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1.
BMC Neurosci ; 16: 67, 2015 Oct 19.
Article in English | MEDLINE | ID: mdl-26480871

ABSTRACT

BACKGROUND: Synaptic dysfunction is a key event in pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD) where synapse loss pathologically correlates with cognitive decline and dementia. Although evidence suggests that aberrant protein production and aggregation are the causative factors in familial subsets of such diseases, drugs singularly targeting these hallmark proteins, such as amyloid-ß, have failed in late stage clinical trials. Therefore, to provide a successful disease-modifying compound and address synaptic dysfunction and memory loss in AD and mixed pathology dementia, we repurposed a clinically proven drug, CMZ, with neuroprotective and anti-inflammatory properties via addition of nitric oxide (NO) and cGMP signaling property. RESULTS: The novel compound, NMZ, was shown to retain the GABAA potentiating actions of CMZ in vitro and sedative activity in vivo. Importantly, NMZ restored LTP in hippocampal slices from AD transgenic mice, whereas CMZ was without effect. NMZ reversed amnestic blockade of acetylcholine receptors by scopolamine as well as NMDA receptor blockade by a benzodiazepine and a NO synthase inhibitor in the step-through passive avoidance (STPA) test of learning and working memory. A PK/PD relationship was developed based on STPA analysis coupled with pharmacokinetic measures of drug levels in the brain: at 1 nM concentration in brain and plasma, NMZ was able to restore memory consolidation in mice. CONCLUSION: Our findings show that NMZ embodies a promising pharmacological approach targeting synaptic dysfunction and opens new avenues for neuroprotective intervention strategies in mixed pathology AD, neurodegeneration, and dementia.


Subject(s)
Alzheimer Disease/drug therapy , Chlormethiazole/analogs & derivatives , Drug Repositioning/methods , GABA-A Receptor Agonists/pharmacology , Hippocampus/drug effects , Long-Term Potentiation/drug effects , Neuroprotective Agents/pharmacology , Nootropic Agents/pharmacology , Animals , CREB-Binding Protein/metabolism , Cyclic GMP/metabolism , Disease Models, Animal , GABA-A Receptor Agonists/pharmacokinetics , Male , Mice , Mice, Transgenic , Neuroprotective Agents/pharmacokinetics , Nitric Oxide/metabolism , Nootropic Agents/pharmacokinetics , Signal Transduction/drug effects , Synapses/drug effects , Synapses/pathology , Xenopus laevis
2.
Hum Mol Genet ; 24(18): 5285-98, 2015 Sep 15.
Article in English | MEDLINE | ID: mdl-26123489

ABSTRACT

Cumulative evidence indicates that the onset and severity of Huntington's disease (HD) symptoms correlate with connectivity deficits involving specific neuronal populations within cortical and basal ganglia circuits. Brain imaging studies and pathological reports further associated these deficits with alterations in cerebral white matter structure and axonal pathology. However, whether axonopathy represents an early pathogenic event or an epiphenomenon in HD remains unknown, nor is clear the identity of specific neuronal populations affected. To directly evaluate early axonal abnormalities in the context of HD in vivo, we bred transgenic YFP(J16) with R6/2 mice, a widely used HD model. Diffusion tensor imaging and fluorescence microscopy studies revealed a marked degeneration of callosal axons long before the onset of motor symptoms. Accordingly, a significant fraction of YFP-positive cortical neurons in YFP(J16) mice cortex were identified as callosal projection neurons. Callosal axon pathology progressively worsened with age and was influenced by polyglutamine tract length in mutant huntingtin (mhtt). Degenerating axons were dissociated from microscopically visible mhtt aggregates and did not result from loss of cortical neurons. Interestingly, other axonal populations were mildly or not affected, suggesting differential vulnerability to mhtt toxicity. Validating these results, increased vulnerability of callosal axons was documented in the brains of HD patients. Observations here provide a structural basis for the alterations in cerebral white matter structure widely reported in HD patients. Collectively, our data demonstrate a dying-back pattern of degeneration for cortical projection neurons affected in HD, suggesting that axons represent an early and potentially critical target for mhtt toxicity.


Subject(s)
Axons/pathology , Brain/pathology , Huntington Disease/metabolism , Huntington Disease/pathology , Aged , Animals , Axons/metabolism , Brain/metabolism , Cerebral Cortex/metabolism , Cerebral Cortex/pathology , Female , Gene Expression , Genes, Reporter , Humans , Huntington Disease/diagnosis , Male , Mice , Mice, Transgenic , Microscopy, Confocal , Middle Aged , Nerve Degeneration/metabolism , Nerve Degeneration/pathology , Neurons/metabolism , Neurons/pathology , Protein Aggregation, Pathological , Serotonin Plasma Membrane Transport Proteins/metabolism , Severity of Illness Index
3.
Br J Pharmacol ; 171(2): 389-402, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24116891

ABSTRACT

BACKGROUND AND PURPOSE: Chlormethiazole (CMZ), a clinical sedative/anxiolytic agent, did not reach clinical efficacy in stroke trials despite neuroprotection demonstrated in numerous animal models. Using CMZ as a lead compound, neuroprotective methiazole (MZ) analogues were developed, and neuroprotection and GABA(A) receptor dependence were studied. EXPERIMENTAL APPROACH: Eight MZs were selected from a novel library, of which two were studied in detail. Neuroprotection, glutamate release, intracellular calcium and response to GABA blockade by picrotoxin were measured in rat primary cortical cultures using four cellular models of neurodegeneration. GABA potentiation was assayed in oocytes expressing the α1ß2γ2 GABA(A) receptor. KEY RESULTS: Neuroprotection against a range of insults was retained even with substantial chemical modification. Dependence on GABAA receptor activity was variable: at the extremes, neuroprotection by GN-28 was universally sensitive to picrotoxin, while GN-38 was largely insensitive. In parallel, effects on extracellular glutamate and intracellular calcium were associated with GABA(A) dependence. Consistent with these findings, GN-28 potentiated α1ß2γ2 GABA(A) function, whereas GN-38 had a weak inhibitory effect. Neuroprotection against moderate dose oligomeric Aß1₋42 was also tolerant to structural changes. CONCLUSIONS AND IMPLICATIONS: The results support the concept that CMZ does not contain a single pharmacophore, rather that broad-spectrum neuroprotection results from a GABA(A)-dependent mechanism represented by GN-28, combined with a mechanism represented in GN-38 that shows the least dependence on GABA(A) receptors. These findings allow further refinement of the neuroprotective pharmacophore and investigation into secondary mechanisms that will assist in identifying MZ-based compounds of use in treating neurodegeneration.


Subject(s)
Chlormethiazole/analogs & derivatives , Chlormethiazole/pharmacology , GABA Agonists/pharmacology , Nerve Degeneration/prevention & control , Neuroprotective Agents/pharmacology , Receptors, GABA-A/drug effects , Animals , Biological Availability , Brain/metabolism , Calcium/metabolism , Cell Hypoxia , Chromatography, High Pressure Liquid , Excitatory Amino Acid Agonists/toxicity , Female , Glucose/deficiency , Glutamic Acid/metabolism , Glutamic Acid/toxicity , Mice , Mice, Inbred C57BL , N-Methylaspartate/toxicity , Neurons/drug effects , Oocytes/drug effects , Pregnancy , Primary Cell Culture , Rats , Rats, Sprague-Dawley , Xenopus laevis , gamma-Aminobutyric Acid/pharmacology
4.
J Med Chem ; 56(15): 6054-68, 2013 Aug 08.
Article in English | MEDLINE | ID: mdl-23834438

ABSTRACT

Hyperactivation of the calcium-dependent cysteine protease calpain 1 (Cal1) is implicated as a primary or secondary pathological event in a wide range of illnesses and in neurodegenerative states, including Alzheimer's disease (AD). E-64 is an epoxide-containing natural product identified as a potent nonselective, calpain inhibitor, with demonstrated efficacy in animal models of AD. By use of E-64 as a lead, three successive generations of calpain inhibitors were developed using computationally assisted design to increase selectivity for Cal1. First generation analogues were potent inhibitors, effecting covalent modification of recombinant Cal1 catalytic domain (Cal1cat), demonstrated using LC-MS/MS. Refinement yielded second generation inhibitors with improved selectivity. Further library expansion and ligand refinement gave three Cal1 inhibitors, one of which was designed as an activity-based protein profiling probe. These were determined to be irreversible and selective inhibitors by kinetics studies comparing full length Cal1 with the general cysteine protease papain.


Subject(s)
Calpain/antagonists & inhibitors , Epoxy Compounds/chemical synthesis , Leucine/analogs & derivatives , Peptidomimetics/chemical synthesis , Calpain/chemistry , Catalytic Domain , Click Chemistry , Computer Simulation , Drug Design , Epoxy Compounds/chemistry , Kinetics , Leucine/chemical synthesis , Leucine/chemistry , Molecular Docking Simulation , Papain/antagonists & inhibitors , Peptidomimetics/chemistry , Stereoisomerism , Structure-Activity Relationship
5.
PLoS One ; 8(6): e65235, 2013.
Article in English | MEDLINE | ID: mdl-23776455

ABSTRACT

Dying-back degeneration of motor neuron axons represents an established feature of familial amyotrophic lateral sclerosis (FALS) associated with superoxide dismutase 1 (SOD1) mutations, but axon-autonomous effects of pathogenic SOD1 remained undefined. Characteristics of motor neurons affected in FALS include abnormal kinase activation, aberrant neurofilament phosphorylation, and fast axonal transport (FAT) deficits, but functional relationships among these pathogenic events were unclear. Experiments in isolated squid axoplasm reveal that FALS-related SOD1 mutant polypeptides inhibit FAT through a mechanism involving a p38 mitogen activated protein kinase pathway. Mutant SOD1 activated neuronal p38 in mouse spinal cord, neuroblastoma cells and squid axoplasm. Active p38 MAP kinase phosphorylated kinesin-1, and this phosphorylation event inhibited kinesin-1. Finally, vesicle motility assays revealed previously unrecognized, isoform-specific effects of p38 on FAT. Axon-autonomous activation of the p38 pathway represents a novel gain of toxic function for FALS-linked SOD1 proteins consistent with the dying-back pattern of neurodegeneration characteristic of ALS.


Subject(s)
Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/pathology , Axonal Transport/drug effects , Nerve Degeneration/pathology , Superoxide Dismutase/toxicity , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Axonal Transport/physiology , Decapodiformes , Immunohistochemistry , Kinesins/antagonists & inhibitors , Kinesins/metabolism , Mass Spectrometry , Mice , Mutation/genetics , Phosphorylation , Spinal Cord/metabolism , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolism , Superoxide Dismutase-1
6.
J Med Chem ; 55(15): 6784-801, 2012 Aug 09.
Article in English | MEDLINE | ID: mdl-22779770

ABSTRACT

Learning and memory deficits in Alzheimer's disease (AD) result from synaptic failure and neuronal loss, the latter caused in part by excitotoxicity and oxidative stress. A therapeutic approach is described that uses NO-chimeras directed at restoration of both synaptic function and neuroprotection. 4-Methylthiazole (MZ) derivatives were synthesized, based upon a lead neuroprotective pharmacophore acting in part by GABA(A) receptor potentiation. MZ derivatives were assayed for protection of primary neurons against oxygen-glucose deprivation and excitotoxicity. Selected neuroprotective derivatives were incorporated into NO-chimera prodrugs, coined nomethiazoles. To provide proof of concept for the nomethiazole drug class, selected examples were assayed for restoration of synaptic function in hippocampal slices from AD-transgenic mice, reversal of cognitive deficits, and brain bioavailability of the prodrug and its neuroprotective MZ metabolite. Taken together, the assay data suggest that these chimeric nomethiazoles may be of use in treatment of multiple components of neurodegenerative disorders, such as AD.


Subject(s)
Neuroprotective Agents/chemical synthesis , Nitric Oxide/chemistry , Prodrugs/chemical synthesis , Thiazoles/chemical synthesis , Alzheimer Disease/drug therapy , Alzheimer Disease/genetics , Alzheimer Disease/psychology , Amyloid beta-Protein Precursor/genetics , Animals , Avoidance Learning/drug effects , Biological Availability , Brain/metabolism , Click Chemistry , Cognition Disorders/drug therapy , Glucose/deficiency , Hippocampus/drug effects , Hippocampus/physiology , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Molecular Mimicry , Mutation , Neurons/cytology , Neurons/drug effects , Neuroprotective Agents/chemistry , Neuroprotective Agents/pharmacology , Oxygen/metabolism , Primary Cell Culture , Prodrugs/chemistry , Prodrugs/pharmacology , Receptors, GABA-A/metabolism , Small Molecule Libraries , Structure-Activity Relationship , Synaptic Transmission/drug effects , Thiazoles/chemistry , Thiazoles/pharmacology , Tissue Distribution
7.
ACS Med Chem Lett ; 2(9): 656-661, 2011 Sep 08.
Article in English | MEDLINE | ID: mdl-21927645

ABSTRACT

Hybrid nitrate drugs have been reported to provide NO bioactivity to ameliorate side effects or to provide ancillary therapeutic activity. Hybrid nitrate selective serotonin reuptake inhibitors (NO-SSRIs) were prepared to improve the therapeutic profile of this drug class. A synthetic strategy for use of a thiocarbamate linker was developed, which in the case of NO-fluoxetine facilitated hydrolysis to fluoxetine at pH 7.4 within 7 hours. In cell culture, NO-SSRIs were weak inhibitors of the serotonin transporter, however, in the forced swimming task (FST) in rats, NO-fluoxetine demonstrated classical antidepressant activity. Comparison of NO-fluoxetine, with fluoxetine, and an NO-chimera nitrate developed for Alzheimer's disease (GT-1061), was made in the step through passive avoidance (STPA) test of learning and memory in rats treated with scopolamine as an amnesic agent. Fluoxetine was inactive, whereas NO-fluoxetine and GT-1061 both restored long-term memory. GT-1061 also produced antidepressant behavior in FST. These data support the potential for NO-SSRIs to overcome the lag in onset of therapeutic action and provide co-therapy of neuropathologies concomitant with depression.

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