ABSTRACT
The molecular mechanisms underlying seizure generation remain elusive, yet they are crucial for developing effective treatments for epilepsy. The current study shows that inhibiting c-Abl tyrosine kinase prevents apoptosis, reduces dendritic spine loss, and maintains N-methyl-d-aspartate (NMDA) receptor subunit 2B (NR2B) phosphorylated in in vitro models of excitotoxicity. Pilocarpine-induced status epilepticus (SE) in mice promotes c-Abl phosphorylation, and disrupting c-Abl activity leads to fewer seizures, increases latency toward SE, and improved animal survival. Currently, clinically used c-Abl inhibitors are non-selective and have poor brain penetration. The allosteric c-Abl inhibitor, neurotinib, used here has favorable potency, selectivity, pharmacokinetics, and vastly improved brain penetration. Neurotinib-administered mice have fewer seizures and improved survival following pilocarpine-SE induction. Our findings reveal c-Abl kinase activation as a key factor in ictogenesis and highlight the impact of its inhibition in preventing the insurgence of epileptic-like seizures in rodents and humans.
Subject(s)
Pilocarpine , Proto-Oncogene Proteins c-abl , Seizures , Animals , Male , Mice , Apoptosis/drug effects , Mice, Inbred C57BL , Neurons/drug effects , Neurons/pathology , Neurons/metabolism , Phosphorylation/drug effects , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-abl/metabolism , Proto-Oncogene Proteins c-abl/antagonists & inhibitors , Pyrimidines/pharmacology , Pyrimidines/therapeutic use , Seizures/chemically induced , Seizures/drug therapy , Seizures/pathology , Status Epilepticus/chemically induced , Status Epilepticus/drug therapy , Status Epilepticus/pathologyABSTRACT
CONTEXT: Thyroid-stimulating hormone (or thyrotropin) receptor (TSHR) could be a selective target for small molecule ligands to treat thyroid cancer (TC). OBJECTIVE: We report a novel, orally efficacious ligand for TSHR that exhibits proliferation inhibitory activity against human TC in vitro and in vivo, and inhibition of metastasis in vivo. METHODS: A35 (NCATS-SM4420; NCGC00241808) was selected from a sublibrary of >200 TSHR ligands. Cell proliferation assays including BrdU incorporation and WST-1, along with molecular docking studies were done. In vivo activity of A35 was assessed in TC cell-derived xenograft (CDX) models with immunocompromised (NSG) mice. Formalin-fixed, paraffin-embedded sections of tumor and lung tissues were observed for the extent of cell death and metastasis. RESULTS: A35 was shown to stimulate cAMP production in some cell types by activating TSHR but not in TC cells, MDA-T32, and MDA-T85. A35 inhibited proliferation of MDA-T32 and MDA-T85 in vitro and in vivo, and pulmonary metastasis of MDA-T85F1 in mice. In vitro, A35 inhibition of proliferation was reduced by a selective TSHR antagonist. Inhibition of CDX tumor growth without decreases in mouse weights and liver function showed A35 to be efficacious without apparent toxicity. Lastly, A35 reduced levels of Ki67 in the tumors and metastatic markers in lung tissues. CONCLUSION: We conclude that A35 is a TSHR-selective inhibitor of TC cell proliferation and metastasis, and suggest that A35 may be a promising lead drug candidate for the treatment of differentiated TC in humans.
Subject(s)
Cell Proliferation , Receptors, Thyrotropin , Thyroid Neoplasms , Xenograft Model Antitumor Assays , Animals , Humans , Mice , Thyroid Neoplasms/drug therapy , Thyroid Neoplasms/pathology , Thyroid Neoplasms/metabolism , Cell Proliferation/drug effects , Receptors, Thyrotropin/antagonists & inhibitors , Receptors, Thyrotropin/metabolism , Ligands , Cell Line, Tumor , Administration, Oral , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Antineoplastic Agents/administration & dosage , Neoplasm Metastasis , Molecular Docking Simulation , Lung Neoplasms/drug therapy , Lung Neoplasms/secondary , Lung Neoplasms/pathology , Lung Neoplasms/metabolism , FemaleABSTRACT
The endoplasmic reticulum is a subcellular organelle key in the control of synthesis, folding, and sorting of proteins. Under endoplasmic reticulum stress, an adaptative unfolded protein response is activated; however, if this activation is prolonged, cells can undergo cell death, in part due to oxidative stress and mitochondrial fragmentation. Here, we report that endoplasmic reticulum stress activates c-Abl tyrosine kinase, inducing its translocation to mitochondria. We found that endoplasmic reticulum stress-activated c-Abl interacts with and phosphorylates the mitochondrial fusion protein MFN2, resulting in mitochondrial fragmentation and apoptosis. Moreover, the pharmacological or genetic inhibition of c-Abl prevents MFN2 phosphorylation, mitochondrial fragmentation, and apoptosis in cells under endoplasmic reticulum stress. Finally, in the amyotrophic lateral sclerosis mouse model, where endoplasmic reticulum and oxidative stress has been linked to neuronal cell death, we demonstrated that the administration of c-Abl inhibitor neurotinib delays the onset of symptoms. Our results uncovered a function of c-Abl in the crosstalk between endoplasmic reticulum stress and mitochondrial dynamics via MFN2 phosphorylation.
ABSTRACT
Background: Growing evidence suggests that the non-receptor tyrosine kinase, c-Abl, plays a significant role in the pathogenesis of Alzheimer's disease (AD). Here, we analyzed the effect of c-Abl on the cognitive performance decline of APPSwe/PSEN1ΔE9 (APP/PS1) mouse model for AD. Methods: We used the conditional genetic ablation of c-Abl in the brain (c-Abl-KO) and pharmacological treatment with neurotinib, a novel allosteric c-Abl inhibitor with high brain penetrance, imbued in rodent's chow. Results: We found that APP/PS1/c-Abl-KO mice and APP/PS1 neurotinib-fed mice had improved performance in hippocampus-dependent tasks. In the object location and Barnes-maze tests, they recognized the displaced object and learned the location of the escape hole faster than APP/PS1 mice. Also, APP/PS1 neurotinib-fed mice required fewer trials to reach the learning criterion in the memory flexibility test. Accordingly, c-Abl absence and inhibition caused fewer amyloid plaques, reduced astrogliosis, and preserved neurons in the hippocampus. Discussion: Our results further validate c-Abl as a target for AD, and the neurotinib, a novel c-Abl inhibitor, as a suitable preclinical candidate for AD therapies.
ABSTRACT
We have developed and characterized a novel D2R antagonist with exceptional GPCR selectivity - ML321. In functional profiling screens of 168 different GPCRs, ML321 showed little activity beyond potent inhibition of the D2R and to a lesser extent the D3R, demonstrating excellent receptor selectivity. The D2R selectivity of ML321 may be related to the fact that, unlike other monoaminergic ligands, ML321 lacks a positively charged amine group and adopts a unique binding pose within the orthosteric binding site of the D2R. PET imaging studies in non-human primates demonstrated that ML321 penetrates the CNS and occupies the D2R in a dose-dependent manner. Behavioral paradigms in rats demonstrate that ML321 can selectively antagonize a D2R-mediated response (hypothermia) while not affecting a D3R-mediated response (yawning) using the same dose of drug, thus indicating exceptional in vivo selectivity. We also investigated the effects of ML321 in animal models that are predictive of antipsychotic efficacy in humans. We found that ML321 attenuates both amphetamine- and phencyclidine-induced locomotor activity and restored pre-pulse inhibition (PPI) of acoustic startle in a dose-dependent manner. Surprisingly, using doses that were maximally effective in both the locomotor and PPI studies, ML321 was relatively ineffective in promoting catalepsy. Kinetic studies revealed that ML321 exhibits slow-on and fast-off receptor binding rates, similar to those observed with atypical antipsychotics with reduced extrapyramidal side effects. Taken together, these observations suggest that ML321, or a derivative thereof, may exhibit â³atypicalâ³ antipsychotic activity in humans with significantly fewer side effects than observed with the currently FDA-approved D2R antagonists.
ABSTRACT
Niemann-Pick type A (NPA) disease is a fatal lysosomal neurodegenerative disorder caused by the deficiency in acid sphingomyelinase (ASM) activity. NPA patients present severe and progressive neurodegeneration starting at an early age. Currently, there is no effective treatment for this disease and NPA patients die between 2 and 3 years of age. NPA is characterized by an accumulation of sphingomyelin in lysosomes and dysfunction in the autophagy-lysosomal pathway. Recent studies show that c-Abl tyrosine kinase activity downregulates autophagy and the lysosomal pathway. Interestingly, this kinase is also activated in other lysosomal neurodegenerative disorders. Here, we describe that c-Abl activation contributes to the mechanisms of neuronal damage and death in NPA disease. Our data demonstrate that: 1) c-Abl is activated in-vitro as well as in-vivo NPA models; 2) imatinib, a clinical c-Abl inhibitor, reduces autophagy-lysosomal pathway alterations, restores autophagy flux, and lowers sphingomyelin accumulation in NPA patient fibroblasts and NPA neuronal models and 3) chronic treatment with nilotinib and neurotinib, two c-Abl inhibitors with differences in blood-brain barrier penetrance and target binding mode, show further benefits. While nilotinib treatment reduces neuronal death in the cerebellum and improves locomotor functions, neurotinib decreases glial activation, neuronal disorganization, and loss in hippocampus and cortex, as well as the cognitive decline of NPA mice. Our results support the participation of c-Abl signaling in NPA neurodegeneration and autophagy-lysosomal alterations, supporting the potential use of c-Abl inhibitors for the clinical treatment of NPA patients.
ABSTRACT
The majority of FDA-approved HCV therapeutics target the viral replicative machinery. An automated high-throughput phenotypic screen identified several small molecules as potent inhibitors of hepatitis C virus replication. Here, we disclose the discovery and optimization of a 4-aminopiperidine (4AP) scaffold targeting the assembly stages of the HCV life cycle. The original screening hit (1) demonstrates efficacy in the HCVcc assay but does not show potency prior to or during viral replication. Colocalization and infectivity studies indicate that the 4AP chemotype inhibits the assembly and release of infectious HCV. Compound 1 acts synergistically with FDA-approved direct-acting antiviral compounds Telaprevir and Daclatasvir, as well as broad spectrum antivirals Ribavirin and cyclosporin A. Following an SAR campaign, several derivatives of the 4AP series have been identified with increased potency against HCV, reduced in vitro toxicity, as well as improved in vitro and in vivo ADME properties.
Subject(s)
Antiviral Agents/pharmacology , Drug Discovery , Hepacivirus/drug effects , Piperidines/pharmacology , Animals , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , Cells, Cultured , Dose-Response Relationship, Drug , Humans , Male , Mice , Microbial Sensitivity Tests , Molecular Structure , Piperidines/chemical synthesis , Piperidines/chemistry , Rats , Structure-Activity Relationship , Virus Replication/drug effectsABSTRACT
Fluoxazolevir is an aryloxazole-based entry inhibitor of hepatitis C virus (HCV). We show that fluoxazolevir inhibits fusion of HCV with hepatic cells by binding HCV envelope protein 1 to prevent fusion. Nine of ten fluoxazolevir resistance-associated substitutions are in envelope protein 1, and four are in a putative fusion peptide. Pharmacokinetic studies in mice, rats and dogs revealed that fluoxazolevir localizes to the liver. A 4-week intraperitoneal regimen of fluoxazolevir in humanized chimeric mice infected with HCV genotypes 1b, 2a or 3 resulted in a 2-log reduction in viraemia, without evidence of drug resistance. In comparison, daclatasvir, an approved HCV drug, suppressed more than 3 log of viraemia but is associated with the emergence of resistance-associated substitutions in mice. Combination therapy using fluoxazolevir and daclatasvir cleared HCV genotypes 1b and 3 in mice. Fluoxazolevir combined with glecaprevir and pibrentasvir was also effective in clearing multidrug-resistant HCV replication in mice. Fluoxazolevir may be promising as the next generation of combination drug cocktails for HCV treatment.
Subject(s)
Antiviral Agents/administration & dosage , Hepacivirus/drug effects , Hepatitis C/drug therapy , Virus Internalization/drug effects , Animals , Carbamates/administration & dosage , Disease Models, Animal , Dogs , Drug Therapy, Combination , Genotype , Hepacivirus/classification , Hepacivirus/genetics , Hepacivirus/physiology , Hepatitis C/virology , Humans , Imidazoles/administration & dosage , Male , Mice , Pyrrolidines/administration & dosage , Rats , Rats, Sprague-Dawley , Valine/administration & dosage , Valine/analogs & derivatives , Viral Envelope Proteins/genetics , Viral Envelope Proteins/metabolismABSTRACT
Th17 cells are critical drivers of autoimmune diseases and immunopathology. There is an unmet need to develop therapies targeting pathogenic Th17 cells for the treatment of autoimmune disorders. Here, we report that anxiolytic FGIN-1-27 inhibits differentiation and pathogenicity of Th17 cells in vitro and in vivo using the experimental autoimmune encephalomyelitis (EAE) model of Th17 cell-driven pathology. Remarkably, we found that the effects of FGIN-1-27 were independent of translocator protein (TSPO), the reported target for this small molecule, and instead were driven by a metabolic switch in Th17 cells that led to the induction of the amino acid starvation response and altered cellular fatty acid composition. Our findings suggest that the small molecule FGIN-1-27 can be re-purposed to relieve autoimmunity by metabolic reprogramming of pathogenic Th17 cells.
Subject(s)
Anti-Anxiety Agents/pharmacology , Autoimmunity/drug effects , Cellular Reprogramming Techniques , Encephalomyelitis, Autoimmune, Experimental , Indoleacetic Acids/pharmacology , Th17 Cells/immunology , Animals , Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/pathology , Encephalomyelitis, Autoimmune, Experimental/therapy , Mice , Mice, Transgenic , Receptors, GABA/immunology , Th17 Cells/pathologyABSTRACT
Hepatitis C virus (HCV) is a small, single-stranded, positive-sense RNA virus that infects more than an estimated 70 million people worldwide. Untreated, persistent HCV infection often results in chronic hepatitis, cirrhosis, or liver failure, with progression to hepatocellular carcinoma. Current anti-HCV regimens comprising direct acting antivirals (DAAs) can provide curative treatment; however, due to high costs there remains a need for effective, shorter-duration, and affordable treatments. Recently, we disclosed anti-HCV activity of the cheap antihistamine chlorcyclizine, targeting viral entry. Following our hit-to-lead optimization campaign, we report evaluation of preclinical in vitro absorption, distribution, metabolism, and excretion properties, and in vivo pharmacokinetic profiles of lead compounds. This led to selection of a new lead compound and evaluation of efficacy in chimeric mice engrafted with primary human hepatocytes infected with HCV. Further development and incorporation of this compound into DAA regimens has the potential to improve treatment efficacy, affordability, and accessibility.
Subject(s)
Antiviral Agents/pharmacology , Hepacivirus/drug effects , Hepatitis C, Chronic/drug therapy , Piperazines/pharmacology , Animals , Carcinoma, Hepatocellular/virology , Cell Line , Genotype , Hepatocytes/virology , Humans , Liver Cirrhosis/virology , Liver Neoplasms/virology , Male , Mice , Mice, SCID , Virus Internalization/drug effectsABSTRACT
The drug 2-hydroxypropyl-ß-cyclodextrin (HPßCD) reduces lysosomal cholesterol accumulation in Niemann-Pick disease, type C (NPC) and has been advanced to human clinical trials. However, its mechanism of action for reducing cholesterol accumulation in NPC cells is uncertain and its molecular target is unknown. We found that methyl-ß-cyclodextrin (MßCD), a potent analog of HPßCD, restored impaired macroautophagy/autophagy flux in Niemann-Pick disease, type C1 (NPC1) cells. This effect was mediated by a direct activation of AMP-activated protein kinase (AMPK), an upstream kinase in the autophagy pathway, through MßCD binding to its ß-subunits. Knockdown of PRKAB1 or PRKAB2 (encoding the AMPK ß1 or ß2 subunit) expression and an AMPK inhibitor abolished MßCD-mediated reduction of cholesterol storage in NPC1 cells. The results demonstrate that AMPK is the molecular target of MßCD and its activation enhances autophagy flux, thereby mitigating cholesterol accumulation in NPC1 cells. The results identify AMPK as an attractive target for drug development to treat NPC.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Autophagy , Niemann-Pick Disease, Type C/drug therapy , Niemann-Pick Disease, Type C/pathology , beta-Cyclodextrins/therapeutic use , Autophagosomes/drug effects , Autophagosomes/metabolism , Autophagy/drug effects , Boron Compounds/metabolism , Cholesterol/metabolism , Endocytosis/drug effects , Enzyme Activation/drug effects , Enzyme Activators/pharmacology , Humans , Kinetics , Models, Biological , Niemann-Pick Disease, Type C/enzymology , Protein Kinase Inhibitors/pharmacology , beta-Cyclodextrins/pharmacologyABSTRACT
Duchenne muscular dystrophy (DMD) is a fatal muscle disease caused by mutations in the dystrophin gene, resulting in a complete loss of the dystrophin protein. Dystrophin is a critical component of the dystrophin glycoprotein complex (DGC), which links laminin in the extracellular matrix to the actin cytoskeleton within myofibers and provides resistance to shear stresses during muscle activity. Loss of dystrophin in DMD patients results in a fragile sarcolemma prone to contraction-induced muscle damage. The α7ß1 integrin is a laminin receptor protein complex in skeletal and cardiac muscle and a major modifier of disease progression in DMD. In a muscle cell-based screen for α7 integrin transcriptional enhancers, we identified a small molecule, SU9516, that promoted increased α7ß1 integrin expression. Here we show that SU9516 leads to increased α7B integrin in murine C2C12 and human DMD patient myogenic cell lines. Oral administration of SU9516 in the mdx mouse model of DMD increased α7ß1 integrin in skeletal muscle, ameliorated pathology, and improved muscle function. We show that these improvements are mediated through SU9516 inhibitory actions on the p65-NF-κB pro-inflammatory and Ste20-related proline alanine rich kinase (SPAK)/OSR1 signaling pathways. This study identifies a first in-class α7 integrin-enhancing small-molecule compound with potential for the treatment of DMD.
Subject(s)
Imidazoles/pharmacology , Indoles/pharmacology , Integrins/metabolism , Muscular Dystrophy, Duchenne/metabolism , Muscular Dystrophy, Duchenne/pathology , Animals , Cell Differentiation/drug effects , Cell Line , Disease Models, Animal , Disease Progression , Female , Fibrosis , Humans , Integrins/agonists , Mice , Mice, Inbred mdx , Models, Biological , Muscle Development/drug effects , Muscle Strength , Muscle, Skeletal/drug effects , Muscle, Skeletal/metabolism , Muscular Dystrophy, Duchenne/drug therapy , Myoblasts, Skeletal/cytology , Myoblasts, Skeletal/drug effects , Myoblasts, Skeletal/metabolism , NF-kappa B/metabolism , Protein Serine-Threonine Kinases/metabolism , Regeneration/drug effects , Signal Transduction/drug effectsABSTRACT
One of the pathological hallmarks of Alzheimer's disease (AD) is the presence of amyloid plaques, which are deposits of misfolded and aggregated amyloid-beta peptide (Aß). The role of the c-Abl tyrosine kinase in Aß-mediated neurodegeneration has been previously reported. Here, we investigated the therapeutic potential of inhibiting c-Abl using imatinib. We developed a novel method, based on a technique used to detect prions (PMCA), to measure minute amounts of misfolded-Aß in the blood of AD transgenic mice. We found that imatinib reduces Aß-oligomers in plasma, which correlates with a reduction of AD brain features such as plaques and oligomers accumulation, neuroinflammation, and cognitive deficits. Cells exposed to imatinib and c-Abl KO mice display decreased levels of ß-CTF fragments, suggesting that an altered processing of the amyloid-beta protein precursor is the most probable mechanism behind imatinib effects. Our findings support the role of c-Abl in Aß accumulation and AD, and propose AD-PMCA as a new tool to evaluate AD progression and screening for drug candidates.
Subject(s)
Alzheimer Disease/blood , Alzheimer Disease/enzymology , Amyloid beta-Peptides/blood , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-abl/antagonists & inhibitors , Proto-Oncogene Proteins c-abl/blood , Alzheimer Disease/pathology , Animals , Cell Line , Hippocampus/drug effects , Hippocampus/metabolism , Hippocampus/pathology , Mice , Mice, Knockout , Mice, TransgenicABSTRACT
Infection with human cytomegalovirus (HCMV) is a threat for pregnant women and immunocompromised hosts. Although limited drugs are available, development of new agents against HCMV is desired. Through screening of the LOPAC library, we identified emetine as HCMV inhibitor. Additional studies confirmed its anti-HCMV activities in human foreskin fibroblasts: EC50-40±1.72 nM, CC50-8±0.56 µM, and selectivity index of 200. HCMV inhibition occurred after virus entry, but before DNA replication, and resulted in decreased expression of viral proteins. Synergistic virus inhibition was achieved when emetine was combined with ganciclovir. In a mouse CMV (MCMV) model, emetine was well-tolerated, displayed long half-life, preferential distribution to tissues over plasma, and effectively suppressed MCMV. Since the in vitro anti-HCMV activity of emetine decreased significantly in low-density cells, a mechanism involving cell cycle regulation was suspected. HCMV inhibition by emetine depended on ribosomal processing S14 (RPS14) binding to MDM2, leading to disruption of HCMV-induced MDM2-p53 and MDM2-IE2 interactions. Irrespective of cell density, emetine induced RPS14 translocation into the nucleus during infection. In infected high-density cells, MDM2 was available for interaction with RPS14, resulting in disruption of MDM2-p53 interaction. However, in low-density cells the pre-existing interaction of MDM2-p53 could not be disrupted, and RPS14 could not interact with MDM2. In high-density cells the interaction of MDM2-RPS14 resulted in ubiquitination and degradation of RPS14, which was not observed in low-density cells. In infected-only or in non-infected emetine-treated cells, RPS14 failed to translocate into the nucleus, hence could not interact with MDM2, and was not ubiquitinated. HCMV replicated similarly in RPS14 knockdown or control cells, but emetine did not inhibit virus replication in the former cell line. The interaction of MDM2-p53 was maintained in infected RPS14 knockdown cells despite emetine treatment, confirming a unique mechanism by which emetine exploits RPS14 to disrupt MDM2-p53 interaction. Summarized, emetine may represent a promising candidate for HCMV therapy alone or in combination with ganciclovir through a novel host-dependent mechanism.
Subject(s)
Antiviral Agents/pharmacology , Cytomegalovirus Infections , Cytomegalovirus/drug effects , Emetine/pharmacology , Animals , Cell Survival/drug effects , Cells, Cultured , Disease Models, Animal , Fluorescent Antibody Technique , Humans , Immunoblotting , Immunoprecipitation , Male , Mice , Mice, Inbred BALB C , Microscopy, Confocal , Polymerase Chain Reaction , Virus Replication/drug effectsABSTRACT
Recently, we reported that chlorcyclizine (CCZ, Rac-2), an over-the-counter antihistamine piperazine drug, possesses in vitro and in vivo activity against hepatitis C virus. Here, we describe structure-activity relationship (SAR) efforts that resulted in the optimization of novel chlorcyclizine derivatives as anti-HCV agents. Several compounds exhibited EC50 values below 10 nM against HCV infection, cytotoxicity selectivity indices above 2000, and showed improved in vivo pharmacokinetic properties. The optimized molecules can serve as lead preclinical candidates for the treatment of hepatitis C virus infection and as probes to study hepatitis C virus pathogenesis and host-virus interaction.
Subject(s)
Drug Discovery , Hepatitis C/drug therapy , Hepatitis C/virology , Piperazines/therapeutic use , Dose-Response Relationship, Drug , Hepacivirus/drug effects , Microbial Sensitivity Tests , Molecular Structure , Piperazines/chemical synthesis , Piperazines/chemistry , Structure-Activity RelationshipABSTRACT
BACKGROUND: Niemann-Pick type C (NPC) disease is a fatal neurodegenerative disorder characterized by the accumulation of free cholesterol in lysosomes. There are currently no effective FDA-approved treatments for NPC, although in the last years the inhibition of histone deacetylases (HDACs) has emerged as a potential treatment for this disease. However, the molecular mechanisms that deregulate HDAC activity in NPC disease are unknown. Previously our group had shown that the proapoptotic tyrosine kinase c-Abl signaling is activated in NPC neurons. Here, we demonstrate that c-Abl activity increases HDAC2 levels inducing neuronal gene repression of key synaptic genes in NPC models. RESULTS: Our data show that: i) HDAC2 levels and activity are increased in NPC neuronal models and in Npc1(-/-) mice; ii) inhibition of c-Abl or c-Abl deficiency prevents the increase of HDAC2 protein levels and activity in NPC neuronal models; iii) c-Abl inhibition decreases the levels of HDAC2 tyrosine phosphorylation; iv) treatment with methyl-ß-cyclodextrin and vitamin E decreases the activation of the c-Abl/HDAC2 pathway in NPC neurons; v) in vivo treatment with two c-Abl inhibitors prevents the increase of HDAC2 protein levels in the brain of Npc1(-/-) mice; and vi) c-Abl inhibition prevents HDAC2 recruitment to the promoter of neuronal genes, triggering an increase in their expression. CONCLUSION: Our data show the involvement of the c-Abl/HDAC2 signaling pathway in the regulation of neuronal gene expression in NPC neuronal models. Thus, inhibition of c-Abl could be a pharmacological target for preventing the deleterious effects of increased HDAC2 levels in NPC disease.
Subject(s)
Histone Deacetylase 2/genetics , Neurons/metabolism , Niemann-Pick Disease, Type C/genetics , Proto-Oncogene Proteins c-abl/genetics , Animals , Cholesterol/genetics , Cholesterol/metabolism , Cyclodextrins/administration & dosage , Disease Models, Animal , Gene Expression Regulation/drug effects , Histone Deacetylase 2/biosynthesis , Humans , Lysosomes/metabolism , Mice , Neurons/pathology , Niemann-Pick Disease, Type C/drug therapy , Niemann-Pick Disease, Type C/pathology , Proto-Oncogene Proteins c-abl/biosynthesis , Signal Transduction/drug effects , Vitamin E/administration & dosageABSTRACT
The efficacy of vaccine adjuvants such as Toll-like receptor agonists (TLRa) can be improved through formulation and delivery approaches. Here, we attached small molecule TLR-7/8a to polymer scaffolds (polymer-TLR-7/8a) and evaluated how different physicochemical properties of the TLR-7/8a and polymer carrier influenced the location, magnitude and duration of innate immune activation in vivo. Particle formation by polymer-TLR-7/8a was the most important factor for restricting adjuvant distribution and prolonging activity in draining lymph nodes. The improved pharmacokinetic profile by particulate polymer-TLR-7/8a was also associated with reduced morbidity and enhanced vaccine immunogenicity for inducing antibodies and T cell immunity. We extended these findings to the development of a modular approach in which protein antigens are site-specifically linked to temperature-responsive polymer-TLR-7/8a adjuvants that self-assemble into immunogenic particles at physiologic temperatures in vivo. Our findings provide a chemical and structural basis for optimizing adjuvant design to elicit broad-based antibody and T cell responses with protein antigens.
Subject(s)
Adjuvants, Immunologic/chemistry , Toll-Like Receptors/agonists , Vaccines/immunology , Animals , Drug Carriers/chemistry , Female , Male , Mice , Mice, Inbred C57BL , T-Lymphocytes/immunologyABSTRACT
Nanoscopy has now become a real procedure in fluorescence microscopy of living cells. The STED/RESOLFT family of nanoscopy approaches has the best prospects for delivering high speed imaging, but the history of STED includes a continuing struggle to reduce the deactivation power applied, along with difficulties in achieving simultaneous multicolor images. In this manuscript, we present a concept for a similar real-time nanoscopy, using a new class of bipartite probes that separate the luminescent and quenching functions into two coupled molecules. In particular, the STAQ (Superresolution via Transiently Activated Quencher) example we show herein employs the excited state absorbance (not ground state) of the partner to accept energy from and quench the luminescent dye. The result is that much less deactivation power is needed for superresolved (â¼50 nm) imaging. Moreover, the TAQ partner excited by the "donut" beam is shown to quench several different visible dyes via the same mechanism, opening the door to easier multicolor imaging. We demonstrate three dyes sharing the same deactivation and show examples of superresolved multicolor images. We suggest STAQ will facilitate the growth of real-time nanoscopy by reducing confounding photodamage within living cells while expanding the nanoscopist's palette.