RESUMEN
Huntington's disease (HD) is a genetic neurodegenerative disease caused by an expanded CAG repeat in the Huntingtin (HTT) gene that encodes for an expanded polyglutamine (polyQ) repeat in exon-1 of the human mutant huntingtin (mHTT) protein. The presence of this polyQ repeat results in neuronal degeneration, for which there is no cure or treatment that modifies disease progression. In previous studies, we have shown that small molecules that bind selectively to σ2R/TMEM97 can have significant neuroprotective effects in models of Alzheimer's disease, traumatic brain injury, and several other neurodegenerative diseases. In the present work, we extend these investigations and show that certain σ2R/TMEM97-selective ligands decrease mHTT-induced neuronal toxicity. We first synthesized a set of compounds designed to bind to σ2R/TMEM97 and determined their binding profiles (Ki values) for σ2R/TMEM97 and other proteins in the central nervous system. Modulators with high affinity and selectivity for σ2R/TMEM97 were then tested in our HD cell model. Primary cortical neurons were cultured in vitro for 7 days and then co-transfected with either a normal HTT construct (Htt N-586-22Q/GFP) or the mHTT construct Htt-N586-82Q/GFP. Transfected neurons were treated with either σ2R/TMEM97 or σ1R modulators for 48 h. After treatment, neurons were fixed and stained with Hoechst, and condensed nuclei were quantified to assess cell death in the transfected neurons. Significantly, σ2R/TMEM97 modulators reduce the neuronal toxicity induced by mHTT, and their neuroprotective effects are not blocked by NE-100, a selective σ1R antagonist known to block neuroprotection by σ1R ligands. These results indicate for the first time that σ2R/TMEM97 modulators can protect neurons from mHTT-induced neuronal toxicity, suggesting that targeting σ2R/TMEM97 may lead to a novel therapeutic approach to treat patients with HD.
Asunto(s)
Enfermedad de Huntington , Enfermedades Neurodegenerativas , Fármacos Neuroprotectores , Animales , Modelos Animales de Enfermedad , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Enfermedad de Huntington/metabolismo , Proteínas de la Membrana/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Neuronas/metabolismo , Fármacos Neuroprotectores/uso terapéutico , Receptores de Neurotransmisores/metabolismo , Receptores sigma/metabolismoRESUMEN
Substituted norbenzomorphans are known to display high affinity and selectivity for the two sigma receptor (σR) subtypes. In order to study the effects of simplifying the structures of these compounds, a scaffold hopping strategy was used to design several novel sets of substituted isoindolines, tetrahydroisoquinolines and tetrahydro-2-benzazepines. The binding affinities of these new compounds for the sigma 1 (σ1R) and sigma 2 (σ2R) receptors were determined, and some analogs were identified that exhibit high affinity (Kiâ¯≤â¯25â¯nM) and significant selectivity (>10-fold) for σ1R or σ2R. The preferred binding modes of selected compounds for the σ1R are predicted by modeling studies, and the nature of substituents on the aromatic ring and the nitrogen atom of the bicyclic skeleton appears to affect the preferred binding orientation of σ1R-preferring ligands.
Asunto(s)
Benzazepinas/química , Benzazepinas/farmacología , Isoindoles/química , Isoindoles/farmacología , Receptores sigma/metabolismo , Tetrahidroisoquinolinas/química , Tetrahidroisoquinolinas/farmacología , Humanos , Ligandos , Simulación del Acoplamiento Molecular , Unión Proteica , Relación Estructura-ActividadRESUMEN
Unnatural uridine diphosphate (UDP)-sugar donors, UDP-4-deoxy-4-fluoro-N-acetylglucosamine (4FGlcNAc) and UDP-4-deoxy-4-fluoro-N-acetylgalactosamine (4FGalNAc), were prepared using both chemical and chemoenzymatic syntheses relying on N-acetylglucosamine-1-phosphate uridylyltransferase (GlmU). The resulting unnatural UDP-sugar donors were then tested as substrates in glycosaminoglycan synthesis catalyzed by various synthases. UDP-4FGlcNAc was transferred onto an acceptor by Pastuerella multocida heparosan synthase 1 and subsequently served as a chain terminator.
Asunto(s)
Glicosaminoglicanos/biosíntesis , Nucleotidiltransferasas/metabolismo , Biocatálisis , Conformación de Carbohidratos , Glicosaminoglicanos/química , Nucleotidiltransferasas/química , Azúcares de Uridina Difosfato/biosíntesis , Azúcares de Uridina Difosfato/químicaRESUMEN
The separation and quantification of glycosaminoglycan (GAG) chains with different levels of sulfation from cells and media, and prepared through chemoenzymatic synthesis or metabolic engineering, pose a major challenge in glycomics analysis. A method for microscale separation and quantification of heparin, heparan sulfate, and heparosan from cells is reported. This separation relies on a mini strong anion exchange spin column eluted stepwise with various concentrations of sodium chloride. Disaccharide analysis by LC-MS was used to monitor the chemical structure of the various GAG chains that were recovered.