RESUMO
The treatment of chronic inflammation with systemically administered anti-inflammatory treatments is associated with moderate-to-severe side effects, and the efficacy of locally administered drugs is short-lived. Here we show that inflammation can be locally suppressed by a fusion protein of the immunosuppressive enzyme indoleamine 2,3-dioxygenase 1 (IDO) and galectin-3 (Gal3). Gal3 anchors IDO to tissue, limiting the diffusion of IDO-Gal3 away from the injection site. In rodent models of endotoxin-induced inflammation, psoriasis, periodontal disease and osteoarthritis, the fusion protein remained in the inflamed tissues and joints for about 1 week after injection, and the amelioration of local inflammation, disease progression and inflammatory pain in the animals were concomitant with homoeostatic preservation of the tissues and with the absence of global immune suppression. IDO-Gal3 may serve as an immunomodulatory enzyme for the control of focal inflammation in other inflammatory conditions.
Assuntos
Galectina 2 , Indolamina-Pirrol 2,3,-Dioxigenase , Animais , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Progressão da DoençaRESUMO
In nature, the precise heterogeneous co-assembly of different protein domains gives rise to supramolecular machines that perform complex functions through the co-integrated activity of the individual protein subunits. A synthetic approach capable of mimicking this process would afford access to supramolecular machines with new or improved functional capabilities. Here we show that the distinct peptide strands of a heterotrimeric α-helical coiled-coil (i.e., peptides "A", "B", and "C") can be used as fusion tags for heterogeneous co-assembly of proteins into supramolecular structures with tunable subunit stoichiometry. In particular, we demonstrate that recombinant fusion of A with NanoLuc luciferase (NL-A), B with superfolder green fluorescent protein (sfGFP-B), and C with mRuby (mRuby-C) enables formation of ternary complexes capable of simultaneously emitting blue, green, and red light via sequential bioluminescence and fluorescence resonance energy transfer (BRET/FRET). Fusion of galectin-3 onto the C-terminus of NL-A, sfGFP-B, and mRuby-C endows the ternary complexes with lactose-binding affinity that can be tuned by varying the number of galectin-3 domains integrated into the complex from one to three, while maintaining BRET/FRET function. The modular nature of the fusion protein design, the precise control of domain stoichiometry, and the multiplicity afforded by the three-stranded coiled-coil scaffold provides access to a greater range of subunit combinations than what is possible with heterodimeric coiled-coils used previously. We envision that access to this expanded range of co-integrated protein domain diversity will be advantageous for future development of designer supramolecular machines for therapeutic, diagnostic, and biotechnology applications.
RESUMO
Success of enzymes as drugs requires that they persist within target tissues over therapeutically effective time frames. Here we report a general strategy to anchor enzymes at injection sites via fusion to galectin-3 (G3), a carbohydrate-binding protein. Fusing G3 to luciferase extended bioluminescence in subcutaneous tissue to ~7 days, whereas unmodified luciferase was undetectable within hours. Engineering G3-luciferase fusions to self-assemble into a trimeric architecture extended bioluminescence in subcutaneous tissue to 14 days, and intramuscularly to 3 days. The longer local half-life of the trimeric assembly was likely due to its higher carbohydrate-binding affinity compared to the monomeric fusion. G3 fusions and trimeric assemblies lacked extracellular signaling activity of wild-type G3 and did not accumulate in blood after subcutaneous injection, suggesting low potential for deleterious off-site effects. G3-mediated anchoring to common tissue glycans is expected to be broadly applicable for improving local pharmacokinetics of various existing and emerging enzyme drugs.
Assuntos
Galectina 3/metabolismo , Luciferases/metabolismo , Polissacarídeos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Espaço Extracelular/metabolismo , Feminino , Galectina 3/genética , Humanos , Injeções Subcutâneas , Células Jurkat , Luciferases/genética , Medições Luminescentes/métodos , Camundongos Endogâmicos C57BL , Músculos/metabolismo , Ligação Proteica , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/farmacocinética , Tela Subcutânea/metabolismoRESUMO
Carbohydrate-modified biomaterials are attractive candidates for disrupting natural protein-glycan binding events because they present ligands in multivalent arrangements that can address the weak affinity of monovalent protein-carbohydrate interactions. However, protein binding depends on physical aspects of immobilized carbohydrate display, such as density and valency, which are often difficult to predict and can vary for different types of biomaterials. Here, we report on protein interactions with ß-sheet peptide nanofibers with tunable immobilized carbohydrate content, which were prepared by co-assembling QQKFQFQFEQQ (Q11) with a glycosylated variant modified with N-acetylglucosamine (GQ11) at different molar ratios. The rate of protein binding increased as carbohydrate density decreased, with nanofibers having a GQ11 : Q11 molar ratio of 1 : 3 reaching equilibrium faster than formulations with a GQ11 mole fraction of 1. Larger proteins demonstrated a lower extent of binding than smaller proteins; however, the optimal range of carbohydrate densities was independent of the protein size. Nanofibers with the highest apparent protein binding affinity inhibited T cell death induced by wheat germ agglutinin (WGA) more effectively than did sub-optimal formulations, because they bound more protein within biologically relevant time frames (min to h). Collectively, these observations suggest that tuning carbohydrate density via co-assembly of glycosylated and non-glycosylated Q11 variants can maximize multivalent avidity effects while minimizing steric penalties. We anticipate that this approach will enable rapid iterative development of biomaterials with optimal activity for inhibiting the protein-glycan interactions implicated in disease progression.
Assuntos
Acetilglucosamina/química , Nanofibras/química , Peptídeos/química , Acetilglucosamina/administração & dosagem , Apoptose/efeitos dos fármacos , Materiais Biocompatíveis/administração & dosagem , Materiais Biocompatíveis/química , Sobrevivência Celular/efeitos dos fármacos , Glicosilação , Humanos , Células Jurkat , Nanofibras/administração & dosagem , Peptídeos/administração & dosagem , Lectinas de Plantas/química , Ligação Proteica , Conformação Proteica em Folha beta , Aglutininas do Germe de Trigo/química , Aglutininas do Germe de Trigo/metabolismoRESUMO
Synthetic materials that can engage the innate and adaptive immune systems are receiving increasing interest to confer protection against onset of future disease, such as pathogen infection, as well as to treat established diseases, such as autoimmunity and cancer. Carbohydrates are integral to various immune-related processes, including inflammation, adaptive memory, and tolerance, through both their non-covalent recognition of carbohydrate-binding proteins and as chemical signatures that distinguish self from non-self. Harnessing the biological activity of carbohydrates, however, was long hindered by the lack of a 'sugar code' defining their structure-function relationships, the difficulty of carbohydrate synthesis, and the weak binding affinity of monovalent carbohydrates for proteins or other biomolecules. The advent of new glycan synthesis approaches, combined with increased understanding of the role of multivalent carbohydrate clusters in immunology, has spurred significant recent growth in the development of multivalent synthetic materials modified with carbohydrates (i.e."glycomaterials") to activate, temper, or inhibit specific immunological processes. In this review, we highlight recent advances in glycomaterials that can inhibit T cell apoptosis, establish antigen-specific tolerance, suppress inflammation, or inhibit viral entry into host cells via non-covalent recognition of carbohydrate-binding proteins. In addition, we survey glycomaterials that can act as vaccines for adaptive immunological recognition and memory of carbohydrate antigens, with a particular emphasis on vaccines against tumor-associated carbohydrate antigens as cancer immunotherapies. In total, these examples demonstrate the enormous potential of glycomaterials to prevent or treat diverse diseases by engaging specific immunological processes.
RESUMO
With a growing number of bioactive protein drugs approved for clinical use each year, there is increasing need for vehicles for localized protein delivery to reduce administered doses, prevent off-target activity, and maintain protein bioactivity. Ideal protein delivery vehicles provide high encapsulation efficiency of bioactive drug, enable fine-tuning of protein release profiles, are biocompatible, and can be administered via minimally-invasive routes. Here we developed an approach to create micron-sized hydrated gels (i.e."microgels") for protein delivery that fulfill these requirements via desolvation of self-assembled ß-sheet peptide nanofibers. Specifically, aqueous solutions of peptide nanofibers were diluted under stirring conditions in a "desolvating agent", such as ethanol, which is miscible with water but poorly solvates peptides. The desolvating agent induced nanofiber physical crosslinking into microgels that retained ß-sheet secondary structure and were stable in aqueous solutions. Microgels did not activate dendritic cells in vitro, suggesting they are biocompatible. Peptide nanofibers and proteins having similar non-solvent immiscibility properties were co-desolvated to produce protein-loaded microgels with loading efficiencies of â¼85%. Encapsulated bioactive proteins rapidly diffused into bulk aqueous media, as expected for hydrated gels. Modifying peptide nanofibers with a protein-binding ligand provided tunable affinity-controlled protein release. Biocompatible microgels formed via desolvation of self-assembled peptide nanofibers are therefore likely to be broadly useful as vehicles for localized delivery of bioactive proteins, as well as other therapeutic molecules.
RESUMO
Galectins are carbohydrate-binding proteins that act as extracellular signaling molecules in various normal and pathological processes. Galectin bioactivity is mediated by specific non-covalent interactions with cell-surface and extracellular matrix (ECM) glycoproteins, which can enhance or inhibit signaling events that influence various cellular behaviors, including adhesion, proliferation, differentiation, and apoptosis. Here, we developed a materials approach to modulate galectin bioactivity by mimicking natural galectin-glycoprotein interactions. Specifically, we created a variant of a peptide that self-assembles into ß-sheet nanofibers under aqueous conditions, QQKFQFQFEQQ (Q11), which has an asparagine residue modified with the monosaccharide N-acetylglucosamine (GlcNAc) at its N-terminus (GlcNAc-Q11). GlcNAc-Q11 self-assembled into ß-sheet nanofibers under similar conditions as Q11. Nanofibrillar GlcNAc moieties were efficiently converted to the galectin-binding disaccharide N-acetyllactosamine (LacNAc) via the enzyme ß-1,4-galactosyltransferase and the sugar donor UDP-galactose, while retaining ß-sheet structure and nanofiber morphology. LacNAc-Q11 nanofibers bound galectin-1 and -3 in a LacNAc concentration-dependent manner, although nanofibers bound galectin-1 with higher affinity than galectin-3. In contrast, galectin-1 bound weakly to GlcNAc-Q11 nanofibers, while no galectin-3 binding to these nanofibers was observed. Galectin-1 binding to LacNAc-Q11 nanofibers was specific because it could be inhibited by excess soluble ß-lactose, a galectin-binding carbohydrate. LacNAc-Q11 nanofibers inhibited galectin-1-mediated apoptosis of Jurkat T cells in a LacNAc concentration-dependent manner, but were unable to inhibit galectin-3 activity, consistent with galectin-binding affinity of the nanofibers. We envision that glycopeptide nanofibers capable of modulating galectin-1 bioactivity will be broadly useful as biomaterials for various medical applications, including cancer therapeutics, immunotherapy, tissue regeneration, and viral prophylaxis.
RESUMO
OBJECTIVES: The bleak prognosis associated with pancreatic cancer (PDAC) drives the need for the development of novel treatment methodologies. Here, we evaluate the applicability of 3 enzyme prodrug therapies for PDAC, which are simultaneously targeted to the tumor, tumor vasculature, and metastases via annexin V. In these therapies, annexin V is fused to an enzyme, creating a fusion protein that converts nontoxic drug precursors, prodrugs, into anticancer compounds while bound to the tumor, therefore mitigating the risk of side effects. METHODS: The binding strength of fusion proteins to the human PDAC cell lines Panc-1 and Capan-1 was measured via streptavidin-horseradish peroxidase binding to biotinylated fusion proteins. Cytotoxic efficacy was evaluated by treatment with saturating concentrations of fusion protein followed by varying concentrations of the corresponding prodrug plus docetaxel. RESULTS: All fusion proteins exhibited strong binding to PDAC cells, with dissociation constants between 0.02 and 1.15 nM. Cytotoxic efficacy was determined to be very good for 2 of the systems, both of which achieved complete cell death on at least 1 cell line at physiologically attainable prodrug concentrations. CONCLUSIONS: Strong binding of fusion proteins to PDAC cells and effective cytotoxicity demonstrate the potential applicability of enzyme prodrug therapy to the treatment of PDAC.