RESUMEN
Oxidative stress is a central part of innate immune-induced neurodegeneration. However, the transcriptomic landscape of central nervous system (CNS) innate immune cells contributing to oxidative stress is unknown, and therapies to target their neurotoxic functions are not widely available. Here, we provide the oxidative stress innate immune cell atlas in neuroinflammatory disease and report the discovery of new druggable pathways. Transcriptional profiling of oxidative stress-producing CNS innate immune cells identified a core oxidative stress gene signature coupled to coagulation and glutathione-pathway genes shared between a microglia cluster and infiltrating macrophages. Tox-seq followed by a microglia high-throughput screen and oxidative stress gene network analysis identified the glutathione-regulating compound acivicin, with potent therapeutic effects that decrease oxidative stress and axonal damage in chronic and relapsing multiple sclerosis models. Thus, oxidative stress transcriptomics identified neurotoxic CNS innate immune populations and may enable discovery of selective neuroprotective strategies.
Asunto(s)
Encefalomielitis Autoinmune Experimental/genética , Perfilación de la Expresión Génica/métodos , Microglía/fisiología , Esclerosis Múltiple/genética , Inflamación Neurogénica/genética , Animales , Antioxidantes/uso terapéutico , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Femenino , Redes Reguladoras de Genes , Ensayos Analíticos de Alto Rendimiento , Humanos , Inmunidad Innata , Isoxazoles/uso terapéutico , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Esclerosis Múltiple/tratamiento farmacológico , Inflamación Neurogénica/tratamiento farmacológico , Estrés Oxidativo , Análisis de Secuencia de ARN , Análisis de la Célula IndividualRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Activation of innate immunity and deposition of blood-derived fibrin in the central nervous system (CNS) occur in autoimmune and neurodegenerative diseases, including multiple sclerosis (MS) and Alzheimer's disease (AD). However, the mechanisms that link disruption of the blood-brain barrier (BBB) to neurodegeneration are poorly understood, and exploration of fibrin as a therapeutic target has been limited by its beneficial clotting functions. Here we report the generation of monoclonal antibody 5B8, targeted against the cryptic fibrin epitope γ377-395, to selectively inhibit fibrin-induced inflammation and oxidative stress without interfering with clotting. 5B8 suppressed fibrin-induced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and the expression of proinflammatory genes. In animal models of MS and AD, 5B8 entered the CNS and bound to parenchymal fibrin, and its therapeutic administration reduced the activation of innate immunity and neurodegeneration. Thus, fibrin-targeting immunotherapy inhibited autoimmunity- and amyloid-driven neurotoxicity and might have clinical benefit without globally suppressing innate immunity or interfering with coagulation in diverse neurological diseases.
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Anticuerpos Monoclonales/inmunología , Fibrinógeno/antagonistas & inhibidores , Enfermedades Neurodegenerativas/inmunología , Animales , Epítopos , Humanos , Inflamación/inmunología , Ratones , RatasRESUMEN
The utility of antigen-binding antibody fragments is often limited by their short half-lives. Half-life extension of such fragments is usually accomplished by attachment or binding to high-molecular-weight carriers that reduce the renal elimination rate. However, the higher hydrodynamic radius results in greater confinement in the vascular compartment and, thus, lower tissue distribution. We have developed a chemically controlled drug delivery system in which the drug is covalently attached to hydrogel microspheres by a self-cleaving ß-eliminative linker; upon subcutaneous injection, the t1/2,ß of the released drug acquires the t1/2 of linker cleavage. In the present work, we compared the pharmacokinetics of an anti-TNFα scFv, the same scFv attached to 40 kDa PEG by a stable linker, and the scFv attached to hydrogel microspheres by a self-cleaving linker. We also developed a general approach for the selective attachment of ß-eliminative linkers to the N-termini of proteins. In rats, the scFv had a t1/2,ß of 4 h and a high volume of distribution at steady state (Vd,SS), suggesting extensive tissue distribution. The PEG-scFv conjugate had an increased t1/2,ß of about 2 days but showed a reduced Vd,SS that was similar to the plasma volume. In contrast, the tissue-penetrable scFv released from the hydrogel system had a t1/2,ß of about 2 weeks. Thus, the cleavable microsphere-scFv conjugate releases its protein cargo with a prolonged half-life comparable to that of most full-length mAbs and in a form that has the high tissue distribution characteristic of smaller mAb fragments. Other antigen-binding antibody fragments should be amenable to the half-life extension approach described here.
RESUMEN
The glucocorticoid receptor initiates the cellular response to glucocorticoid steroid hormones in vertebrates. Co-regulator proteins dock to the receptor in response to hormone binding and potentiate the transcriptional activity of the receptor by modifying DNA and recruiting essential transcription factors like RNA polymerase II. Hormones and co-regulators bind at distinct sites in the ligand binding domain yet function cooperatively to mediate transcriptional control. This study reveals and quantifies energetic coupling between two binding sites using purified components. Using a library of peptides taken from co-regulator proteins, we determine the pattern of co-regulator binding to the glucocorticoid receptor ligand binding domain. We show that peptides from co-regulators differ in their effects on hormone binding and kinetics. Peptides from DAX1 and SRC1 bind with similar affinity, but DAX1 binding is coupled to hormone binding, and SRC1 is not. Mechanistic details of co-regulator binding and coupling to the hormone binding pocket are uncovered by analysis of properties endowed by mutation of a key residue in the allosteric network connecting the sites.
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Glucocorticoides/metabolismo , Receptores de Glucocorticoides/metabolismo , Regulación Alostérica , Secuencia de Aminoácidos , Humanos , Cinética , Datos de Secuencia Molecular , Unión Proteica , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
We have developed a chemically-controlled drug delivery system in which a drug is covalently attached via a carbamate to hydrogel microspheres using a ß-eliminative linker; rate-determining proton removal from a CH bond adjacent to an electron withdrawing group results in a ß-elimination to cleave the carbamate and release the drug. After subcutaneous injection of the hydrogel-drug conjugate, the drug is slowly released into the systemic circulation and acquires an elimination t1/2,ß that matches the t1/2 of linker cleavage. A similar ß-eliminative linker with a slower cleavage rate is installed into crosslinks of the polymer to trigger gel degradation after drug release. We have now prepared ß-eliminative linkers that contain deuterium in place of the hydrogen whose removal initiates cleavage. In vitro model systems of drug release and degelation show large primary deuterium kinetic isotope effects of kH/kDâ¯~â¯2.5 to 3.5. Using a deuterated linker to attach the peptide octreotide to hydrogel-microspheres, the in vivo t1/2,ß of the drug was increased from ~1.5 to 4.5â¯weeks in the rat. Similarly, the in vivo time to biodegradation of hydrogels with deuterium-containing crosslinks could be extended ~2.5-fold compared to hydrogen-containing counterparts. Thus, the use of primary deuterium kinetic isotope effects in a single platform technology can control rates of ß-elimination reactions in drug release and polymer biodegradation rates.
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Deuterio/química , Sistemas de Liberación de Medicamentos , Octreótido/administración & dosificación , Polímeros/química , Animales , Reactivos de Enlaces Cruzados/química , Portadores de Fármacos/química , Liberación de Fármacos , Semivida , Hidrogeles , Octreótido/química , Octreótido/farmacocinética , RatasRESUMEN
Prostate cancer is a leading killer of men in the industrialized world. Underlying this disease is the aberrant action of the androgen receptor (AR). AR is distinguished from other nuclear receptors in that after hormone binding, it preferentially responds to a specialized set of coactivators bearing aromatic-rich motifs, while responding poorly to coactivators bearing the leucine-rich "NR box" motifs favored by other nuclear receptors. Under normal conditions, interactions with these AR-specific coactivators through aromatic-rich motifs underlie targeted gene transcription. However, during prostate cancer, abnormal association with such coactivators, as well as with coactivators containing canonical leucine-rich motifs, promotes disease progression. To understand the paradox of this unusual selectivity, we have derived a complete set of peptide motifs that interact with AR using phage display. Binding affinities were measured for a selected set of these peptides and their interactions with AR determined by X-ray crystallography. Structures of AR in complex with FxxLF, LxxLL, FxxLW, WxxLF, WxxVW, FxxFF, and FxxYF motifs reveal a changing surface of the AR coactivator binding interface that permits accommodation of both AR-specific aromatic-rich motifs and canonical leucine-rich motifs. Induced fit provides perfect mating of the motifs representing the known family of AR coactivators and suggests a framework for the design of AR coactivator antagonists.
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Regulación Neoplásica de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/química , Receptores Androgénicos/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteínas Portadoras/química , Cristalografía por Rayos X , Proteínas del Citoesqueleto/química , Proteínas de Unión al ADN/química , Humanos , Cinética , Proteínas con Dominio LIM , Leucina/química , Masculino , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/química , Coactivador 2 del Receptor Nuclear/química , Coactivadores de Receptor Nuclear , Proteínas Oncogénicas/química , Biblioteca de Péptidos , Péptidos/química , Neoplasias de la Próstata/metabolismo , Unión Proteica , Conformación Proteica , Estructura Secundaria de Proteína , Receptores de Estrógenos/química , Receptores de Glucocorticoides/química , Homología de Secuencia de Aminoácido , Resonancia por Plasmón de Superficie , Receptores beta de Hormona Tiroidea/química , Factores de Tiempo , Factores de Transcripción/químicaRESUMEN
We have developed a chemically controlled very long-acting delivery system to support once-monthly administration of a peptidic GLP-1R agonist. Initially, the prototypical GLP-1R agonist exenatide was covalently attached to hydrogel microspheres by a self-cleaving ß-eliminative linker; after subcutaneous injection in rats, the peptide was slowly released into the systemic circulation. However, the short serum exenatide half-life suggested its degradation in the subcutaneous depot. We found that exenatide undergoes deamidation at Asn28 with an in vitro and in vivo half-life of approximately 2 weeks. The [Gln28]exenatide variant and exenatide showed indistinguishable GLP-1R agonist activities as well as pharmacokinetic and pharmacodynamic effects in rodents; however, unlike exenatide, [Gln28]exenatide is stable for long periods. Two different hydrogel-[Gln28]exenatide conjugates were prepared using ß-eliminative linkers with different cleavage rates. After subcutaneous injection in rodents, the serum half-lives for the released [Gln28]exenatide from the two conjugates were about 2 weeks and one month. Two monthly injections of the latter in the Zucker diabetic fatty rat showed pharmacodynamic effects indistinguishable from two months of continuously infused exenatide. Pharmacokinetic simulations indicate that the delivery system should serve well as a once-monthly GLP-1R agonist for treatment of type 2 diabetes in humans.
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Diabetes Mellitus Tipo 2/tratamiento farmacológico , Sistemas de Liberación de Medicamentos , Receptor del Péptido 1 Similar al Glucagón/agonistas , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Hipoglucemiantes/administración & dosificación , Microesferas , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato/administración & dosificación , Hidrogel de Polietilenoglicol-Dimetacrilato/farmacocinética , Hipoglucemiantes/farmacocinética , Estructura Molecular , Factores de TiempoRESUMEN
Fragment-based lead discovery complements high-throughput screening and computer-aided drug design for the discovery of small-molecule inhibitors of protein-protein interactions. Fragments are molecules with molecular masses ca 280 Da or smaller, and are generally screened using structural or biophysical approaches. Several methods of fragment-based screening are feasible for any soluble protein that can be expressed and purified; specific techniques also have size limitations and/or require multiple milligrams of protein. This chapter describes some of the most common fragment-discovery methods, including surface plasmon resonance, nuclear magnetic resonance, differential scanning fluorimetry, and X-ray crystallography.