RESUMEN
Interleukin (IL)-23 and IL-17 are well-validated therapeutic targets in autoinflammatory diseases. Antibodies targeting IL-23 and IL-17 have shown clinical efficacy but are limited by high costs, safety risks, lack of sustained efficacy, and poor patient convenience as they require parenteral administration. Here, we present designed miniproteins inhibiting IL-23R and IL-17 with antibody-like, low picomolar affinities at a fraction of the molecular size. The minibinders potently block cell signaling in vitro and are extremely stable, enabling oral administration and low-cost manufacturing. The orally administered IL-23R minibinder shows efficacy better than a clinical anti-IL-23 antibody in mouse colitis and has a favorable pharmacokinetics (PK) and biodistribution profile in rats. This work demonstrates that orally administered de novo-designed minibinders can reach a therapeutic target past the gut epithelial barrier. With high potency, gut stability, and straightforward manufacturability, de novo-designed minibinders are a promising modality for oral biologics.
Asunto(s)
Colitis , Interleucina-17 , Células Th17 , Animales , Administración Oral , Ratones , Humanos , Ratas , Colitis/tratamiento farmacológico , Interleucina-17/metabolismo , Interleucina-17/antagonistas & inhibidores , Células Th17/inmunología , Receptores de Interleucina/metabolismo , Receptores de Interleucina/antagonistas & inhibidores , Ratones Endogámicos C57BL , Masculino , Interleucina-23/metabolismo , Interleucina-23/antagonistas & inhibidores , Distribución Tisular , Femenino , Ratas Sprague-DawleyRESUMEN
The de novo design of polar protein-protein interactions is challenging because of the thermodynamic cost of stripping water away from the polar groups. Here, we describe a general approach for designing proteins which complement exposed polar backbone groups at the edge of beta sheets with geometrically matched beta strands. We used this approach to computationally design small proteins that bind to an exposed beta sheet on the human transferrin receptor (hTfR), which shuttles interacting proteins across the blood-brain barrier (BBB), opening up avenues for drug delivery into the brain. We describe a design which binds hTfR with a 20 nM Kd, is hyperstable, and crosses an in vitro microfluidic organ-on-a-chip model of the human BBB. Our design approach provides a general strategy for creating binders to protein targets with exposed surface beta edge strands.
Asunto(s)
Ingeniería de Proteínas/métodos , Receptores de Transferrina/metabolismo , Receptores de Transferrina/fisiología , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Sistemas de Liberación de Medicamentos , Humanos , Proteínas/metabolismo , Transferrina/metabolismoRESUMEN
In all cell types, protein homeostasis, or "proteostasis," is maintained by sophisticated quality control networks that regulate protein synthesis, folding, trafficking, aggregation, disaggregation, and degradation. In one notable example, Escherichia coli employ a proteostasis system that determines whether substrates of the twin-arginine translocation (Tat) pathway are correctly folded and thus suitable for transport across the tightly sealed cytoplasmic membrane. Herein, we review growing evidence that the Tat translocase itself discriminates folded proteins from those that are misfolded and/or aggregated, preferentially exporting only the former. Genetic suppressors that inactivate this mechanism have recently been isolated and provide direct evidence for the participation of the Tat translocase in structural proofreading of its protein substrates. We also discuss how this discriminatory "folding sensor" has been exploited for the discovery of structural probes (e.g., sequence mutations, pharmacologic chaperones, intracellular antibodies) that modulate the folding and solubility of virtually any protein-of-interest, including those associated with aggregation diseases (e.g., α-synuclein, amyloid-ß protein). Taken together, these studies highlight the utility of engineered bacteria for rapidly and inexpensively uncovering potent anti-aggregation factors.
Asunto(s)
Amiloide/análisis , Técnicas Biosensibles/métodos , Amiloide/metabolismo , Anticuerpos/metabolismo , Biotecnología , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Ensayos Analíticos de Alto Rendimiento , Espacio Intracelular/química , Espacio Intracelular/metabolismo , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/metabolismo , Modelos Moleculares , Técnicas de Sonda Molecular , Unión Proteica , Pliegue de ProteínaRESUMEN
Low expression, poor solubility, and polyspecificity are significant obstacles that have impeded the development of antibodies discovered from in vitro display libraries. Current biophysical characterization tools that identify these 'developability' problems are typically only applied after the discovery process, and thus limited to perhaps a few hundred candidates. We report a flow cytometric assay using a polyspecificity reagent (PSR) that allows for the identification and counter selection of polyspecific antibodies both during and after the selection process. The reported assay correlates well with cross-interaction chromatography, a surrogate for antibody solubility, as well as a baculovirus particle enzyme-linked immunosorbent assay, a surrogate for in vivo clearance. However, unlike these assays, PSR labeling is compatible both with screening of individual antibodies as well as selections of large antibody libraries. To this end, we demonstrate the ability to counter-select against polyspecificity while enriching for antigen affinity from a diverse antibody library, which enables simultaneous evolution of both antigen binding and superior non-target-related properties during the discovery process.
Asunto(s)
Anticuerpos/genética , Técnicas de Visualización de Superficie Celular/métodos , Citometría de Flujo , Levaduras/citología , Anticuerpos/química , Anticuerpos/inmunología , Línea Celular , Membrana Celular/metabolismo , Reacciones Cruzadas , Evolución Molecular Dirigida , Humanos , Estabilidad Proteica , Solubilidad , TemperaturaRESUMEN
The "flavin destructase" enzyme BluB catalyzes the unprecedented conversion of flavin mononucleotide (FMN) to 5,6-dimethylbenzimidazole (DMB), a component of vitamin B(12). Because of its unusual chemistry, the mechanism of this transformation has remained elusive. This study reports the identification of 12 mutant forms of BluB that have severely reduced catalytic function, though most retain the ability to bind flavin. The "flavin destructase" BluB is an unusual enzyme that fragments the flavin cofactor FMNH(2) in the presence of oxygen to produce 5,6-dimethylbenzimidazole (DMB), the lower axial ligand of vitamin B(12) (cobalamin). Despite the similarities in sequence and structure between BluB and the nitroreductase and flavin oxidoreductase enzyme families, BluB is the only enzyme known to fragment a flavin isoalloxazine ring. To explore the catalytic residues involved in this unusual reaction, mutants of BluB impaired in DMB biosynthesis were identified in a genetic screen in the bacterium Sinorhizobium meliloti. Of the 16 unique point mutations identified in the screen, the majority were located in conserved residues in the active site or in the unique "lid" domain proposed to shield the active site from solvent. Steady-state enzyme assays of 12 purified mutant proteins showed a significant reduction in DMB synthesis in all of the mutants, with eight completely defective in DMB production. Ten of these mutants have weaker binding affinities for both oxidized and reduced FMN, though only two have a significant effect on complex stability. These results implicate several conserved residues in BluB's unique ability to fragment FMNH(2) and demonstrate the sensitivity of BluB's active site to structural perturbations. This work lays the foundation for mechanistic studies of this enzyme and further advances our understanding of the structure-function relationship of BluB.