RESUMO
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.
Assuntos
Colite , Interleucina-17 , Células Th17 , Animais , Administração Oral , Camundongos , Humanos , Ratos , Colite/tratamento farmacológico , Interleucina-17/metabolismo , Interleucina-17/antagonistas & inibidores , Células Th17/imunologia , Receptores de Interleucina/metabolismo , Receptores de Interleucina/antagonistas & inibidores , Camundongos Endogâmicos C57BL , Masculino , Interleucina-23/metabolismo , Interleucina-23/antagonistas & inibidores , Distribuição Tecidual , Feminino , Ratos Sprague-DawleyRESUMO
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.
Assuntos
Diferenciação Celular , Fatores de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos , Transdução de Sinais , Animais , Humanos , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Camundongos , Ligantes , Cálcio/metabolismo , Sistema de Sinalização das MAP QuinasesRESUMO
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.
Assuntos
Ligação de Hidrogênio , Modelos Moleculares , Proteínas , Proteínas/química , Proteínas/metabolismo , Cristalografia por Raios X , Conformação Proteica , Dobramento de Proteína , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Sítios de Ligação , Peptídeos/química , Peptídeos/metabolismoRESUMO
There is an increasing recognition of the importance of diagnosing genetic conditions with an ever-growing list of genetic testing options. However, most providers do not have formal genetics training, which makes choosing the most appropriate test to order challenging. Our project sought to improve cytogenetic testing utilization in a tertiary care neonatal intensive care unit (NICU) through utilizing quality improvement techniques, specifically the Model for Improvement framework with rapid Plan-Do-Study-Act cycles. Our project utilized various interventions including the implementation of a NICU genetic testing algorithm. Interventions demonstrated improvement in all areas, specifically a 92% reduction in unnecessary cytogenetic testing with improvement in the diagnostic rate. Our work also resulted in a 59% decrease in charges with an estimated projected savings of $21,000 per year. Quality improvement can minimize redundancies and inefficiencies in genetic testing in a Level IV NICU in a large tertiary care children's hospital and result in substantial cost-savings.
RESUMO
The continued evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has compromised neutralizing antibody responses elicited by prior infection or vaccination and abolished the utility of most monoclonal antibody therapeutics. We previously described a computationally-designed, homotrimeric miniprotein inhibitor, designated TRI2-2, that protects mice against pre-Omicron SARS-CoV-2 variants. Here, we show that TRI2-2 exhibits pan neutralization of variants that evolved during the 4.5 years since the emergence of SARS-CoV-2 and protects mice against BQ.1.1, XBB.1.5 and BA.2.86 challenge when administered post-exposure by an intranasal route. The resistance of TRI2-2 to viral escape and its direct delivery to the upper airways rationalize a path toward clinical advancement.
RESUMO
Over the past few decades, the development of potent and safe immune-activating adjuvant technologies has become the heart of intensive research in the constant fight against highly mutative and immune evasive viruses such as influenza, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and human immunodeficiency virus (HIV). Herein, we developed a highly modular saponin-based nanoparticle platform incorporating Toll-like receptor agonists (TLRas) including TLR1/2a, TLR4a, and TLR7/8a adjuvants and their mixtures. These various TLRa-saponin nanoparticle adjuvant constructs induce unique acute cytokine and immune-signaling profiles, leading to specific T helper responses that could be of interest depending on the target disease for prevention. In a murine vaccine study, the adjuvants greatly improved the potency, durability, breadth, and neutralization of both COVID-19 and HIV vaccine candidates, suggesting the potential broad application of these adjuvant constructs to a range of different antigens. Overall, this work demonstrates a modular TLRa-SNP adjuvant platform that could improve the design of vaccines and affect modern vaccine development.
Assuntos
Adjuvantes Imunológicos , Vacinas contra COVID-19 , Nanopartículas , SARS-CoV-2 , Saponinas , Agonistas do Receptor Semelhante a Toll , Animais , Humanos , Camundongos , Adjuvantes Imunológicos/farmacologia , Adjuvantes de Vacinas/química , Vacinas contra a AIDS/imunologia , COVID-19/prevenção & controle , COVID-19/imunologia , COVID-19/virologia , Vacinas contra COVID-19/imunologia , Citocinas/metabolismo , Nanopartículas/química , Saponinas/farmacologia , Saponinas/química , Saponinas/imunologia , SARS-CoV-2/imunologiaRESUMO
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.
Assuntos
Síndrome da Liberação de Citocina , Interleucina-6 , Humanos , Síndrome da Liberação de Citocina/tratamento farmacológico , Interleucina-6/metabolismo , Interleucina-6/antagonistas & inibidores , Cristalografia por Raios X , Receptores de Interleucina-6/antagonistas & inibidores , Receptores de Interleucina-6/metabolismo , Interleucina-1/metabolismo , Interleucina-1/antagonistas & inibidores , Proteína Antagonista do Receptor de Interleucina 1/farmacologia , Proteína Antagonista do Receptor de Interleucina 1/química , Proteína Antagonista do Receptor de Interleucina 1/metabolismo , Desenho de Fármacos , Receptor gp130 de Citocina/metabolismo , Receptor gp130 de Citocina/antagonistas & inibidores , Receptor gp130 de Citocina/química , Ligação Proteica , Transdução de Sinais/efeitos dos fármacos , Receptores Tipo I de Interleucina-1/antagonistas & inibidores , Receptores Tipo I de Interleucina-1/metabolismoRESUMO
While there has been progress in the de novo design of small globular miniproteins (50-65 residues) to bind to primarily concave regions of a target protein surface, computational design of minibinders to convex binding sites remains an outstanding challenge due to low level of overall shape complementarity. Here, we describe a general approach to generate computationally designed proteins which bind to convex target sites that employ geometrically matching concave scaffolds. We used this approach to design proteins binding to TGFßRII, CTLA-4 and PD-L1 which following experimental optimization have low nanomolar to picomolar affinities and potent biological activity. Co-crystal structures of the TGFßRII and CTLA-4 binders in complex with the receptors are in close agreement with the design models. Our approach provides a general route to generating very high affinity binders to convex protein target sites.