RESUMEN
The WHO declared the COVID-19 outbreak a public health emergency of international concern. The causative agent of this acute respiratory disease is a newly emerged coronavirus, named SARS-CoV-2, which originated in China in late 2019. Exposure to SARS-CoV-2 leads to multifaceted disease outcomes from asymptomatic infection to severe pneumonia, acute respiratory distress and potentially death. Understanding the host immune response is crucial for the development of interventional strategies. Humoral responses play an important role in defending viral infections and are therefore of particular interest. With the aim to resolve SARS-CoV-2-specific humoral immune responses at the epitope level, we screened clinically well-characterized sera from COVID-19 patients with mild and severe disease outcome using high-density peptide microarrays covering the entire proteome of SARS-CoV-2. Moreover, we determined the longevity of epitope-specific antibody responses in a longitudinal approach. Here we present IgG and IgA-specific epitope signatures from COVID-19 patients, which may serve as discriminating prognostic or predictive markers for disease outcome and/or could be relevant for intervention strategies.
Asunto(s)
COVID-19/inmunología , Epítopos/inmunología , Proteoma/inmunología , SARS-CoV-2/inmunología , Adulto , Anticuerpos Antivirales/inmunología , Femenino , Humanos , Inmunidad Humoral , Inmunoglobulina A/inmunología , Inmunoglobulina G/inmunología , MasculinoRESUMEN
Discovery and optimization of potency and selectivity of a non-Zn-chelating MMP-13 inhibitor with the aid of protein co-crystal structural information is reported. This inhibitor was observed to have a binding mode distinct from previously published MMP-13 inhibitors. Potency and selectivity were improved by extending the hit structure out from the active site into the S1' pocket.
Asunto(s)
Quelantes/farmacología , Metaloproteinasa 13 de la Matriz/metabolismo , Inhibidores de la Metaloproteinasa de la Matriz , Inhibidores de Proteasas/farmacología , Dominio Catalítico , Quelantes/química , Metaloproteinasa 13 de la Matriz/química , Modelos Moleculares , Inhibidores de Proteasas/química , Unión Proteica , Relación Estructura-ActividadRESUMEN
The over-expression and aggregation of α-synuclein (αSyn) are linked to the onset and pathology of Parkinson's disease. Native monomeric αSyn exists in an intrinsically disordered ensemble of interconverting conformations, which has made its therapeutic targeting by small molecules highly challenging. Nonetheless, here we successfully target the monomeric structural ensemble of αSyn and thereby identify novel drug-like small molecules that impact multiple pathogenic processes. Using a surface plasmon resonance high-throughput screen, in which monomeric αSyn is incubated with microchips arrayed with tethered compounds, we identified novel αSyn interacting drug-like compounds. Because these small molecules could impact a variety of αSyn forms present in the ensemble, we tested representative hits for impact on multiple αSyn malfunctions in vitro and in cells including aggregation and perturbation of vesicular dynamics. We thereby identified a compound that inhibits αSyn misfolding and is neuroprotective, multiple compounds that restore phagocytosis impaired by αSyn overexpression, and a compound blocking cellular transmission of αSyn. Our studies demonstrate that drug-like small molecules that interact with native αSyn can impact a variety of its pathological processes. Thus, targeting the intrinsically disordered ensemble of αSyn offers a unique approach to the development of small molecule research tools and therapeutics for Parkinson's disease.
Asunto(s)
Bibliotecas de Moléculas Pequeñas/farmacología , alfa-Sinucleína/metabolismo , Amiloide/antagonistas & inhibidores , Amiloide/metabolismo , Línea Celular , Transferencia Resonante de Energía de Fluorescencia , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Proteínas Intrínsecamente Desordenadas/metabolismo , Fagocitosis/efectos de los fármacos , Pliegue de Proteína , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/toxicidad , Resonancia por Plasmón de Superficie , alfa-Sinucleína/química , alfa-Sinucleína/efectos de los fármacosRESUMEN
Libraries of small molecules were searched for Fc-fragment selective binders to a recombinant human antibody ("MDJ8â³, IgG(1)-subtype, κ-light chain) via SPR-based screening of chemical microarrays. Identified hit structures were immobilised on NHS-activated Sepharose for the determination of MDJ8 binding and selectivity versus typical proteineous impurities represented by the spend cell culture supernatant. Columns were packed and the most promising ligands further characterized in terms of binding constants, binding kinetics, as well as dynamic and equilibrium binding capacities. The performance of the best ligand, 2A10, was compared to standard Protein A chromatography. Using ligand 2A10 antibody capture from unprocessed cell culture supernatants was possible at similar recovery yield (>90%), purity (>80%), and eluting concentration (approximately 1 g/L) as with Protein A. Affinity constants (K(d)) of 2A10 were an order of magnitude higher than for the Protein A material, but still in the nM-range, while maximum binding capacities and binding kinetics were in the same order of magnitude. Ligand 2A10 was also able to capture a murine monoclonal antibody, again with similar efficiency as Protein A, as well as a number of humanised therapeutic antibodies. Antibody elution from the 2A10 column was possible using the Protein A standard protocol, i.e. 100mM glycine HCl pH 3.0, but also at near physiological pH, when some organic solvent or modifiers were present. Ligand 2A10 thus constitutes a cheaper, more robust alternative to Protein A as possible generic antibody binder. Moreover, the outlined approach to ligand selection could in principle by used to create suitable affinity ligands for other high value biotech products.
Asunto(s)
Anticuerpos/aislamiento & purificación , Cromatografía de Afinidad/métodos , Análisis por Micromatrices/métodos , Animales , Anticuerpos/metabolismo , Electroforesis en Gel de Poliacrilamida , Humanos , Concentración de Iones de Hidrógeno , Concentración Osmolar , Unión Proteica , Estabilidad Proteica , Conejos , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Sefarosa/análogos & derivados , Sefarosa/química , Bibliotecas de Moléculas PequeñasRESUMEN
The goal of this study was to explore the applicability of surface plasmon resonance (SPR)-based fragment screening to identify compounds that bind to factor VIIa (FVIIa). Based on pharmacophore models virtual screening approaches, we selected fragments anticipated to have a reasonable chance of binding to the S1-binding pocket of FVIIa and immobilized these compounds on microarrays. In affinity fingerprinting experiments, a number of compounds were identified to be specifically interacting with FVIIa and shown to fall into four structural classes. The results demonstrate that the chemical microarray technology platform using SPR detection generates unique chemobiological information that is useful for de novo discovery and lead development and allows the detection of weak interactions with ligands of low molecular weight.