RESUMEN
The mechanisms of HLA-DM-catalyzed peptide exchange remain uncertain. Here we found that all stages of the interaction of HLA-DM with HLA-DR were dependent on the occupancy state of the peptide-binding groove. High-affinity peptides were protected from removal by HLA-DM through two mechanisms: peptide binding induced the dissociation of a long-lived complex of empty HLA-DR and HLA-DM, and high-affinity HLA-DR-peptide complexes bound HLA-DM only very slowly. Nonbinding covalent HLA-DR-peptide complexes were converted into efficient HLA-DM binders after truncation of an N-terminal peptide segment that emptied the P1 pocket and disrupted conserved hydrogen bonds to HLA-DR. HLA-DM thus binds only to HLA-DR conformers in which a critical part of the binding site is already vacant because of spontaneous peptide motion.
Asunto(s)
Antígenos HLA-D/metabolismo , Antígeno HLA-DR2/metabolismo , Proteínas Mutantes/metabolismo , Fragmentos de Péptidos/metabolismo , Animales , Presentación de Antígeno , Células CHO , Catálisis , Cricetinae , Cricetulus , Antígenos HLA-D/química , Antígenos HLA-D/genética , Antígeno HLA-DR2/química , Antígeno HLA-DR2/genética , Humanos , Modelos Químicos , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Unión Proteica , Resonancia por Plasmón de Superficie , Transgenes/genéticaRESUMEN
Pridopidine has demonstrated improvement in Huntington Disease (HD) motor symptoms as measured by secondary endpoints in clinical trials. Originally described as a dopamine stabilizer, this mechanism is insufficient to explain the clinical and preclinical effects of pridopidine. This study therefore explored pridopidine's potential mechanisms of action. The effect of pridopidine versus sham treatment on genome-wide expression profiling in the rat striatum was analysed and compared to the pathological expression profile in Q175 knock-in (Q175 KI) vs Q25 WT mouse models. A broad, unbiased pathway analysis was conducted, followed by testing the enrichment of relevant pathways. Pridopidine upregulated the BDNF pathway (P = 1.73E-10), and its effect on BDNF secretion was sigma 1 receptor (S1R) dependent. Many of the same genes were independently found to be downregulated in Q175 KI mice compared to WT (5.2e-7 < P < 0.04). In addition, pridopidine treatment upregulated the glucocorticoid receptor (GR) response, D1R-associated genes and the AKT/PI3K pathway (P = 1E-10, P = 0.001, P = 0.004, respectively). Pridopidine upregulates expression of BDNF, D1R, GR and AKT/PI3K pathways, known to promote neuronal plasticity and survival, as well as reported to demonstrate therapeutic benefit in HD animal models. Activation of S1R, necessary for its effect on the BDNF pathway, represents a core component of the mode of action of pridopidine. Since the newly identified pathways are downregulated in neurodegenerative diseases, including HD, these findings suggest that pridopidine may exert neuroprotective effects beyond its role in alleviating some symptoms of HD.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/biosíntesis , Cuerpo Estriado/metabolismo , Enfermedad de Huntington/tratamiento farmacológico , Fármacos Neuroprotectores/administración & dosificación , Piperidinas/administración & dosificación , Animales , Factor Neurotrófico Derivado del Encéfalo/genética , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/patología , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/genética , Genoma , Humanos , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Ratones , Fármacos Neuroprotectores/metabolismo , Ratas , Receptores de Dopamina D5/biosíntesis , Receptores de Dopamina D5/genética , Receptores de Glucocorticoides/biosíntesis , Receptores de Glucocorticoides/genética , Transducción de Señal/efectos de los fármacosRESUMEN
It is known that differentiation of Th17 cells is promoted by activation of STAT3 and inhibited by activation of STAT1. Although both transcription factors are activated by several cytokines, including IL-6, IL-21, and IL-27, each of these cytokines has a very different effect on Th17 differentiation, ranging from strong induction (IL-6) to strong inhibition (IL-27). To determine the molecular basis for these differences, we measured STAT3 and STAT1 activation profiles for IL-6, IL-21, and IL-27, as well as for cytokine pairs over time. We found that the ratio of activated STAT3/activated STAT1 is crucial in determining whether cytokines promote or inhibit Th17 differentiation. IL-6 and IL-21 induced p-STAT3/p-STAT1 ratios > 1, leading to the promotion of Th17 differentiation, whereas IL-27 or IL-6+IL-27 induced p-STAT3/p-STAT1 ratios < 1, resulting in inhibition of Th17 differentiation. Consistent with these findings, we show that IL-27 induces sufficient p-STAT3 to promote Th17 differentiation in the absence of STAT1. Furthermore, IL-27-induced STAT1-deficient T cells were indistinguishable from bona fide highly proinflammatory Th17 cells because they induced severe experimental autoimmune encephalomyelitis upon adoptive transfer. Our results suggest that the ratio of p-STAT3/p-STAT1 induced by a cytokine or cytokine pairs can be used to predict whether they induce a competent Th17-differentiation program.
Asunto(s)
Interleucina-27/farmacología , Factor de Transcripción STAT1/fisiología , Transducción de Señal/fisiología , Células Th17/efectos de los fármacos , Traslado Adoptivo , Animales , Diferenciación Celular/efectos de los fármacos , Interleucina-6/farmacología , Interleucinas/farmacología , Ratones , Ratones Endogámicos C57BL , Factor de Transcripción STAT3/fisiología , Células Th17/citologíaRESUMEN
Copaxone (glatiramer acetate, GA), a structurally and compositionally complex polypeptide nonbiological drug, is an effective treatment for multiple sclerosis, with a well-established favorable safety profile. The short antigenic polypeptide sequences comprising therapeutically active epitopes in GA cannot be deciphered with state-of-the-art methods; and GA has no measurable pharmacokinetic profile and no validated pharmacodynamic markers. The study reported herein describes the use of orthogonal standard and high-resolution physicochemical and biological tests to characterize GA and a U.S. Food and Drug Administration-approved generic version of GA, Glatopa (USA-FoGA). While similarities were observed with low-resolution or destructive tests, differences between GA and USA-FoGA were measured with high-resolution methods applied to an intact mixture, including variations in surface charge and a unique, high-molecular-weight, hydrophobic polypeptide population observed only in some USA-FoGA lots. Consistent with published reports that modifications in physicochemical attributes alter immune-related processes, genome-wide expression profiles of ex vivo activated splenocytes from mice immunized with either GA or USA-FoGA showed that 7-11% of modulated genes were differentially expressed and enriched for immune-related pathways. Thus, differences between USA-FoGA and GA may include variations in antigenic epitopes that differentially activate immune responses. We propose that the assays reported herein should be considered during the regulatory assessment process for nonbiological complex drugs such as GA.
Asunto(s)
Medicamentos Genéricos/farmacología , Expresión Génica/efectos de los fármacos , Acetato de Glatiramer/farmacología , Fenómenos del Sistema Inmunológico/efectos de los fármacos , Animales , Células Cultivadas , Fenómenos Químicos , Medicamentos Genéricos/química , Medicamentos Genéricos/farmacocinética , Femenino , Perfilación de la Expresión Génica/métodos , Acetato de Glatiramer/química , Acetato de Glatiramer/farmacocinética , Humanos , Fenómenos del Sistema Inmunológico/genética , Inmunosupresores/química , Inmunosupresores/farmacocinética , Inmunosupresores/uso terapéutico , Ratones Endogámicos BALB C , Microscopía de Fuerza Atómica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción de Señal/inmunología , Bazo/citología , Bazo/efectos de los fármacos , Bazo/metabolismo , Equivalencia TerapéuticaRESUMEN
Glatiramer acetate (Copaxone®; GA) is a non-biological complex drug for multiple sclerosis. GA modulated thousands of genes in genome-wide expression studies conducted in THP-1 cells and mouse splenocytes. Comparing GA with differently-manufactured glatiramoid Polimunol (Synthon) in mice yielded hundreds of differentially expressed probesets, including biologically-relevant genes (e.g. Il18, adj p<9e-6) and pathways. In human monocytes, 700+ probesets differed between Polimunol and GA, enriching for 130+ pathways including response to lipopolysaccharide (adj. p<0.006). Key differences were confirmed by qRT-PCR (splenocytes) or proteomics (THP-1). These studies demonstrate the complexity of GA's mechanisms of action, and may help inform therapeutic equivalence assessment.
Asunto(s)
Acetato de Glatiramer/química , Acetato de Glatiramer/farmacología , Bazo/efectos de los fármacos , Bazo/fisiología , Adyuvantes Inmunológicos/química , Adyuvantes Inmunológicos/farmacología , Adyuvantes Inmunológicos/uso terapéutico , Animales , Línea Celular , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Acetato de Glatiramer/uso terapéutico , Humanos , Inmunosupresores/química , Inmunosupresores/farmacología , Inmunosupresores/uso terapéutico , Ratones , Ratones Endogámicos BALB C , Monocitos/efectos de los fármacos , Monocitos/fisiología , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/inmunologíaRESUMEN
To generate new insights into the biology of Alzheimer's Disease (AD), we developed methods to combine and reuse a wide variety of existing data sets in new ways. We first identified genes consistently associated with AD in each of four separate expression studies, and confirmed this result using a fifth study. We next developed algorithms to search hundreds of thousands of Gene Expression Omnibus (GEO) data sets, identifying a link between an AD-associated gene (NEUROD6) and gender. We therefore stratified patients by gender along with APOE4 status, and analyzed multiple SNP data sets to identify variants associated with AD. SNPs in either the region of NEUROD6 or SNAP25 were significantly associated with AD, in APOE4+ females and APOE4+ males, respectively. We developed algorithms to search Connectivity Map (CMAP) data for medicines that modulate AD-associated genes, identifying hypotheses that warrant further investigation for treating specific AD patient subsets. In contrast to other methods, this approach focused on integrating multiple gene expression datasets across platforms in order to achieve a robust intersection of disease-affected genes, and then leveraging these results in combination with genetic studies in order to prioritize potential genes for targeted therapy.
Asunto(s)
Enfermedad de Alzheimer/genética , Apolipoproteína E4/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteína 25 Asociada a Sinaptosomas/genética , Algoritmos , Enfermedad de Alzheimer/tratamiento farmacológico , Bases de Datos de Proteínas , Femenino , Regulación de la Expresión Génica/genética , Predisposición Genética a la Enfermedad , Humanos , Masculino , Polimorfismo de Nucleótido Simple/genética , Factores SexualesRESUMEN
Glatiramer Acetate (GA) has provided safe and effective treatment for multiple sclerosis (MS) patients for two decades. It acts as an antigen, yet the precise mechanism of action remains to be fully elucidated, and no validated pharmacokinetic or pharmacodynamic biomarkers exist. In order to better characterize GA's biological impact, genome-wide expression studies were conducted with a human monocyte (THP-1) cell line. Consistent with previous literature, branded GA upregulated anti-inflammatory markers (e.g. IL10), and modulated multiple immune-related pathways. Despite some similarities, significant differences were observed between expression profiles induced by branded GA and Probioglat, a differently-manufactured glatiramoid purported to be a generic GA. Key results were verified using qRT-PCR. Genes (e.g. CCL5, adj. p < 4.1 × 10(-5)) critically involved in pro-inflammatory pathways (e.g. response to lipopolysaccharide, adj. p = 8.7 × 10(-4)) were significantly induced by Probioglat compared with branded GA. Key genes were also tested and confirmed at the protein level, and in primary human monocytes. These observations suggest differential biological impact by the two glatiramoids and warrant further investigation.
Asunto(s)
Acetato de Glatiramer/farmacología , Transcriptoma/efectos de los fármacos , Línea Celular , Quimiocinas/genética , Quimiocinas/metabolismo , Humanos , Metaloproteinasas de la Matriz/genética , Metaloproteinasas de la Matriz/metabolismo , Monocitos/citología , Monocitos/efectos de los fármacos , Monocitos/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Regulación hacia Arriba/efectos de los fármacosRESUMEN
For decades, policies regarding generic medicines have sought to provide patients with economical access to safe and effective drugs, while encouraging the development of new therapies. This balance is becoming more challenging for physicians and regulators as biologics and non-biological complex drugs (NBCDs) such as glatiramer acetate demonstrate remarkable efficacy, because generics for these medicines are more difficult to assess. We sought to develop computational methods that use transcriptional profiles to compare branded medicines to generics, robustly characterizing differences in biological impact. We combined multiple computational methods to determine whether differentially expressed genes result from random variation, or point to consistent differences in biological impact of the generic compared to the branded medicine. We applied these methods to analyze gene expression data from mouse splenocytes exposed to either branded glatiramer acetate or a generic. The computational methods identified extensive evidence that branded glatiramer acetate has a more consistent biological impact across batches than the generic, and has a distinct impact on regulatory T cells and myeloid lineage cells. In summary, we developed a computational pipeline that integrates multiple methods to compare two medicines in an innovative way. This pipeline, and the specific findings distinguishing branded glatiramer acetate from a generic, can help physicians and regulators take appropriate steps to ensure safety and efficacy.
Asunto(s)
Medicamentos Genéricos/farmacología , Perfilación de la Expresión Génica , Péptidos/farmacología , Animales , Biomarcadores/metabolismo , Linaje de la Célula/efectos de los fármacos , Linaje de la Célula/genética , Factores de Transcripción Forkhead/metabolismo , Acetato de Glatiramer , Sistema Inmunológico/efectos de los fármacos , Sistema Inmunológico/metabolismo , Ratones , Monocitos/citología , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
The reason why IL-6 induces a pro-inflammatory response, while IL-10 induces an anti-inflammatory response, despite both cytokines activating the same transcription factor, STAT3, is not well understood. It is known that IL-6 induces a transient STAT3 signal and that IL-10 induces a sustained STAT3 signal due to the STAT3-induced inhibitor SOCS3's ability to bind to the IL-6R and not the IL-10R. We sought to develop a general transcriptional network that is capable of translating sustained signals into one response, while translating transient signals into a second response. The general structure of such a network is that the transcription factor STAT3 can induce both an inflammatory response and an anti-inflammatory response by inducing two different genes. The anti-inflammatory gene can bind to and inhibit the inflammatory gene's production and the inflammatory gene can bind to its own promoter and induce its own transcription in the absence of the signal. One prediction that can be made from such a network is that in SOCS3-/- mice, where IL-6 induces a sustained STAT3 signal, that IL-6 would act as an anti-inflammatory cytokine, which has indeed been observed experimentally in the literature.
Asunto(s)
Redes Reguladoras de Genes/fisiología , Inflamación/metabolismo , Interleucina-6/metabolismo , Modelos Biológicos , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/fisiología , Activación Transcripcional/fisiología , Animales , Simulación por Computador , Retroalimentación Fisiológica/fisiología , Interleucina-10/metabolismo , Ratones , Ratones Noqueados , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/genética , Proteínas Supresoras de la Señalización de Citocinas/metabolismoRESUMEN
DM catalyzes the exchange of peptides bound to Class II major histocompatibility complex (MHC) molecules. Because the dissociation and association components of the overall reaction are difficult to separate, a detailed mechanism of DM catalysis has long resisted elucidation. UV irradiation of DR molecules loaded with a photocleavable peptide (caged Class II MHC molecules) enabled synchronous and verifiable evacuation of the peptide-binding groove and tracking of early binding events in real time by fluorescence polarization. Empty DR molecules generated by photocleavage rapidly bound peptide but quickly resolved into species with substantially slower binding kinetics. DM formed a complex with empty DR molecules that bound peptide with even faster kinetics than empty DR molecules just having lost their peptide cargo. Mathematical models demonstrate that the peptide association rate of DR molecules is substantially higher in the presence of DM. We therefore unequivocally establish that DM contributes directly to peptide association through formation of a peptide-loading complex between DM and empty Class II MHC. This complex rapidly acquires a peptide analogous to the MHC class I peptide-loading complex.