolutionary advantage of these protein based biopolymers with the limitless versatility of synthetic chemistry to merge the best of both worlds.
Assuntos
Desenho de Fármacos , Nanoestruturas/química , Polímeros/química , Proteínas/química , Modelos Moleculares , Dobramento de ProteínaRESUMO
A disulfide intercalator toolbox was developed for site-specific attachment of a broad variety of functional groups to proteins or peptides under mild, physiological conditions. The peptide hormone somatostatin (SST) served as model compound for intercalation into the available disulfide functionalization schemes starting from the intercalator or the reactive SST precursor before or after bioconjugation. A tetrazole-SST derivative was obtained that undergoes photoinduced cycloaddition in mammalian cells, which was monitored by live-cell imaging.
Assuntos
Dissulfetos/química , Substâncias Intercalantes/química , Somatostatina/química , Linhagem Celular Tumoral , Química Click , Reação de Cicloadição , Dendrímeros/química , Doxorrubicina/química , Humanos , Microscopia Confocal , Somatostatina/metabolismo , Tetrazóis/química , Raios UltravioletaRESUMO
In the ideal case, a precise synthesis yields molecules with a constitutional as well as a conformational perfectness. Such a case of precision is demonstrated by the synthesis of semi-rigid amphiphilic polyphenylene dendrimers (PPDs). Polar sulfonate groups are precisely placed on their periphery in such a manner that patches of polar and non-polar regions are created. Key structural features are the semi-rigid framework and shape-persistent nature of PPDs since the limited flexibility introduces a nano-phase-separated amphiphilic rim of the dendrimer. This results in both attractive and repulsive interactions with a given solvent. Frustrated solvent structures then lead to a remarkable solubility in solvents of different polarity such as toluene, methanol, and water or their mixtures. Water solubility combined with defined surface structuring and variable hydrophobicity of PPDs that resemble the delicate surface textures of proteins are important prerequisites for their biological and medical applications based upon cellular internalization.
Assuntos
Dendrímeros/química , Polímeros/química , Interações Hidrofóbicas e Hidrofílicas , Tamanho da Partícula , Solventes/química , Propriedades de SuperfícieRESUMO
The modulation of protein uptake and activity in response to physiological changes forms an integral part of smart protein therapeutics. We describe herein the self-assembly of a pH-responsive dendrimer shell onto the surface of active enzymes (trypsin, papain, DNaseâ I) as a supramolecular protecting group to form a hybrid dendrimer-enzyme complex. The attachment is based on the interaction between boronic acid and salicyl hydroxamate, thus allowing the macromolecular assembly to respond to changes in pH between 5.0 and 7.4 in a highly reversible fashion. Catalytic activity is efficiently blocked in the presence of the dendrimer shell but is quantitatively restored upon shell degradation under acidic conditions. Unlike the native proteases, the hybrid constructs are shown to be efficiently taken up by A549 cells and colocalized in the acidic compartments. The programmed intracellular release of the proteases induced cytotoxicity, thereby uncovering a new avenue for precision biotherapeutics.
Assuntos
Dendrímeros/química , Precursores Enzimáticos/química , Substâncias Macromoleculares/síntese química , Humanos , Substâncias Macromoleculares/química , Modelos Moleculares , Estrutura MolecularRESUMO
In contrast to biological cell membranes, it is still a major challenge for synthetic membranes to efficiently separate ions and small molecules due to their similar sizes in the sub-nanometer range. Inspired by biological ion channels with their unique channel wall chemistry that facilitates ion sieving by ion-channel interactions, the first free-standing, ultrathin (10-17 nm) nanomembranes composed entirely of polydopamine (PDA) are reported here as ion and molecular sieves. These nanomembranes are obtained via an easily scalable electropolymerization strategy and provide nanochannels with various amine and phenolic hydroxyl groups that offer a favorable chemical environment for ion-channel electrostatic and hydrogen bond interactions. They exhibit remarkable selectivity for monovalent ions over multivalent ions and larger species with K+/Mg2+ of ≈4.2, K+/[Fe(CN)6]3- of ≈10.3, and K+/Rhodamine B of ≈273.0 in a pressure-driven process, as well as cyclic reversible pH-responsive gating properties. Infrared spectra reveal hydrogen bond formation between hydrated multivalent ions and PDA, which prevents the transport of multivalent ions and facilitates high selectivity. Chemically rich, free-standing, and pH-responsive PDA nanomembranes with specific interaction sites are proposed as customizable high-performance sieves for a wide range of challenging separation requirements.
RESUMO
A facile, noncovalent solid-phase immobilization platform is described to assemble Janus-like supramolecular fusion proteins that are responsive to external stimuli. A chemically postmodified transporter protein, DHSA, is fused with (imino)biotinylated cargo proteins via an avidin adaptor with a high degree of spatial control. Notably, the derived heterofusion proteins are able to cross cellular membranes, dissociate at acidic pH due to the iminobiotin linker and preserve the enzymatic activity of the cargo proteins ß-galactosidase and the enzymatic subunit of Clostridium botulinum C2 toxin. The mix-and-match strategy described herein opens unique opportunities to access macromolecular architectures of high structural definition and biological activity, thus complementing protein ligation and recombinant protein expression techniques.
Assuntos
Toxinas Botulínicas/metabolismo , Albumina Sérica/metabolismo , beta-Galactosidase/metabolismo , Animais , Biotina/química , Biotina/metabolismo , Toxinas Botulínicas/química , Linhagem Celular Tumoral , Humanos , Concentração de Íons de Hidrogênio , Substâncias Macromoleculares/química , Maleimidas/química , Maleimidas/metabolismo , Modelos Moleculares , Estrutura Molecular , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Albumina Sérica/química , beta-Galactosidase/químicaRESUMO
We describe the synthesis of a core-shell biohybrid consisting of a human serum albumin (HSA) core that serves as a reservoir for lipophilic molecules and a cationized shell region consisting of ethynyl-G2.0-PAMAM or ethynyl-G3.0-PAMAM dendrons. The binding capacity of lipophilic guests was quantified applying electron paramagnetic resonance (EPR) spectroscopy, and five to six out of seven pockets were still available compared with HSA. The attachment of ethynyl-G2.0-PAMAM dendrons to HSA yielded a nontoxic core-shell macromolecule that was clearly uptaken by A549 human epithelial cells due to the presence of the dendritic PAMAM shell. Significantly higher loading of doxorubicin was observed for dendronized G2-DHSA compared with the native protein due to the availability of binding pockets of the HSA core, and interaction with the dendritic shell. Dendronized G2-DHSA-doxorubicin displayed significant cytotoxicity resulting from high drug loading and high stability under different conditions, thus demonstrating its great potential as a transporter for drug molecules.
Assuntos
Dendrímeros/química , Dendrímeros/metabolismo , Portadores de Fármacos/farmacologia , Albumina Sérica/farmacologia , Carcinoma/tratamento farmacológico , Caspase 3/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 7/efeitos dos fármacos , Caspase 7/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Doxorrubicina/química , Doxorrubicina/farmacologia , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Espectroscopia de Ressonância de Spin Eletrônica , Células Epiteliais/metabolismo , Humanos , Albumina Sérica/químicaRESUMO
The synthesis of biohybrid materials with tailored functional properties represents a topic of emerging interest. Combining proteins as natural, macromolecular building blocks, and synthetic polymers opens access to giant brush-like biopolymers of high structural definition. The properties of these precision polypeptide copolymers can be tailored through various chemical modifications along their polypeptide backbone, which expands the repertoire of known protein-based materials to address biomedical applications. In this article, the synthetic strategies for the design of precision biopolymers from proteins through amino acid specific conjugation reagents are highlighted and the different functionalization strategies, their characterization, and applications are discussed.
Assuntos
Biopolímeros/química , Precursores de Proteínas/químicaRESUMO
With the advent of chemical strategies that allow the design of smart bioconjugates, peptide- and protein-drug conjugates are emerging as highly efficient therapeutics to overcome limitations of conventional treatment, as exemplified by antibody-drug conjugates (ADCs). While targeting peptides serve similar roles as antibodies to recognize overexpressed receptors on diseased cell surfaces, peptide-drug conjugates suffer from poor stability and bioavailability due to their low molecular weights. Through a combination of a supramolecular protein-based assembly platform and a pH-responsive linker, the authors devise herein the convenient assembly of a trivalent protein-drug conjugate. The conjugate should ideally possess distinct features of ADCs such as 1) recognition sites that recognize cell receptor and are arranged on 2) distinct locations on a high molecular weight protein scaffold, 3) a stimuli-responsive linker, as well as 4) an attached payload such as a drug molecule. These AD-like conjugates target cancer cells that overexpress somatostatin receptors, can enable controlled release in the microenvironment of cancer cells through a new pH-responsive biotin linker, and exhibit stability in biological media.