Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 10 de 10
Filtrar
1.
Proc Natl Acad Sci U S A ; 120(46): e2306129120, 2023 Nov 14.
Artículo en Inglés | MEDLINE | ID: mdl-37939083

RESUMEN

Controlling the biodistribution of protein- and nanoparticle-based therapeutic formulations remains challenging. In vivo library selection is an effective method for identifying constructs that exhibit desired distribution behavior; library variants can be selected based on their ability to localize to the tissue or compartment of interest despite complex physiological challenges. Here, we describe further development of an in vivo library selection platform based on self-assembling protein nanoparticles encapsulating their own mRNA genomes (synthetic nucleocapsids or synNCs). We tested two distinct libraries: a low-diversity library composed of synNC surface mutations (45 variants) and a high-diversity library composed of synNCs displaying miniproteins with binder-like properties (6.2 million variants). While we did not identify any variants from the low-diversity surface library that yielded therapeutically relevant changes in biodistribution, the high-diversity miniprotein display library yielded variants that shifted accumulation toward lungs or muscles in just two rounds of in vivo selection. Our approach should contribute to achieving specific tissue homing patterns and identifying targeting ligands for diseases of interest.


Asunto(s)
Biblioteca de Péptidos , Proteínas , Distribución Tisular , Nucleocápside , Mutación
2.
Trends Biochem Sci ; 46(11): 863-865, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-34456123

RESUMEN

Viruses are fascinating molecular machines that inspire many therapeutic design efforts. Tetter, Terasaka, Steinauer et al. recently reported the laboratory evolution of a synthetic nucleocapsid from a bacterial enzyme. Their work sheds light on the potential origin of viruses and points the way to improved nanotechnology platforms.


Asunto(s)
Cápside , Virus , Proteínas de la Cápside , Nanotecnología , Virión
3.
Bioconjug Chem ; 33(11): 2018-2034, 2022 11 16.
Artículo en Inglés | MEDLINE | ID: mdl-35487503

RESUMEN

Despite remarkable advances over the past several decades, many therapeutic nanomaterials fail to overcome major in vivo delivery barriers. Controlling immunogenicity, optimizing biodistribution, and engineering environmental responsiveness are key outstanding delivery problems for most nanotherapeutics. However, notable exceptions exist including some lipid and polymeric nanoparticles, some virus-based nanoparticles, and nanoparticle vaccines where immunogenicity is desired. Self-assembling protein nanoparticles offer a powerful blend of modularity and precise designability to the field, and have the potential to solve many of the major barriers to delivery. In this review, we provide a brief overview of key designable features of protein nanoparticles and their implications for therapeutic delivery applications. We anticipate that protein nanoparticles will rapidly grow in their prevalence and impact as clinically relevant delivery platforms.


Asunto(s)
Nanopartículas , Distribución Tisular , Proteínas/uso terapéutico , Polímeros , Sistemas de Liberación de Medicamentos
4.
bioRxiv ; 2023 Jun 12.
Artículo en Inglés | MEDLINE | ID: mdl-37398153

RESUMEN

The RGD (Arg-Gly-Asp)-binding integrins αvß6 and αvß8 are clinically validated cancer and fibrosis targets of considerable therapeutic importance. Compounds that can discriminate between the two closely related integrin proteins and other RGD integrins, stabilize specific conformational states, and have sufficient stability enabling tissue restricted administration could have considerable therapeutic utility. Existing small molecules and antibody inhibitors do not have all of these properties, and hence there is a need for new approaches. Here we describe a method for computationally designing hyperstable RGD-containing miniproteins that are highly selective for a single RGD integrin heterodimer and conformational state, and use this strategy to design inhibitors of αvß6 and αvß8 with high selectivity. The αvß6 and αvß8 inhibitors have picomolar affinities for their targets, and >1000-fold selectivity over other RGD integrins. CryoEM structures are within 0.6-0.7Å root-mean-square deviation (RMSD) to the computational design models; the designed αvß6 inhibitor and native ligand stabilize the open conformation in contrast to the therapeutic anti-αvß6 antibody BG00011 that stabilizes the bent-closed conformation and caused on-target toxicity in patients with lung fibrosis, and the αvß8 inhibitor maintains the constitutively fixed extended-closed αvß8 conformation. In a mouse model of bleomycin-induced lung fibrosis, the αvß6 inhibitor potently reduced fibrotic burden and improved overall lung mechanics when delivered via oropharyngeal administration mimicking inhalation, demonstrating the therapeutic potential of de novo designed integrin binding proteins with high selectivity.

5.
Nat Commun ; 14(1): 5660, 2023 09 13.
Artículo en Inglés | MEDLINE | ID: mdl-37704610

RESUMEN

The RGD (Arg-Gly-Asp)-binding integrins αvß6 and αvß8 are clinically validated cancer and fibrosis targets of considerable therapeutic importance. Compounds that can discriminate between homologous αvß6 and αvß8 and other RGD integrins, stabilize specific conformational states, and have high thermal stability could have considerable therapeutic utility. Existing small molecule and antibody inhibitors do not have all these properties, and hence new approaches are needed. Here we describe a generalized method for computationally designing RGD-containing miniproteins selective for a single RGD integrin heterodimer and conformational state. We design hyperstable, selective αvß6 and αvß8 inhibitors that bind with picomolar affinity. CryoEM structures of the designed inhibitor-integrin complexes are very close to the computational design models, and show that the inhibitors stabilize specific conformational states of the αvß6 and the αvß8 integrins. In a lung fibrosis mouse model, the αvß6 inhibitor potently reduced fibrotic burden and improved overall lung mechanics, demonstrating the therapeutic potential of de novo designed integrin binding proteins with high selectivity.


Asunto(s)
Integrinas , Fibrosis Pulmonar , Animales , Ratones , Membrana Celular , Microscopía por Crioelectrón , Modelos Animales de Enfermedad
6.
Adv Healthc Mater ; 11(9): e2101651, 2022 05.
Artículo en Inglés | MEDLINE | ID: mdl-34706166

RESUMEN

Peptide-based cancer vaccines offer production and safety advantages but have had limited clinical success due to their intrinsic instability, rapid clearance, and low cellular uptake. Nanoparticle-based delivery vehicles can improve the in vivo stability and cellular uptake of peptide antigens. Here, a well-defined, self-assembling mannosylated polymer is developed for anticancer peptide antigen delivery. The amphiphilic polymer is prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization, and the peptide antigens are conjugated to the pH-sensitive hydrophobic block through the reversible disulfide linkage for selective release after cell entry. The polymer-peptide conjugates self-assemble into sub-100 nm micelles at physiological pH and dissociate at endosomal pH. The mannosylated micellar corona increases the accumulation of vaccine cargoes in the draining inguinal lymph nodes and facilitates nanoparticle uptake by professional antigen presenting cells. In vivo studies demonstrate that the mannosylated micelle formulation improves dendritic cell activation and enhances antigen-specific T cell responses, resulting in higher antitumor immunity in tumor-bearing mice compared to free peptide antigen. The mannosylated polymer is therefore a simple and promising platform for the delivery of peptide cancer vaccines.


Asunto(s)
Vacunas contra el Cáncer , Neoplasias , Animales , Antígenos , Sistemas de Liberación de Medicamentos , Ratones , Micelas , Neoplasias/terapia , Péptidos , Polímeros/química , Vacunas de Subunidad
7.
Nat Commun ; 13(1): 1825, 2022 04 05.
Artículo en Inglés | MEDLINE | ID: mdl-35383176

RESUMEN

Influenza virus neuraminidase (NA) is a major antiviral drug target and has recently reemerged as a key target of antibody-mediated protective immunity. Here we show that recombinant NAs across non-bat subtypes adopt various tetrameric conformations, including an "open" state that may help explain poorly understood variations in NA stability across viral strains and subtypes. We use homology-directed protein design to uncover the structural principles underlying these distinct tetrameric conformations and stabilize multiple recombinant NAs in the "closed" state, yielding two near-atomic resolution structures of NA by cryo-EM. In addition to enhancing thermal stability, conformational stabilization improves affinity to protective antibodies elicited by viral infection, including antibodies targeting a quaternary epitope and the broadly conserved catalytic site. Stabilized NAs can also be integrated into viruses without affecting fitness. Our findings provide a deeper understanding of NA structure, stability, and antigenicity, and establish design strategies for reinforcing the conformational integrity of recombinant NA proteins.


Asunto(s)
Neuraminidasa , Orthomyxoviridae/enzimología , Proteínas Virales , Anticuerpos Antivirales , Epítopos , Neuraminidasa/química , Proteínas Recombinantes/química , Proteínas Virales/química
8.
Science ; 369(6511): 1637-1643, 2020 09 25.
Artículo en Inglés | MEDLINE | ID: mdl-32820060

RESUMEN

Precise cell targeting is challenging because most mammalian cell types lack a single surface marker that distinguishes them from other cells. A solution would be to target cells using specific combinations of proteins present on their surfaces. In this study, we design colocalization-dependent protein switches (Co-LOCKR) that perform AND, OR, and NOT Boolean logic operations. These switches activate through a conformational change only when all conditions are met, generating rapid, transcription-independent responses at single-cell resolution within complex cell populations. We implement AND gates to redirect T cell specificity against tumor cells expressing two surface antigens while avoiding off-target recognition of single-antigen cells, and three-input switches that add NOT or OR logic to avoid or include cells expressing a third antigen. Thus, de novo designed proteins can perform computations on the surface of cells, integrating multiple distinct binding interactions into a single output.


Asunto(s)
Computadores Moleculares , Ingeniería de Proteínas/métodos , Proteínas/química , Antígenos de Superficie/química , Membrana Celular/química , Conformación Proteica
9.
Adv Drug Deliv Rev ; 130: 39-49, 2018 05.
Artículo en Inglés | MEDLINE | ID: mdl-29964079

RESUMEN

Targeting ligands are used in drug delivery to improve drug distribution to desired cells or tissues and to facilitate cellular entry. In vivo biopanning, whereby billions of potential ligand sequences are screened in biologically-relevant and complex conditions, is a powerful method for identification of novel target ligands. This tool has impacted drug delivery technologies and expanded our arsenal of therapeutics and diagnostics. Within this review we will discuss current in vivo panning technologies and ways that these technologies can be improved to advance next-generation drug delivery strategies.


Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Sistemas de Liberación de Medicamentos/tendencias , Diseño de Fármacos , Aptámeros de Nucleótidos/síntesis química , Aptámeros de Nucleótidos/química , Humanos , Ligandos , Especificidad por Sustrato
10.
PLoS One ; 11(1): e0147494, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-26799494

RESUMEN

Two-component signaling (2CS) systems enable bacterial cells to respond to changes in their local environment, often using a membrane-bound sensor protein and a cytoplasmic responder protein to regulate gene expression. Previous work has shown that Escherichia coli's natural EnvZ/OmpR 2CS could be modified to construct a light-sensing bacterial photography system. The resulting bacterial photographs, or "coliroids," rely on a phosphotransfer reaction between Cph8, a synthetic version of EnvZ that senses red light, and OmpR. Gene expression changes can be visualized through upregulation of a LacZ reporter gene by phosphorylated OmpR. Unfortunately, basal LacZ expression leads to a detectable reporter signal even when cells are grown in the light, diminishing the contrast of the coliroids. We performed site-directed mutagenesis near the phosphotransfer site of Cph8 to isolate mutants with potentially improved image contrast. Five mutants were examined, but only one of the mutants, T541S, increased the ratio of dark/light gene expression, as measured by ß-galactosidase activity. The ratio changed from 2.57 fold in the starting strain to 5.59 in the T541S mutant. The ratio decreased in the four other mutant strains we examined. The phenotype observed in the T541S mutant strain may arise because the serine sidechain is chemically similar but physically smaller than the threonine sidechain. This may minimally change the protein's local structure, but may be less sterically constrained when compared to threonine, resulting in a higher probability of a phosphotransfer event. Our initial success pairing synthetic biology and site-directed mutagenesis to optimize the bacterial photography system's performance encourages us to imagine further improvements to the performance of this and other synthetic systems, especially those based on 2CS signaling.


Asunto(s)
Mutagénesis Sitio-Dirigida/métodos , Transducción de Señal/genética , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Escherichia coli/genética , Escherichia coli/fisiología , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/fisiología , Regulación Bacteriana de la Expresión Génica/genética , Operón Lac/genética , Operón Lac/fisiología , Luz , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/fisiología , Organismos Modificados Genéticamente/genética , Organismos Modificados Genéticamente/fisiología , Fotograbar , Transactivadores/genética , Transactivadores/fisiología , beta-Galactosidasa/metabolismo
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA