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
Many growth factors and cytokines signal by binding to the extracellular domains of their receptors and driving association and transphosphorylation of the receptor intracellular tyrosine kinase domains, initiating downstream signaling cascades. To enable systematic exploration of how receptor valency and geometry affect signaling outcomes, we designed cyclic homo-oligomers with up to 8 subunits using repeat protein building blocks that can be modularly extended. By incorporating a de novo-designed fibroblast growth factor receptor (FGFR)-binding module into these scaffolds, we generated a series of synthetic signaling ligands that exhibit potent valency- and geometry-dependent Ca2+ release and mitogen-activated protein kinase (MAPK) pathway activation. The high specificity of the designed agonists reveals distinct roles for two FGFR splice variants in driving arterial endothelium and perivascular cell fates during early vascular development. Our designed modular assemblies should be broadly useful for unraveling the complexities of signaling in key developmental transitions and for developing future therapeutic applications.
Asunto(s)
Diferenciación Celular , Factores de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento de Fibroblastos , Transducción de Señal , Animales , Humanos , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Ratones , Ligandos , Calcio/metabolismo , Sistema de Señalización de MAP QuinasasRESUMEN
We use computational design coupled with experimental characterization to systematically investigate the design principles for macrocycle membrane permeability and oral bioavailability. We designed 184 6-12 residue macrocycles with a wide range of predicted structures containing noncanonical backbone modifications and experimentally determined structures of 35; 29 are very close to the computational models. With such control, we show that membrane permeability can be systematically achieved by ensuring all amide (NH) groups are engaged in internal hydrogen bonding interactions. 84 designs over the 6-12 residue size range cross membranes with an apparent permeability greater than 1 × 10-6 cm/s. Designs with exposed NH groups can be made membrane permeable through the design of an alternative isoenergetic fully hydrogen-bonded state favored in the lipid membrane. The ability to robustly design membrane-permeable and orally bioavailable peptides with high structural accuracy should contribute to the next generation of designed macrocycle therapeutics.
Asunto(s)
Amidas , Péptidos , Amidas/química , Hidrógeno , Enlace de Hidrógeno , Lípidos , Péptidos/químicaRESUMEN
Respiratory syncytial virus (RSV) is a worldwide public health concern for which no vaccine is available. Elucidation of the prefusion structure of the RSV F glycoprotein and its identification as the main target of neutralizing antibodies have provided new opportunities for development of an effective vaccine. Here, we describe the structure-based design of a self-assembling protein nanoparticle presenting a prefusion-stabilized variant of the F glycoprotein trimer (DS-Cav1) in a repetitive array on the nanoparticle exterior. The two-component nature of the nanoparticle scaffold enabled the production of highly ordered, monodisperse immunogens that display DS-Cav1 at controllable density. In mice and nonhuman primates, the full-valency nanoparticle immunogen displaying 20 DS-Cav1 trimers induced neutralizing antibody responses â¼10-fold higher than trimeric DS-Cav1. These results motivate continued development of this promising nanoparticle RSV vaccine candidate and establish computationally designed two-component nanoparticles as a robust and customizable platform for structure-based vaccine design.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Virus Sincitiales Respiratorios/inmunología , Vacunación/métodos , Animales , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Antivirales/inmunología , Caveolina 1 , Línea Celular , Células HEK293 , Humanos , Ratones , Ratones Endogámicos BALB C , Nanopartículas/uso terapéutico , Cultivo Primario de Células , Virus Sincitiales Respiratorios/patogenicidad , Vacunas/inmunología , Proteínas Virales de Fusión/inmunología , Proteínas Virales de Fusión/metabolismo , Proteínas Virales de Fusión/fisiologíaRESUMEN
Many peptide hormones form an α-helix on binding their receptors1-4, and sensitive methods for their detection could contribute to better clinical management of disease5. De novo protein design can now generate binders with high affinity and specificity to structured proteins6,7. However, the design of interactions between proteins and short peptides with helical propensity is an unmet challenge. Here we describe parametric generation and deep learning-based methods for designing proteins to address this challenge. We show that by extending RFdiffusion8 to enable binder design to flexible targets, and to refining input structure models by successive noising and denoising (partial diffusion), picomolar-affinity binders can be generated to helical peptide targets by either refining designs generated with other methods, or completely de novo starting from random noise distributions without any subsequent experimental optimization. The RFdiffusion designs enable the enrichment and subsequent detection of parathyroid hormone and glucagon by mass spectrometry, and the construction of bioluminescence-based protein biosensors. The ability to design binders to conformationally variable targets, and to optimize by partial diffusion both natural and designed proteins, should be broadly useful.
Asunto(s)
Diseño Asistido por Computadora , Aprendizaje Profundo , Péptidos , Proteínas , Técnicas Biosensibles , Difusión , Glucagón/química , Glucagón/metabolismo , Mediciones Luminiscentes , Espectrometría de Masas , Hormona Paratiroidea/química , Hormona Paratiroidea/metabolismo , Péptidos/química , Péptidos/metabolismo , Estructura Secundaria de Proteína , Proteínas/química , Proteínas/metabolismo , Especificidad por Sustrato , Modelos MolecularesRESUMEN
General approaches for designing sequence-specific peptide-binding proteins would have wide utility in proteomics and synthetic biology. However, designing peptide-binding proteins is challenging, as most peptides do not have defined structures in isolation, and hydrogen bonds must be made to the buried polar groups in the peptide backbone1-3. Here, inspired by natural and re-engineered protein-peptide systems4-11, we set out to design proteins made out of repeating units that bind peptides with repeating sequences, with a one-to-one correspondence between the repeat units of the protein and those of the peptide. We use geometric hashing to identify protein backbones and peptide-docking arrangements that are compatible with bidentate hydrogen bonds between the side chains of the protein and the peptide backbone12. The remainder of the protein sequence is then optimized for folding and peptide binding. We design repeat proteins to bind to six different tripeptide-repeat sequences in polyproline II conformations. The proteins are hyperstable and bind to four to six tandem repeats of their tripeptide targets with nanomolar to picomolar affinities in vitro and in living cells. Crystal structures reveal repeating interactions between protein and peptide interactions as designed, including ladders of hydrogen bonds from protein side chains to peptide backbones. By redesigning the binding interfaces of individual repeat units, specificity can be achieved for non-repeating peptide sequences and for disordered regions of native proteins.
Asunto(s)
Péptidos , Ingeniería de Proteínas , Proteínas , Secuencia de Aminoácidos , Modelos Moleculares , Péptidos/química , Péptidos/metabolismo , Proteínas/química , Proteínas/metabolismo , Ingeniería de Proteínas/métodos , Enlace de Hidrógeno , Unión Proteica , Pliegue de Proteína , Conformación ProteicaRESUMEN
The design of proteins that bind to a specific site on the surface of a target protein using no information other than the three-dimensional structure of the target remains a challenge1-5. Here we describe a general solution to this problem that starts with a broad exploration of the vast space of possible binding modes to a selected region of a protein surface, and then intensifies the search in the vicinity of the most promising binding modes. We demonstrate the broad applicability of this approach through the de novo design of binding proteins to 12 diverse protein targets with different shapes and surface properties. Biophysical characterization shows that the binders, which are all smaller than 65 amino acids, are hyperstable and, following experimental optimization, bind their targets with nanomolar to picomolar affinities. We succeeded in solving crystal structures of five of the binder-target complexes, and all five closely match the corresponding computational design models. Experimental data on nearly half a million computational designs and hundreds of thousands of point mutants provide detailed feedback on the strengths and limitations of the method and of our current understanding of protein-protein interactions, and should guide improvements of both. Our approach enables the targeted design of binders to sites of interest on a wide variety of proteins for therapeutic and diagnostic applications.
Asunto(s)
Proteínas Portadoras , Proteínas , Aminoácidos/metabolismo , Sitios de Unión , Proteínas Portadoras/metabolismo , Unión Proteica , Proteínas/químicaRESUMEN
Naturally occurring protein switches have been repurposed for the development of biosensors and reporters for cellular and clinical applications1. However, the number of such switches is limited, and reengineering them is challenging. Here we show that a general class of protein-based biosensors can be created by inverting the flow of information through de novo designed protein switches in which the binding of a peptide key triggers biological outputs of interest2. The designed sensors are modular molecular devices with a closed dark state and an open luminescent state; analyte binding drives the switch from the closed to the open state. Because the sensor is based on the thermodynamic coupling of analyte binding to sensor activation, only one target binding domain is required, which simplifies sensor design and allows direct readout in solution. We create biosensors that can sensitively detect the anti-apoptosis protein BCL-2, the IgG1 Fc domain, the HER2 receptor, and Botulinum neurotoxin B, as well as biosensors for cardiac troponin I and an anti-hepatitis B virus antibody with the high sensitivity required to detect these molecules clinically. Given the need for diagnostic tools to track the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)3, we used the approach to design sensors for the SARS-CoV-2 spike protein and antibodies against the membrane and nucleocapsid proteins. The former, which incorporates a de novo designed spike receptor binding domain (RBD) binder4, has a limit of detection of 15 pM and a luminescence signal 50-fold higher than the background level. The modularity and sensitivity of the platform should enable the rapid construction of sensors for a wide range of analytes, and highlights the power of de novo protein design to create multi-state protein systems with new and useful functions.
Asunto(s)
Anticuerpos Antivirales/análisis , Técnicas Biosensibles/métodos , Virus de la Hepatitis B/inmunología , SARS-CoV-2/química , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/análisis , Troponina I/análisis , Anticuerpos Antivirales/inmunología , Técnicas Biosensibles/normas , Toxinas Botulínicas/análisis , Proteínas de la Nucleocápside de Coronavirus/inmunología , Inmunoglobulina G/análisis , Inmunoglobulina G/inmunología , Límite de Detección , Luminiscencia , Fosfoproteínas/inmunología , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Receptor ErbB-2/análisis , Sensibilidad y Especificidad , Proteínas de la Matriz Viral/inmunologíaRESUMEN
Influenza vaccines that confer broad and durable protection against diverse viral strains would have a major effect on global health, as they would lessen the need for annual vaccine reformulation and immunization1. Here we show that computationally designed, two-component nanoparticle immunogens2 induce potently neutralizing and broadly protective antibody responses against a wide variety of influenza viruses. The nanoparticle immunogens contain 20 haemagglutinin glycoprotein trimers in an ordered array, and their assembly in vitro enables the precisely controlled co-display of multiple distinct haemagglutinin proteins in defined ratios. Nanoparticle immunogens that co-display the four haemagglutinins of licensed quadrivalent influenza vaccines elicited antibody responses in several animal models against vaccine-matched strains that were equivalent to or better than commercial quadrivalent influenza vaccines, and simultaneously induced broadly protective antibody responses to heterologous viruses by targeting the subdominant yet conserved haemagglutinin stem. The combination of potent receptor-blocking and cross-reactive stem-directed antibodies induced by the nanoparticle immunogens makes them attractive candidates for a supraseasonal influenza vaccine candidate with the potential to replace conventional seasonal vaccines3.
Asunto(s)
Anticuerpos ampliamente neutralizantes/inmunología , Virus de la Influenza A/clasificación , Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Gripe Humana/inmunología , Gripe Humana/prevención & control , Nanomedicina , Nanopartículas , Animales , Modelos Animales de Enfermedad , Femenino , Hurones/inmunología , Hurones/virología , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Humanos , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H3N2 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/química , Gripe Humana/virología , Masculino , Ratones , Ratones Endogámicos BALB C , Modelos MolecularesRESUMEN
In natural proteins, structured loops have central roles in molecular recognition, signal transduction and enzyme catalysis. However, because of the intrinsic flexibility and irregularity of loop regions, organizing multiple structured loops at protein functional sites has been very difficult to achieve by de novo protein design. Here we describe a solution to this problem that designs tandem repeat proteins with structured loops (9-14 residues) buttressed by extensive hydrogen bonding interactions. Experimental characterization shows that the designs are monodisperse, highly soluble, folded and thermally stable. Crystal structures are in close agreement with the design models, with the loops structured and buttressed as designed. We demonstrate the functionality afforded by loop buttressing by designing and characterizing binders for extended peptides in which the loops form one side of an extended binding pocket. The ability to design multiple structured loops should contribute generally to efforts to design new protein functions.
Asunto(s)
Enlace de Hidrógeno , Modelos Moleculares , Proteínas , Proteínas/química , Proteínas/metabolismo , Cristalografía por Rayos X , Conformación Proteica , Pliegue de Proteína , Ingeniería de Proteínas/métodos , Secuencia de Aminoácidos , Sitios de Unión , Péptidos/química , Péptidos/metabolismoRESUMEN
Natural photosystems couple light harvesting to charge separation using a 'special pair' of chlorophyll molecules that accepts excitation energy from the antenna and initiates an electron-transfer cascade. To investigate the photophysics of special pairs independently of the complexities of native photosynthetic proteins, and as a first step toward creating synthetic photosystems for new energy conversion technologies, we designed C2-symmetric proteins that hold two chlorophyll molecules in closely juxtaposed arrangements. X-ray crystallography confirmed that one designed protein binds two chlorophylls in the same orientation as native special pairs, whereas a second designed protein positions them in a previously unseen geometry. Spectroscopy revealed that the chlorophylls are excitonically coupled, and fluorescence lifetime imaging demonstrated energy transfer. The cryo-electron microscopy structure of a designed 24-chlorophyll octahedral nanocage with a special pair on each edge closely matched the design model. The results suggest that the de novo design of artificial photosynthetic systems is within reach of current computational methods.
Asunto(s)
Clorofila , Clorofila/química , Clorofila/metabolismo , Cristalografía por Rayos X , Modelos Moleculares , Fotosíntesis , Transferencia de Energía , Microscopía por Crioelectrón , Conformación Proteica , Complejos de Proteína Captadores de Luz/química , Complejos de Proteína Captadores de Luz/metabolismoRESUMEN
Transmembrane channels and pores have key roles in fundamental biological processes1 and in biotechnological applications such as DNA nanopore sequencing2-4, resulting in considerable interest in the design of pore-containing proteins. Synthetic amphiphilic peptides have been found to form ion channels5,6, and there have been recent advances in de novo membrane protein design7,8 and in redesigning naturally occurring channel-containing proteins9,10. However, the de novo design of stable, well-defined transmembrane protein pores that are capable of conducting ions selectively or are large enough to enable the passage of small-molecule fluorophores remains an outstanding challenge11,12. Here we report the computational design of protein pores formed by two concentric rings of α-helices that are stable and monodisperse in both their water-soluble and their transmembrane forms. Crystal structures of the water-soluble forms of a 12-helical pore and a 16-helical pore closely match the computational design models. Patch-clamp electrophysiology experiments show that, when expressed in insect cells, the transmembrane form of the 12-helix pore enables the passage of ions across the membrane with high selectivity for potassium over sodium; ion passage is blocked by specific chemical modification at the pore entrance. When incorporated into liposomes using in vitro protein synthesis, the transmembrane form of the 16-helix pore-but not the 12-helix pore-enables the passage of biotinylated Alexa Fluor 488. A cryo-electron microscopy structure of the 16-helix transmembrane pore closely matches the design model. The ability to produce structurally and functionally well-defined transmembrane pores opens the door to the creation of designer channels and pores for a wide variety of applications.
Asunto(s)
Simulación por Computador , Genes Sintéticos/genética , Canales Iónicos/química , Canales Iónicos/genética , Modelos Moleculares , Biología Sintética , Línea Celular , Microscopía por Crioelectrón , Cristalografía por Rayos X , Conductividad Eléctrica , Escherichia coli/genética , Escherichia coli/metabolismo , Hidrazinas , Canales Iónicos/metabolismo , Transporte Iónico , Liposomas/metabolismo , Técnicas de Placa-Clamp , Porinas/química , Porinas/genética , Porinas/metabolismo , Ingeniería de Proteínas , Estructura Secundaria de Proteína , Solubilidad , Agua/químicaRESUMEN
We describe a de novo computational approach for designing proteins that recapitulate the binding sites of natural cytokines, but are otherwise unrelated in topology or amino acid sequence. We use this strategy to design mimics of the central immune cytokine interleukin-2 (IL-2) that bind to the IL-2 receptor ßγc heterodimer (IL-2Rßγc) but have no binding site for IL-2Rα (also called CD25) or IL-15Rα (also known as CD215). The designs are hyper-stable, bind human and mouse IL-2Rßγc with higher affinity than the natural cytokines, and elicit downstream cell signalling independently of IL-2Rα and IL-15Rα. Crystal structures of the optimized design neoleukin-2/15 (Neo-2/15), both alone and in complex with IL-2Rßγc, are very similar to the designed model. Neo-2/15 has superior therapeutic activity to IL-2 in mouse models of melanoma and colon cancer, with reduced toxicity and undetectable immunogenicity. Our strategy for building hyper-stable de novo mimetics could be applied generally to signalling proteins, enabling the creation of superior therapeutic candidates.
Asunto(s)
Diseño de Fármacos , Interleucina-15/inmunología , Interleucina-2/inmunología , Imitación Molecular , Receptores de Interleucina-2/agonistas , Receptores de Interleucina-2/inmunología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/inmunología , Simulación por Computador , Cristalografía por Rayos X , Modelos Animales de Enfermedad , Humanos , Interleucina-15/uso terapéutico , Interleucina-2/uso terapéutico , Subunidad alfa del Receptor de Interleucina-2/inmunología , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Melanoma/tratamiento farmacológico , Melanoma/inmunología , Ratones , Modelos Moleculares , Estabilidad Proteica , Receptores de Interleucina-2/metabolismo , Transducción de Señal/inmunologíaRESUMEN
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
De novo protein design holds promise for creating small stable proteins with shapes customized to bind therapeutic targets. We describe a massively parallel approach for designing, manufacturing and screening mini-protein binders, integrating large-scale computational design, oligonucleotide synthesis, yeast display screening and next-generation sequencing. We designed and tested 22,660 mini-proteins of 37-43 residues that target influenza haemagglutinin and botulinum neurotoxin B, along with 6,286 control sequences to probe contributions to folding and binding, and identified 2,618 high-affinity binders. Comparison of the binding and non-binding design sets, which are two orders of magnitude larger than any previously investigated, enabled the evaluation and improvement of the computational model. Biophysical characterization of a subset of the binder designs showed that they are extremely stable and, unlike antibodies, do not lose activity after exposure to high temperatures. The designs elicit little or no immune response and provide potent prophylactic and therapeutic protection against influenza, even after extensive repeated dosing.
Asunto(s)
Diseño de Fármacos , Gripe Humana/tratamiento farmacológico , Gripe Humana/prevención & control , Terapia Molecular Dirigida/métodos , Ingeniería de Proteínas/métodos , Proteínas/química , Proteínas/uso terapéutico , Toxinas Botulínicas/clasificación , Toxinas Botulínicas/metabolismo , Simulación por Computador , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Calor , Humanos , Gripe Humana/metabolismo , Simulación de Dinámica Molecular , Unión Proteica , Estabilidad Proteica , Proteínas/inmunología , Proteínas/metabolismo , TemperaturaRESUMEN
The glycoside hydrolases encoded by the human gut microbiome play an integral role in processing a variety of exogenous and endogenous glycoconjugates. Here we present three structurally and functionally distinct ß-glucuronidase (GUS) glycoside hydrolases from a single human gut commensal microbe, Bacteroides uniformis We show using nine crystal structures, biochemical, and biophysical data that whereas these three proteins share similar overall folds, they exhibit different structural features that create three structurally and functionally unique enzyme active sites. Notably, quaternary structure plays an important role in creating distinct active site features that are hard to predict via structural modeling methods. The enzymes display differential processing capabilities toward glucuronic acid-containing polysaccharides and SN-38-glucuronide, a metabolite of the cancer drug irinotecan. We also demonstrate that GUS-specific and nonselective inhibitors exhibit varying potencies toward each enzyme. Together, these data highlight the diversity of GUS enzymes within a single Bacteroides gut commensal and advance our understanding of how structural details impact the specific roles microbial enzymes play in processing drug-glucuronide and glycan substrates.
Asunto(s)
Bacteroides/enzimología , Microbioma Gastrointestinal , Glucuronidasa/química , Glucuronidasa/metabolismo , Isoenzimas/química , Isoenzimas/metabolismo , Secuencia de Aminoácidos , Dominio Catalítico , Inhibidores Enzimáticos/farmacología , Ácido Glucárico/análogos & derivados , Glucuronidasa/antagonistas & inhibidores , Humanos , Conformación ProteicaRESUMEN
Broadly neutralizing antibodies targeting a highly conserved region in the hemagglutinin (HA) stem protect against influenza infection. Here, we investigate the protective efficacy of a protein (HB36.6) computationally designed to bind with high affinity to the same region in the HA stem. We show that intranasal delivery of HB36.6 affords protection in mice lethally challenged with diverse strains of influenza independent of Fc-mediated effector functions or a host antiviral immune response. This designed protein prevents infection when given as a single dose of 6.0 mg/kg up to 48 hours before viral challenge and significantly reduces disease when administered as a daily therapeutic after challenge. A single dose of 10.0 mg/kg HB36.6 administered 1-day post-challenge resulted in substantially better protection than 10 doses of oseltamivir administered twice daily for 5 days. Thus, binding of HB36.6 to the influenza HA stem region alone, independent of a host response, is sufficient to reduce viral infection and replication in vivo. These studies demonstrate the potential of computationally designed binding proteins as a new class of antivirals for influenza.
Asunto(s)
Anticuerpos Antivirales/inmunología , Proteínas Portadoras/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Virus de la Influenza A/inmunología , Gripe Humana/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Citocinas/metabolismo , Humanos , Virus de la Influenza A/genética , Gripe Humana/virología , Ratones , Modelos Moleculares , Mutación , Replicación ViralRESUMEN
Selection of aptamers from nucleic acid libraries by in vitro evolution represents a powerful method of identifying high-affinity ligands for a broad range of molecular targets. Nevertheless, a sizeable fraction of proteins remain difficult targets due to inherently limited chemical diversity of nucleic acids. We have exploited synthetic nucleotide modifications that confer protein-like diversity on a nucleic acid scaffold, resulting in a new generation of binding reagents called SOMAmers (Slow Off-rate Modified Aptamers). Here we report a unique crystal structure of a SOMAmer bound to its target, platelet-derived growth factor B (PDGF-BB). The SOMAmer folds into a compact structure and exhibits a hydrophobic binding surface that mimics the interface between PDGF-BB and its receptor, contrasting sharply with mainly polar interactions seen in traditional protein-binding aptamers. The modified nucleotides circumvent the intrinsic diversity constraints of natural nucleic acids, thereby greatly expanding the structural vocabulary of nucleic acid ligands and considerably broadening the range of accessible protein targets.
Asunto(s)
Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Proteínas Proto-Oncogénicas c-sis/metabolismo , Técnica SELEX de Producción de Aptámeros/métodos , Secuencias de Aminoácidos/genética , Becaplermina , Cristalografía por Rayos X , Cartilla de ADN/genética , Datos de Secuencia Molecular , Estructura Molecular , Fosforilación , Unión Proteica , Proteínas Proto-Oncogénicas c-sis/química , Análisis de Secuencia de ADN , Temperatura de TransiciónRESUMEN
Cytokine release syndrome (CRS), commonly known as cytokine storm, is an acute systemic inflammatory response that is a significant global health threat. Interleukin-6 (IL-6) and interleukin-1 (IL-1) are key pro-inflammatory cytokines involved in CRS and are hence critical therapeutic targets. Current antagonists, such as tocilizumab and anakinra, target IL-6R/IL-1R but have limitations due to their long half-life and systemic anti-inflammatory effects, making them less suitable for acute or localized treatments. Here we present the de novo design of small protein antagonists that prevent IL-1 and IL-6 from interacting with their receptors to activate signaling. The designed proteins bind to the IL-6R, GP130 (an IL-6 co-receptor), and IL-1R1 receptor subunits with binding affinities in the picomolar to low-nanomolar range. X-ray crystallography studies reveal that the structures of these antagonists closely match their computational design models. In a human cardiac organoid disease model, the IL-1R antagonists demonstrated protective effects against inflammation and cardiac damage induced by IL-1ß. These minibinders show promise for administration via subcutaneous injection or intranasal/inhaled routes to mitigate acute cytokine storm effects.
Asunto(s)
Síndrome de Liberación de Citoquinas , Interleucina-6 , Humanos , Síndrome de Liberación de Citoquinas/tratamiento farmacológico , Interleucina-6/metabolismo , Interleucina-6/antagonistas & inhibidores , Cristalografía por Rayos X , Receptores de Interleucina-6/antagonistas & inhibidores , Receptores de Interleucina-6/metabolismo , Interleucina-1/metabolismo , Interleucina-1/antagonistas & inhibidores , Proteína Antagonista del Receptor de Interleucina 1/farmacología , Proteína Antagonista del Receptor de Interleucina 1/química , Proteína Antagonista del Receptor de Interleucina 1/metabolismo , Diseño de Fármacos , Receptor gp130 de Citocinas/metabolismo , Receptor gp130 de Citocinas/antagonistas & inhibidores , Receptor gp130 de Citocinas/química , Unión Proteica , Transducción de Señal/efectos de los fármacos , Receptores Tipo I de Interleucina-1/antagonistas & inhibidores , Receptores Tipo I de Interleucina-1/metabolismoRESUMEN
There remains a critical need for new antibiotics against multi-drug-resistant Gram-negative bacteria, a major global threat that continues to impact mortality rates. Lipoprotein signal peptidase II is an essential enzyme in the lipoprotein biosynthetic pathway of Gram-negative bacteria, making it an attractive target for antibacterial drug discovery. Although natural inhibitors of LspA have been identified, such as the cyclic depsipeptide globomycin, poor stability and production difficulties limit their use in a clinical setting. We harness computational design to generate stable de novo cyclic peptide analogues of globomycin. Only 12 peptides needed to be synthesized and tested to yield potent inhibitors, avoiding costly preparation of large libraries and screening campaigns. The most potent analogues showed comparable or better antimicrobial activity than globomycin in microdilution assays against ESKAPE-E pathogens. This work highlights computational design as a general strategy to combat antibiotic resistance.