ABSTRACT
Since the largest and most fatal Ebola virus epidemic during 2014-2016, there have been several consecutive filoviral outbreaks in recent years, including those in 2021, 2022, and 2023. Ongoing outbreak prevalence and limited FDA-approved filoviral therapeutics emphasize the need for novel small molecule treatments. Here, we showcase the structure-activity relationship development of N-substituted pyrrole-based heterocycles and their potent, submicromolar entry inhibition against diverse filoviruses in a target-based pseudovirus assay. Inhibitor antiviral activity was validated using replication-competent Ebola, Sudan, and Marburg viruses. Mutational analysis was used to map the targeted region within the Ebola virus glycoprotein. Antiviral counter-screen and phospholipidosis assays were performed to demonstrate the reduced off-target activity of these filoviral entry inhibitors. Favorable antiviral potency, selectivity, and drug-like properties of the N-substituted pyrrole-based heterocycles support their potential as broad-spectrum antifiloviral treatments.
Subject(s)
Antiviral Agents , Ebolavirus , Pyrroles , Virus Internalization , Pyrroles/pharmacology , Pyrroles/chemistry , Pyrroles/chemical synthesis , Antiviral Agents/pharmacology , Antiviral Agents/chemistry , Antiviral Agents/chemical synthesis , Humans , Structure-Activity Relationship , Ebolavirus/drug effects , Virus Internalization/drug effects , Heterocyclic Compounds/pharmacology , Heterocyclic Compounds/chemistry , Heterocyclic Compounds/chemical synthesis , Filoviridae/drug effects , Marburgvirus/drug effectsABSTRACT
The influenza A virus (IAV) is a highly contagious virus that causes pandemics and seasonal epidemics, which are major public health issues. Current anti-influenza therapeutics are limited partly due to the continuous emergence of drug-resistant IAV strains; thus, there is an unmet need to develop novel anti-influenza therapies. Here, we present a novel imidazo[1,2-a]pyrimidine scaffold that targets group 2 IAV entry. We have explored three different regions of the lead compound, and we have developed a series of small molecules that have nanomolar activity against oseltamivir-sensitive and -resistant forms of group 2 IAVs. These small molecules target hemagglutinin (HA), which mediates the viral entry process. Mapping a known small-molecule-binding cavity of the HA structure with resistant mutants suggests that these molecules bind to that cavity and block HA-mediated membrane fusion.
Subject(s)
Influenza A virus , Influenza, Human , Humans , Influenza A virus/metabolism , Oseltamivir , Hemagglutinin Glycoproteins, Influenza Virus/metabolism , Hemagglutinins , Influenza, Human/drug therapy , Structure-Activity Relationship , Pyrimidines/pharmacology , Antiviral Agents/pharmacology , Antiviral Agents/chemistryABSTRACT
To evaluate the differences in action of commercially available 2-oxoglutarate mimetics and "branched-tail" oxyquinoline inhibitors of hypoxia-inducible factor prolyl hydroxylase (HIF PHD), the inhibitors' IC50 values in the activation of HIF1 ODD-luciferase reporter were selected for comparative transcriptomics. Structure-activity relationship and computer modeling for the oxyquinoline series of inhibitors led to the identification of novel inhibitors, which were an order of magnitude more active in the reporter assay than roxadustat and vadadustat. Unexpectedly, 2-methyl-substitution in the oxyquinoline core of the best HIF PHD inhibitor was found to be active in the reporter assay and almost equally effective in the pretreatment paradigm of the oxygen-glucose deprivation in vitro model. Comparative transcriptomic analysis of the signaling pathways induced by HIF PHD inhibitors showed high potency of the two novel oxyquinoline inhibitors (#4896-3249 and #5704-0720) at 2 µM concentrations matching the effect of 30 µM roxadustat and 500 µM dimethyl oxalyl glycine in inducing HIF1 and HIF2-linked pathways. The two oxyquinoline inhibitors exerted the same activation of HIF-triggered glycolytic pathways but opposite effects on signaling pathways linked to alternative substrates of HIF PHD 1 and 3, such as p53, NF-κB, and ATF4. This finding can be interpreted as the specificity of the 2-methyl-substitute variant for HIF PHD2.
ABSTRACT
Aspirin is a desired leaving group in prodrugs aimed at treatment of neurodegeneration and other conditions. A library of aspirin derivatives of various scaffolds potentially activating Nrf2 has been tested in Neh2-luc reporter assay which screens for direct Nrf2 protein stabilizers working via disruption of Nrf2-Keap1 interaction. Most aspirin prodrugs had a pro-alkylating or pro-oxidant motif in the structure and, therefore, were toxic at high concentrations. However, among the active compounds, we identified a molecule resembling a well-known Nrf2 displacement activator, bis-1,4-(4-methoxybenzenesulfonamidyl) naphthalene (NMBSA). The direct comparison of the newly identified compound with NMBSA and its improved analog in the reporter assay showed no quenching with N-acetyl cysteine, thus pointing to Nrf2 stabilization mechanism without cysteine alkylation. The potency of the newly identified compound in the reporter assay was much stronger than NMBSA, despite its inhibitory action in the commercial fluorescence polarization assay was observed only in the millimolar range. Molecular docking predicted that mono-deacetylation of the novel prodrug should generate a potent displacement activator. The time-course of reporter activation with the novel prodrug had a pronounced lag-period pointing to a plausible intracellular transformation leading to an active product. Treatment of the novel prodrug with blood plasma or cell lysate demonstrated stepwise deacetylation as judge by liquid chromatography-mass spectrometry (LC-MS). Hence, the esterase-catalyzed hydrolysis of the prodrug liberates only acetyl groups from aspirin moiety and generates a potent Nrf2 activator. The discovered mechanism of prodrug activation makes the newly identified compound a promising lead for future optimization studies.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Aspirin/pharmacology , NF-E2-Related Factor 2/metabolism , Prodrugs/pharmacology , Cell Line, Tumor , Dose-Response Relationship, Drug , Humans , NF-E2-Related Factor 2/agonists , Protein Structure, TertiaryABSTRACT
Nipah virus (NiV) and Hendra virus (HeV) are highly pathogenic, bat-borne paramyxoviruses in the genus Henipavirus that cause severe and often fatal acute respiratory and/or neurologic diseases in humans and livestock. There are currently no approved antiviral therapeutics or vaccines for use in humans to treat or prevent NiV or HeV infection. To facilitate development of henipavirus antivirals, a high-throughput screening (HTS) platform was developed based on a well-characterized recombinant version of the nonpathogenic Henipavirus, Cedar virus (rCedV). Using reverse genetics, a rCedV encoding firefly luciferase (rCedV-Luc) was rescued and its utility evaluated for high-throughput antiviral compound screening. The luciferase reporter gene signal kinetics of rCedV-Luc in different human cell lines was characterized and validated as an authentic real-time measure of viral growth. The rCedV-Luc platform was optimized as an HTS assay that demonstrated high sensitivity with robust Z' scores, excellent signal-to-background ratios and coefficients of variation. Eight candidate compounds that inhibited rCedV replication were identified for additional validation and demonstrated that 4 compounds inhibited authentic NiV-Bangladesh replication. Further evaluation of 2 of the 4 validated compounds in a 9-point dose response titration demonstrated potent antiviral activity against NiV-Bangladesh and HeV, with minimal cytotoxicity. This rCedV reporter can serve as a surrogate yet authentic BSL-2 henipavirus platform that will dramatically accelerate drug candidate identification in the development of anti-henipavirus therapies.
Subject(s)
Antiviral Agents/pharmacology , Henipavirus Infections/drug therapy , Henipavirus/drug effects , High-Throughput Screening Assays , Viral Envelope Proteins/metabolism , Cell Line , Genes, Reporter , Henipavirus/physiology , Henipavirus Infections/virology , Humans , Luciferases/genetics , Luciferases/metabolism , Recombination, Genetic , Viral Envelope Proteins/genetics , Virus Internalization/drug effects , Virus Replication/drug effectsABSTRACT
Influenza A virus (IAV) causes seasonal epidemics and occasional but devastating pandemics, which are major public health concerns. The putative antiviral therapeutics are useful for the treatment of influenza, however, the emerging resistant strains necessitate a constant search for new drug candidates. Here we report the discovery of a novel antiviral agent, compound CBS1194, which was identified by a parallel high-throughput screening (HTS) campaign using two retroviral pseudotypes bearing H7 or H5 hemagglutinins (HAs). Subsequent analyses demonstrated that CBS1194 is specific to IAVs of group 2, while it has no effect against those of group 1. In a time-of-addition assay, CBS1194 showed a significant inhibitory effect during the early phase of viral infection. In addition, HA-mediated hemolysis can be inhibited by CBS1194 treatment, indicating that this compound may target the HA stalk region, which is responsible for membrane fusion. Escape mutant analyses and in silico docking further revealed that CBS1194 fits into a pocket near the fusion peptide, causing steric hindrance that blocks the low-pH induced rearrangement of HA. In summary, our study identifies a novel fusion inhibitor of group 2 IAVs, which has the potential as lead compound for further development.
Subject(s)
Antiviral Agents/pharmacology , Drug Discovery , Hemagglutinins/metabolism , High-Throughput Screening Assays , Influenza A virus/drug effects , Small Molecule Libraries/pharmacology , A549 Cells , Animals , Dogs , HEK293 Cells , Hemagglutinins/genetics , Humans , Influenza A virus/chemistry , Influenza, Human/virology , Madin Darby Canine Kidney Cells , Molecular Docking Simulation , Virus Internalization/drug effectsABSTRACT
Calpain inhibitors have been proposed as drug candidates for neurodegenerative disorders, with ABT-957 entering clinical trials for Alzheimer's disease and mild cognitive impairment. The structure of ABT-957 was very recently disclosed, and trials were terminated owing to inadequate CNS concentrations to obtain a pharmacodynamic effect. The multistep synthesis of an α-ketoamide peptidomimetic inhibitor series potentially including ABT-957 was optimized to yield diastereomerically pure compounds that are potent and selective for calpain-1 over papain and cathepsins B and K. As the final oxidation step, with its optimized synthesis protocol, does not alter the configuration of the substrate, the synthesis of the diastereomeric pair (R)-1-benzyl-N-((S)-4-((4-fluorobenzyl)amino)-3,4-dioxo-1-phenylbutan-2-yl)-5-oxopyrrolidine-2-carboxamide (1 c) and (R)-1-benzyl-N-((R)-4-((4-fluorobenzyl)amino)-3,4-dioxo-1-phenylbutan-2-yl)-5-oxopyrrolidine-2-carboxamide (1 g) was feasible. This allowed the exploration of stereoselective inhibition of calpain-1, with 1 c (IC50 =78â nM) being significantly more potent than 1 g. Moreover, inhibitor 1 c restored cognitive function in amnestic mice.
Subject(s)
Amnesia/drug therapy , Calpain/antagonists & inhibitors , Glycoproteins/pharmacology , Neuroprotective Agents/pharmacology , Pyrrolidines/pharmacology , Amnesia/chemically induced , Amnesia/metabolism , Animals , Calpain/metabolism , Glycoproteins/chemical synthesis , Glycoproteins/chemistry , Mice , Molecular Structure , Neuroprotective Agents/chemical synthesis , Neuroprotective Agents/chemistry , Pyrrolidines/chemical synthesis , Pyrrolidines/chemistry , Scopolamine , StereoisomerismABSTRACT
HA plays a critical role in influenza infection and, thus HA is a potential target for antivirals. Recently, our laboratories have described a novel fusion inhibitor, termed CBS1117, with EC50 â¼3 µM against group 1 HA. In this work, we characterize the binding properties of CBS1117 to avian H5 HA by x-ray crystallography, NMR, and mutagenesis. The x-ray structure of the complex shows that the compound binds near the HA fusion peptide, a region that plays a critical role in HA-mediated fusion. NMR studies demonstrate binding of CBS1117 to H5 HA in solution and show extensive hydrophobic contacts between the compound and HA surface. Mutagenesis studies further support the location of the compound binding site proximal to the HA fusion peptide and identify additional amino acids that are important to compound binding. Together, this work gives new insights into the CBS1117 mechanism of action and can be exploited to further optimize this compound and better understand the group specific activity of small-molecule inhibitors of HA-mediated entry.
Subject(s)
Antiviral Agents/chemistry , Hemagglutinins/ultrastructure , Animals , Antiviral Agents/pharmacology , Binding Sites/drug effects , Birds/virology , Crystallography, X-Ray/methods , Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Hemagglutinin Glycoproteins, Influenza Virus/metabolism , Hemagglutinins/metabolism , Humans , Influenza A Virus, H5N1 Subtype/chemistry , Influenza A Virus, H5N1 Subtype/drug effects , Influenza in Birds/metabolism , Influenza, Human/metabolism , Models, Molecular , Orthomyxoviridae Infections , Virus Internalization/drug effectsABSTRACT
The recent Ebola epidemics in West Africa underscore the great need for effective and practical therapies for future Ebola virus outbreaks. We have discovered a new series of remarkably potent small molecule inhibitors of Ebola virus entry. These 4-(aminomethyl)benzamide-based inhibitors are also effective against Marburg virus. Synthetic routes to these compounds allowed for the preparation of a wide variety of structures, including a conformationally restrained subset of indolines (compounds 41-50). Compounds 20, 23, 32, 33, and 35 are superior inhibitors of Ebola (Mayinga) and Marburg (Angola) infectious viruses. Representative compounds (20, 32, and 35) have shown good metabolic stability in plasma and liver microsomes (rat and human), and 32 did not inhibit CYP3A4 nor CYP2C9. These 4-(aminomethyl)benzamides are suitable for further optimization as inhibitors of filovirus entry, with the potential to be developed as therapeutic agents for the treatment and control of Ebola virus infections.
Subject(s)
Antiviral Agents/pharmacology , Benzamides/pharmacology , Hemorrhagic Fever, Ebola/virology , Marburg Virus Disease/virology , Virus Internalization/drug effects , A549 Cells , Animals , Antiviral Agents/chemistry , Benzamides/chemistry , Chlorocebus aethiops , Cytochrome P-450 CYP3A Inhibitors/chemistry , Cytochrome P-450 CYP3A Inhibitors/pharmacology , Drug Evaluation, Preclinical , Humans , Microsomes, Liver/drug effects , Molecular Docking Simulation , Structure-Activity Relationship , Toremifene/chemistry , Toremifene/metabolism , Toremifene/pharmacology , Vero Cells , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/metabolismABSTRACT
Vaccination is the most prevalent prophylactic means for controlling seasonal influenza infections. However, an effective vaccine usually takes at least 6 months to develop for the circulating strains. Therefore, new therapeutic options are needed for the acute treatment of influenza infections to control this virus and prevent epidemics/pandemics from developing. We have discovered fast-acting, orally bioavailable acylated 4-aminopiperidines with an effective mechanism of action targeting viral hemagglutinin (HA). Our data show that these compounds are potent entry inhibitors of influenza A viruses. We present docking studies that suggest an HA binding site for these inhibitors on H5N1. Compound 16 displayed a significant decrease of viral titer when evaluated in the infectious assays with influenza virus H1N1 (A/Puerto Rico/8/1934) or H5N1 (A/Vietnam/1203/2004) strains and the oseltamivir-resistant strain with the most common H274Y mutation. In addition, compound 16 showed significant synergistic activity with oseltamivir in vitro.
Subject(s)
Antiviral Agents/pharmacology , Influenza A Virus, H1N1 Subtype/drug effects , Influenza A Virus, H5N1 Subtype/drug effects , Oseltamivir/pharmacology , Piperidines/pharmacology , Virus Internalization/drug effects , Animals , Antiviral Agents/chemical synthesis , Antiviral Agents/metabolism , Binding Sites , Dogs , Drug Synergism , Hemagglutinins, Viral/chemistry , Hemagglutinins, Viral/metabolism , Humans , Influenza A Virus, H1N1 Subtype/chemistry , Influenza A Virus, H5N1 Subtype/chemistry , Madin Darby Canine Kidney Cells , Mice , Microbial Sensitivity Tests , Microsomes, Liver/metabolism , Molecular Docking Simulation , Molecular Structure , Piperidines/chemical synthesis , Piperidines/metabolism , Protein Binding , Small Molecule Libraries/chemical synthesis , Small Molecule Libraries/pharmacology , Structure-Activity RelationshipABSTRACT
Activation of HIF-1α and Nrf2 is a primary component of cellular response to oxidative stress, and activation of HIF-1α and Nrf2 provides neuroprotection in models of neurodegenerative disorders, including ischemic stroke, Alzheimer's and Parkinson's diseases. Screening a library of CNS-targeted drugs using novel reporters for HIF-1α and Nrf2 elevation in neuronal cells revealed histone deacetylase (HDAC) inhibitors as potential activators of these pathways. We report the identification of phenylhydroxamates as single agents exhibiting tripartite inhibition of HDAC6, inhibition of HIF-1 prolyl hydroxylase (PHD), and activation of Nrf2. Two superior tripartite agents, ING-6 and ING-66, showed neuroprotection against various cellular insults, associated with stabilization of both Nrf2 and HIF-1, and expression of their respective target genes in vitro and in vivo. Discovery of the innate ability of phenylhydroxamate HDAC inhibitors to activate Nrf2 and HIF provides a novel route to multifunctional neuroprotective agents and cautions against HDAC6 selective inhibitors as chemical probes of specific HDAC isoform function.
Subject(s)
Histone Deacetylase 6/metabolism , NF-E2-Related Factor 2/metabolism , Oxidative Stress/drug effects , Histone Deacetylase Inhibitors/pharmacology , Humans , Hydroxylamines/pharmacology , Neurodegenerative Diseases/drug therapy , Neurons/drug effects , Neuroprotective Agents/pharmacologyABSTRACT
L-Ascorbate (L-Asc), but not D-isoascorbate (D-Asc) and N-acetylcysteine (NAC) suppress HIF1 ODD-luc reporter activation induced by various inhibitors of HIF prolyl hydroxylase (PHD). The efficiency of suppression by L-Asc was sensitive to the nature of HIF PHD inhibitor chosen for reporter activation. In particular, the inhibitors developed to compete with alpha-ketoglutarate (αKG), were less sensitive to suppression by the physiological range of L-Asc (40-100⯵M) than those having a strong iron chelation motif. Challenging those HIF activators in the reporter system with D-Asc demonstrated that the D-isomer, despite exhibiting the same reducing potency with respect to ferric iron, had almost no effect compared to L-Asc. Similarly, no effect on reporter activation was observed with cell-permeable reducing agent NAC up to 1â¯mM. Docking of L-Asc and D-Asc acid into the HIF PHD2 crystal structure showed interference of Tyr310 with respect to D-Asc. This suggests that L-Asc is not merely a reducing agent preventing enzyme inactivation. Rather, the overall results identify L-Asc as a co-substrate of HIF PHD that may compete for the binding site of αKG in the enzyme active center. This conclusion is in agreement with the results obtained recently in cell-based systems for TET enzymes and jumonji histone demethylases, where L-Asc has been proposed to act as a co-substrate and not as a reducing agent preventing enzyme inactivation.
Subject(s)
Ascorbic Acid/metabolism , Prolyl Hydroxylases/metabolism , Prolyl Hydroxylases/pharmacology , Ascorbic Acid/chemistry , Cell Line, Tumor , Humans , Prolyl-Hydroxylase Inhibitors/pharmacology , Protein Binding , StereoisomerismABSTRACT
Background Nitric oxide (NO) has been heavily implicated in migraine. Nitroglycerin is a prototypic NO-donor, and triggers migraine in humans. However, nitroglycerin also induces oxidative/nitrosative stress and is a source of peroxynitrite - factors previously linked with migraine etiology. Soluble guanylyl cyclase (sGC) is the high affinity NO receptor in the body, and the aim of this study was to identify the precise role of sGC in acute and chronic migraine. Methods We developed a novel brain-bioavailable sGC stimulator (VL-102), and tested its hyperalgesic properties in mice. We also determined the effect of VL-102 on c-fos and calcitonin gene related peptide (CGRP) immunoreactivity within the trigeminovascular complex. In addition, we also tested the known sGC inhibitor, ODQ, within the chronic nitroglycerin migraine model. Results VL-102-evoked acute and chronic mechanical cephalic and hind-paw allodynia in a dose-dependent manner, which was blocked by the migraine medications sumatriptan, propranolol, and topiramate. In addition, VL-102 also increased c-fos and CGRP expressing cells within the trigeminovascular complex. Importantly, ODQ completely inhibited acute and chronic hyperalgesia induced by nitroglycerin. ODQ also blocked hyperalgesia already established by chronic nitroglycerin, implicating this pathway in migraine chronicity. Conclusions These results indicate that nitroglycerin causes migraine-related pain through stimulation of the sGC pathway, and that super-activation of this receptor may be an important component for the maintenance of chronic migraine. This work opens the possibility for negative sGC modulators as novel migraine therapies.
Subject(s)
Hyperalgesia/chemically induced , Hyperalgesia/enzymology , Migraine Disorders/enzymology , Soluble Guanylyl Cyclase/physiology , Adrenergic beta-Antagonists/administration & dosage , Adrenergic beta-Antagonists/therapeutic use , Allosteric Regulation , Animals , Anticonvulsants/administration & dosage , Anticonvulsants/therapeutic use , Calcitonin Gene-Related Peptide/biosynthesis , Disease Models, Animal , Enzyme Inhibitors/administration & dosage , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/therapeutic use , Female , Hyperalgesia/drug therapy , Male , Mice , Mice, Inbred C57BL , Migraine Disorders/chemically induced , Migraine Disorders/drug therapy , Migraine Disorders/etiology , Molecular Targeted Therapy , Nitric Oxide/adverse effects , Nitric Oxide/metabolism , Nitric Oxide Donors/pharmacology , Nitroglycerin/pharmacology , Oxadiazoles/administration & dosage , Oxadiazoles/metabolism , Oxadiazoles/therapeutic use , Propranolol/administration & dosage , Propranolol/therapeutic use , Proto-Oncogene Proteins c-fos/biosynthesis , Quinoxalines/administration & dosage , Quinoxalines/metabolism , Quinoxalines/therapeutic use , Serotonin 5-HT1 Receptor Agonists/administration & dosage , Serotonin 5-HT1 Receptor Agonists/therapeutic use , Soluble Guanylyl Cyclase/metabolism , Sumatriptan/administration & dosage , Sumatriptan/therapeutic use , Topiramate/administration & dosage , Topiramate/therapeutic useABSTRACT
UNLABELLED: A promising approach to neurotherapeutics involves activating the nuclear-factor-E2-related factor 2 (Nrf2)/antioxidant response element signaling, which regulates expression of antioxidant, anti-inflammatory, and cytoprotective genes. Tecfidera, a putative Nrf2 activator, is an oral formulation of dimethylfumarate (DMF) used to treat multiple sclerosis. We compared the effects of DMF and its bioactive metabolite monomethylfumarate (MMF) on Nrf2 signaling and their ability to block 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced experimental Parkinson's disease (PD). We show that in vitro DMF and MMF activate the Nrf2 pathway via S-alkylation of the Nrf2 inhibitor Keap1 and by causing nuclear exit of the Nrf2 repressor Bach1. Nrf2 activation by DMF but not MMF was associated with depletion of glutathione, decreased cell viability, and inhibition of mitochondrial oxygen consumption and glycolysis rates in a dose-dependent manner, whereas MMF increased these activities in vitro However, both DMF and MMF upregulated mitochondrial biogenesis in vitro in an Nrf2-dependent manner. Despite the in vitro differences, both DMF and MMF exerted similar neuroprotective effects and blocked MPTP neurotoxicity in wild-type but not in Nrf2 null mice. Our data suggest that DMF and MMF exhibit neuroprotective effects against MPTP neurotoxicity because of their distinct Nrf2-mediated antioxidant, anti-inflammatory, and mitochondrial functional/biogenetic effects, but MMF does so without depleting glutathione and inhibiting mitochondrial and glycolytic functions. Given that oxidative damage, neuroinflammation, and mitochondrial dysfunction are all implicated in PD pathogenesis, our results provide preclinical evidence for the development of MMF rather than DMF as a novel PD therapeutic. SIGNIFICANCE STATEMENT: Almost two centuries since its first description by James Parkinson, Parkinson's disease (PD) remains an incurable disease with limited symptomatic treatment. The current study provides preclinical evidence that a Food and Drug Administration-approved drug, dimethylfumarate (DMF), and its metabolite monomethylfumarate (MMF) can block nigrostriatal dopaminergic neurodegeneration in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of PD. We elucidated mechanisms by which DMF and its active metabolite MMF activates the redox-sensitive transcription factor nuclear-factor-E2-related factor 2 (Nrf2) to upregulate antioxidant, anti-inflammatory, mitochondrial biosynthetic and cytoprotective genes to render neuroprotection via distinct S-alkylating properties and depletion of glutathione. Our data suggest that targeting Nrf2-mediated gene transcription using MMF rather than DMF is a promising approach to block oxidative stress, neuroinflammation, and mitochondrial dysfunction for therapeutic intervention in PD while minimizing side effects.
Subject(s)
Fumarates/therapeutic use , NF-E2-Related Factor 2/metabolism , Neuroprotective Agents/therapeutic use , Parkinsonian Disorders/chemically induced , Parkinsonian Disorders/drug therapy , Signal Transduction/drug effects , 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine/pharmacology , Animals , Antigens, CD/metabolism , Cell Line, Transformed , Disease Models, Animal , Dose-Response Relationship, Drug , Fumarates/pharmacology , Gene Expression Regulation/drug effects , Gene Expression Regulation/genetics , Humans , Maleates/pharmacology , Mice , Mice, Inbred C57BL , Mice, Knockout , NF-E2-Related Factor 2/genetics , Parkinsonian Disorders/prevention & control , Rats , Tyrosine/analogs & derivatives , Tyrosine/pharmacologyABSTRACT
We aimed to investigate the mechanistic, functional, and therapeutic role of glycogen synthase kinase 3ß (GSK-3ß) in the regulation and activation of the proinflammatory oncogenic transcription factor nuclear factor of activated T cells (NFATc2) in pancreatic cancer. IHC, qPCR, immunoblotting, immunofluorescence microscopy, and proliferation assays were used to analyze mouse and human tissues and cell lines. Protein-protein interactions and promoter regulation were analyzed by coimmunoprecipitation, DNA pulldown, reporter, and ChIP assays. Preclinical assays were performed using a variety of pancreatic cancer cells lines, xenografts, and a genetically engineered mouse model (GEMM). GSK-3ß-dependent SP2 phosphorylation mediates NFATc2 protein stability in the nucleus of pancreatic cancer cells stimulating pancreatic cancer growth. In addition to protein stabilization, GSK-3ß also maintains NFATc2 activation through a distinct mechanism involving stabilization of NFATc2-STAT3 complexes independent of SP2 phosphorylation. For NFATc2-STAT3 complex formation, GSK-3ß-mediated phosphorylation of STAT3 at Y705 is required to stimulate euchromatin formation of NFAT target promoters, such as cyclin-dependent kinase-6, which promotes tumor growth. Finally, preclinical experiments suggest that targeting the NFATc2-STAT3-GSK-3ß module inhibits proliferation and tumor growth and interferes with inflammation-induced pancreatic cancer progression in Kras(G12D) mice. In conclusion, we describe a novel mechanism by which GSK-3ß fine-tunes NFATc2 and STAT3 transcriptional networks to integrate upstream signaling events that govern pancreatic cancer progression and growth. Furthermore, the therapeutic potential of GSK-3ß is demonstrated for the first time in a relevant Kras and inflammation-induced GEMM for pancreatic cancer.
Subject(s)
Glycogen Synthase Kinase 3 beta/metabolism , Inflammation/metabolism , NFATC Transcription Factors/metabolism , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Signal Transduction , Animals , Binding Sites , Cell Line, Tumor , Disease Models, Animal , Disease Progression , Gene Expression , Genes, ras , Humans , Mice , Mice, Knockout , Multiprotein Complexes/metabolism , NFATC Transcription Factors/genetics , Nucleotide Motifs , Pancreatic Neoplasms/genetics , Phosphorylation , Protein Binding , Protein Stability , STAT3 Transcription Factor/metabolismABSTRACT
Flavonoids are known to trigger the intrinsic genetic adaptive programs to hypoxic or oxidative stress via estrogen receptor engagement or upstream kinase activation. To reveal specific structural requirements for direct stabilization of the transcription factors responsible for triggering the antihypoxic and antioxidant programs, we studied flavones, isoflavones and catechols including dihydroxybenzoate, didox, levodopa, and nordihydroguaiaretic acid (NDGA), using novel luciferase-based reporters specific for the first step in HIF1 or Nrf2 protein stabilization. Distinct structural requirements for either transcription factor stabilization have been found: as expected, these requirements for activation of HIF ODD-luc reporter correlate with in silico binding to HIF prolyl hydroxylase. By contrast, stabilization of Nrf2 requires the presence of 3,4-dihydroxy- (catechol) groups. Thus, only some but not all flavonoids are direct activators of the hypoxic and antioxidant genetic programs. NDGA from the Creosote bush resembles the best flavonoids in their ability to directly stabilize HIF1 and Nrf2 and is superior with respect to LOX inhibition thus favoring this compound over others. Given much higher bioavailability and stability of NDGA than any flavonoid, NDGA has been tested in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-animal model of Parkinson's Disease and demonstrated neuroprotective effects.
ABSTRACT
The histone deacetylases (HDACs) occur in 11 different isoforms, and these enzymes regulate the activity of a large number of proteins involved in cancer initiation and progression. The discovery of isoform-selective HDAC inhibitors (HDACIs) is desirable, as it is likely that such compounds would avoid some of the undesirable side effects found with the first-generation inhibitors. A series of HDACIs previously reported by us were found to display some selectivity for HDAC6 and to induce cell-cycle arrest and apoptosis in pancreatic cancer cells. In the present work, we show that structural modification of these isoxazole-based inhibitors leads to high potency and selectivity for HDAC6 over HDAC1-3 and HDAC10, while unexpectedly abolishing their ability to block cell growth. Three inhibitors with lower HDAC6 selectivity inhibit the growth of cell lines BxPC3 and L3.6pl, and they only induce apoptosis in L3.6pl cells. We conclude that HDAC6 inhibition alone is insufficient for disruption of cell growth, and that some degree of classâ 1 HDAC inhibition is required. Moreover, the highly selective HDAC6Is reported herein that are weakly cytotoxic may find use in cancer immune system reactivation.
Subject(s)
Antineoplastic Agents/pharmacology , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylases/metabolism , Pancreatic Neoplasms/pathology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Histone Deacetylase 6 , Histone Deacetylase Inhibitors/chemical synthesis , Histone Deacetylase Inhibitors/chemistry , Humans , Molecular Structure , Pancreatic Neoplasms/enzymology , Structure-Activity RelationshipABSTRACT
Glycogen synthase kinase-3 (GSK-3), a constitutively active serine/threonine kinase, is a key regulator of numerous cellular processes ranging from glycogen metabolism to cell-cycle regulation and proliferation. Consistent with its involvement in many pathways, it has also been implicated in the pathogenesis of various human diseases, including type II diabetes, Alzheimer disease, bipolar disorder, inflammation, and cancer. Consequently, it is recognized as an attractive target for the development of new drugs. In the present study, we investigated the effect of both pharmacologic and genetic inhibition of GSK-3 in two different renal cancer cell lines. We have shown potent antiproliferative activity of 9-ING-41, a maleimide-based GSK-3 inhibitor. The antiproliferative activity is most likely caused by G(0)-G(1) and G(2)-M phase arrest as evident from cell-cycle analysis. We have established that inhibition of GSK-3 imparted a differentiated phenotype in renal cancer cells. We have also shown that GSK-3 inhibition induced autophagy, likely as a result of imbalanced energy homeostasis caused by impaired glucose metabolism. In addition, we have demonstrated the antitumor activity of 9-ING-41 in two different subcutaneous xenograft renal cell carcinoma tumor models. To our knowledge, this is the first report describing autophagy induction due to GSK-3 inhibition in renal cancer cells.
Subject(s)
Cell Differentiation/physiology , Glucose/metabolism , Glycogen Synthase Kinase 3/metabolism , Kidney Neoplasms/metabolism , Animals , Autophagy/drug effects , Blotting, Western , Cell Cycle Checkpoints/drug effects , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Glycogen Synthase Kinase 3/antagonists & inhibitors , Glycogen Synthase Kinase 3/genetics , Glycogen Synthase Kinase 3 beta , Humans , Indoles/chemistry , Indoles/pharmacology , Kidney Neoplasms/drug therapy , Kidney Neoplasms/genetics , Male , Maleimides/chemistry , Maleimides/pharmacology , Mice, Nude , Microscopy, Confocal , Molecular Structure , RNA Interference , Xenograft Model Antitumor AssaysABSTRACT
Inhibition of GSK-3ß has been well documented to account for the behavioral actions of the mood stabilizer lithium in various animal models of mood disorders. Recent studies have showed that genetic or pharmacological inhibition of GSK-3ß resulted in anxiolytic-like and pro-social behavior. In our ongoing efforts to develop GSK-3ß inhibitors for the treatment of mood disorders, SAR studies on maleimide-based compounds were undertaken. We present herein for the first time that some of these GSK-3ß inhibitors, in particular analogues 1 and 9, were able to stimulate progesterone production in the MA-10 mouse tumor Leydig cell model of steroidogenesis without any significant toxicity. These two compounds were tested in the SmartCube behavioral assay and showed anxiolytic-like signatures following daily dose administration (50 mg/kg, ip) for 13 days. Taken together, these results support the hypothesis that GSK-3ß inhibition could influence neuroactive steroid production thereby mediating the modulation of anxiety-like behavior in vivo.
Subject(s)
Glycogen Synthase Kinase 3/antagonists & inhibitors , Maleimides/chemistry , Maleimides/pharmacology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Steroids/biosynthesis , Adenosine Triphosphate/metabolism , Animals , Binding, Competitive , Cell Line, Tumor , Humans , Maleimides/metabolism , Mice , Protein Kinase Inhibitors/metabolismABSTRACT
The discovery of upregulated glycogen synthase kinase-3 (GSK-3) in various pathological conditions has led to the development of a host of chemically diverse small molecule GSK-3 inhibitors, such as BIP-135. GSK-3 inhibition emerged as an alternative therapeutic target for treating spinal muscular atrophy (SMA) when a number of GSK-3 inhibitors were shown to elevate survival motor neuron (SMN) levels in vitro and to rescue motor neurons when their intrinsic SMN level was diminished by SMN-specific short hairpin RNA (shRNA). Despite their cellular potency, the in vivo efficacy of GSK-3 inhibitors has yet to be evaluated in an animal model of SMA. Herein, we disclose that a potent and reasonably selective GSK-3 inhibitor, namely BIP-135, was tested in a transgenic Δ7 SMA KO mouse model of SMA, and found to prolong the median survival of these animals. In addition, this compound was shown to elevate the SMN protein level in SMA patient-derived fibroblast cells as determined by western blot, and was neuroprotective in a cell-based, SMA-related model of oxidative stress-induced neurodegeneration.