ABSTRACT
Inhibition of human ornithine aminotransferase interferes with glutamine and proline metabolism in hepatocellular carcinoma, depriving tumors of essential nutrients. A proposed mechanism-based inhibitor containing a bicyclo[3.1.1]heptanol warhead is reported herein. The proposed inactivation mechanism involves a novel α-iminol rearrangement. The synthesis of the proposed inhibitor features an asymmetric intramolecular Mannich reaction, utilizing a chiral sulfinamide. This study presents a novel approach toward the synthesis of functionalized bicyclo[3.1.1]heptanes and highlights an underutilized method to access enantiopure exocyclic amines.
Subject(s)
Carboxylic Acids , Stereoisomerism , Carboxylic Acids/chemistry , Molecular Structure , Bridged Bicyclo Compounds/chemistry , Bridged Bicyclo Compounds/chemical synthesis , HumansABSTRACT
Human ornithine aminotransferase (hOAT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, has been shown to play an essential role in the metabolic reprogramming and progression of hepatocellular carcinoma (HCC). HCC accounts for approximately 75% of primary liver cancers and is within the top three causes of cancer death worldwide. As a result of treatment limitations, the overall 5-year survival rate for all patients with HCC is under 20%. The prevalence of HCC necessitates continued development of novel and effective treatment methods. In recent years, the therapeutic potential of selective inactivation of hOAT has been demonstrated for the treatment of HCC. Inspired by previous increased selectivity for hOAT by the expansion of the cyclopentene ring scaffold to a cyclohexene, we designed, synthesized, and evaluated a series of novel fluorinated cyclohexene analogues and identified (R)-3-amino-5,5-difluorocyclohex-1-ene-1-carboxylic acid as a time-dependent inhibitor of hOAT. Structural and mechanistic studies have elucidated the mechanism of inactivation of hOAT by 5, resulting in a PLP-inactivator adduct tightly bound to the active site of the enzyme. Intact protein mass spectrometry, 19F NMR spectroscopy, transient state kinetic studies, and X-ray crystallography were used to determine the structure of the final adduct and elucidate the mechanisms of inactivation. Interestingly, despite the highly electrophilic intermediate species conferred by fluorine and structural evidence of solvent accessibility in the hOAT active site, Lys292 and water did not participate in nucleophilic addition during the inactivation mechanism of hOAT by 5. Instead, rapid aromatization to yield the final adduct was favored.
Subject(s)
Drug Design , Enzyme Inhibitors , Ornithine-Oxo-Acid Transaminase , Humans , Ornithine-Oxo-Acid Transaminase/metabolism , Ornithine-Oxo-Acid Transaminase/chemistry , Ornithine-Oxo-Acid Transaminase/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/chemical synthesis , Carboxylic Acids/chemistry , Carboxylic Acids/chemical synthesis , Carboxylic Acids/pharmacology , Cyclohexenes/chemistry , Cyclohexenes/chemical synthesis , Cyclohexenes/pharmacology , Cyclohexenes/metabolism , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/metabolism , Crystallography, X-Ray , Models, MolecularABSTRACT
In our efforts to develop inhibitors selective for neuronal nitric oxide synthase (nNOS) over endothelial nitric oxide synthase (eNOS), we found that nNOS can undergo conformational changes in response to inhibitor binding that does not readily occur in eNOS. One change involves movement of a conserved tyrosine, which hydrogen bonds to one of the heme propionates, but in the presence of an inhibitor, changes conformation, enabling part of the inhibitor to hydrogen bond with the heme propionate. This movement does not occur as readily in eNOS and may account for the reason why these inhibitors bind more tightly to nNOS. A second structural change occurs upon the binding of a second inhibitor molecule to nNOS, displacing the pterin cofactor. Binding of this second site inhibitor requires structural changes at the dimer interface, which also occurs more readily in nNOS than in eNOS. Here, we used a combination of crystallography, mutagenesis, and computational methods to better understand the structural basis for these differences in NOS inhibitor binding. Computational results show that a conserved tyrosine near the primary inhibitor binding site is anchored more tightly in eNOS than in nNOS, allowing for less flexibility of this residue. We also find that the inefficiency of eNOS to bind a second inhibitor molecule is likely due to the tighter dimer interface in eNOS compared with nNOS. This study provides a better understanding of how subtle structural differences in NOS isoforms can result in substantial dynamic differences that can be exploited in the development of isoform-selective inhibitors.
Subject(s)
Nitric Oxide Synthase Type III , Nitric Oxide Synthase , Nitric Oxide Synthase/metabolism , Nitric Oxide Synthase Type III/genetics , Nitric Oxide Synthase Type III/chemistry , Nitric Oxide Synthase Type I , Protein Isoforms/chemistry , Crystallography, X-Ray , Enzyme Inhibitors/pharmacology , Heme/chemistry , Tyrosine , Nitric OxideABSTRACT
Toxoplasma gondii causes morbidity, mortality, and disseminates widely via cat sexual stages. Here, we find T. gondii ornithine aminotransferase (OAT) is conserved across phyla. We solve TgO/GABA-AT structures with bound inactivators at 1.55 Å and identify an inactivator selective for TgO/GABA-AT over human OAT and GABA-AT. However, abrogating TgO/GABA-AT genetically does not diminish replication, virulence, cyst-formation, or eliminate cat's oocyst shedding. Increased sporozoite/merozoite TgO/GABA-AT expression led to our study of a mutagenized clone with oocyst formation blocked, arresting after forming male and female gametes, with "Rosetta stone"-like mutations in genes expressed in merozoites. Mutations are similar to those in organisms from plants to mammals, causing defects in conception and zygote formation, affecting merozoite capacitation, pH/ionicity/sodium-GABA concentrations, drawing attention to cyclic AMP/PKA, and genes enhancing energy or substrate formation in TgO/GABA-AT-related-pathways. These candidates potentially influence merozoite's capacity to make gametes that fuse to become zygotes, thereby contaminating environments and causing disease.
ABSTRACT
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with no cure, and current treatment options are very limited. Previously, we performed a high-throughput screen to identify small molecules that inhibit protein aggregation caused by a mutation in the gene that encodes superoxide dismutase 1 (SOD1), which is responsible for about 25% of familial ALS. This resulted in three hit series of compounds that were optimized over several years to give three compounds that were highly active in a mutant SOD1 ALS model. Here we identify the target of two of the active compounds (6 and 7) with the use of photoaffinity labeling, chemical biology reporters, affinity purification, proteomic analysis, and fluorescent/cellular thermal shift assays. Evidence is provided to demonstrate that these two pyrazolone compounds directly interact with 14-3-3-E and 14-3-3-Q isoforms, which have chaperone activity and are known to interact with mutant SOD1G93A aggregates and become insoluble in the subcellular JUNQ compartment, leading to apoptosis. Because protein aggregation is the hallmark of all neurodegenerative diseases, knowledge of the target compounds that inhibit protein aggregation allows for the design of more effective molecules for the treatment of ALS and possibly other neurodegenerative diseases.
ABSTRACT
A series of potent, selective, and highly permeable human neuronal nitric oxide synthase inhibitors (hnNOS), based on a difluorobenzene ring linked to a 2-aminopyridine scaffold with different functionalities at the 4-position, is reported. In our efforts to develop novel nNOS inhibitors for the treatment of neurodegenerative diseases, we discovered 17, which showed excellent potency toward both rat (Ki 15 nM) and human nNOS (Ki 19 nM), with 1075-fold selectivity over human eNOS and 115-fold selectivity over human iNOS. 17 also showed excellent permeability (Pe = 13.7 × 10-6 cm s-1), a low efflux ratio (ER 0.48), along with good metabolic stability in mouse and human liver microsomes, with half-lives of 29 and >60 min, respectively. X-ray cocrystal structures of inhibitors bound with three NOS enzymes, namely, rat nNOS, human nNOS, and human eNOS, revealed detailed structure-activity relationships for the observed potency, selectivity, and permeability properties of the inhibitors.
Subject(s)
Enzyme Inhibitors , Nitric Oxide Synthase , Rats , Mice , Humans , Animals , Nitric Oxide Synthase Type I , Nitric Oxide Synthase/chemistry , Nitric Oxide Synthase/metabolism , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/chemistry , Structure-Activity Relationship , Nitric OxideABSTRACT
Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease in which the motor neuron circuitry displays progressive degeneration, affecting mostly the motor neurons in the brain and in the spinal cord. There are no effective cures, albeit three drugs, riluzole, edaravone, and AMX0035 (a combination of sodium phenylbutyrate and taurursodiol), have been approved by the Food and Drug Administration, with limited improvement in patients. There is an urgent need to build better and more effective treatment strategies for ALS. Since the disease is very heterogenous, numerous approaches have been explored, such as targeting genetic mutations, decreasing oxidative stress and excitotoxicity, enhancing mitochondrial function and protein degradation mechanisms, and inhibiting neuroinflammation. In addition, various chemical libraries or previously identified drugs have been screened for potential repurposing in the treatment of ALS. Here, we review previous drug discovery efforts targeting a variety of cellular pathologies that occur from genetic mutations that cause ALS, such as mutations in SOD1, C9orf72, FUS, and TARDP-43 genes. These mutations result in protein aggregation, which causes neuronal degeneration. Compounds used to target cellular pathologies that stem from these mutations are discussed and comparisons among different preclinical models are presented. Because the drug discovery landscape for ALS and other motor neuron diseases is changing rapidly, we also offer recommendations for a novel, more effective, direction in ALS drug discovery that could accelerate translation of effective compounds from animals to patients.
Subject(s)
Amyotrophic Lateral Sclerosis , Neurodegenerative Diseases , Animals , Humans , Amyotrophic Lateral Sclerosis/drug therapy , Amyotrophic Lateral Sclerosis/genetics , Neurodegenerative Diseases/metabolism , Disease Models, Animal , Motor Neurons/metabolism , Motor Neurons/pathologyABSTRACT
An improved synthesis of 4-methyl-7-(3-((methylamino)methyl)phenethyl)quinolin-2-amine (1) is reported. A scalable, rapid, and efficient methodology was developed to access this compound with an overall yield of 35%, which is 5.9-fold higher than the previous report. The key differences in the improved synthesis are a high yielding quinoline synthesis by a Knorr reaction, a copper-mediated Sonogashira coupling to the internal alkyne in excellent yield, and a crucial deprotection of the N-acetyl and N-Boc groups achieved under acidic conditions in a single step rather than a poor yielding quinoline N-oxide strategy, basic deprotection conditions, and low yielding copper-free conditions that were reported in the previous report. Compound 1, which previously was shown to inhibit IFN-γ-induced tumor growth in a human melanoma xenograft mouse model, was found to inhibit the growth of metastatic melanoma, glioblastoma, and hepatocellular carcinoma in vitro.
Subject(s)
Melanoma , Nitric Oxide Synthase , Mice , Humans , Animals , Nitric Oxide Synthase Type I , Enzyme Inhibitors/pharmacology , Cells, Cultured , Nitric OxideABSTRACT
Ornithine aminotransferase (OAT) is overexpressed in hepatocellular carcinoma (HCC), and we previously showed that inactivation of OAT inhibits the growth of HCC. Recently, we found that (3S,4S)-3-amino-4-fluorocyclopentenecarboxylic acid (5) was a potent inactivator of γ-aminobutyric acid aminotransferase (GABA-AT), proceeding by an enamine mechanism. Here we describe our investigations into the activity and mechanism of 5 as an inactivator of human OAT. We have found that 5 exhibits 10-fold less inactivation efficiency (kinact/KI) against hOAT than GABA-AT. A comprehensive mechanistic study was carried out to understand its inactivation mechanism with hOAT. pKa and electrostatic potential calculations were performed to further support the notion that the α,ß-unsaturated alkene of 5 is critical for enhancing acidity and nucleophilicity of the corresponding intermediates and ultimately responsible for the improved inactivation efficiency of 5 over the corresponding saturated analogue (4). Intact protein mass spectrometry and the crystal structure complex with hOAT provide evidence to conclude that 5 mainly inactivates hOAT through noncovalent interactions, and that, unlike with GABA-AT, covalent binding with hOAT is a minor component of the total inhibition which is unique relative to other monofluoro-substituted derivatives. Furthermore, based on the results of transient-state measurements and free energy calculations, it is suggested that the α,ß-unsaturated carboxylate group of PLP-bound 5 may be directly involved in the inactivation cascade by forming an enolate intermediate. Overall, compound 5 exhibits unusual structural conversions which are catalyzed by specific residues within hOAT, ultimately leading to an enamine mechanism-based inactivation of hOAT through noncovalent interactions and covalent modification.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Humans , Amino Acids/pharmacology , Enzyme Inhibitors/pharmacology , Ornithine-Oxo-Acid Transaminase/chemistry , Ornithine-Oxo-Acid Transaminase/metabolism , gamma-Aminobutyric Acid , Carboxylic Acids/pharmacology , Carboxylic Acids/chemistry , OrnithineABSTRACT
A series of potent, selective, and highly permeable human neuronal nitric oxide synthase inhibitors (hnNOS) based on the 2-aminopyridine scaffold with a shortened amino sidechain is reported. A rapid and simple protocol was developed to access these inhibitors in excellent yields. Neuronal nitric oxide synthase (nNOS) is a novel therapeutic target for the treatment of various neurological disorders. The major challenges in designing nNOS inhibitors in humans focus on potency, selectivity over other isoforms of nitric oxide synthases (NOSs), and blood-brain barrier permeability. In this context, we discovered a promising inhibitor, 6-(3-(4,4-difluoropiperidin-1-yl)propyl)-4-methylpyridin-2-amine dihydrochloride, that exhibits excellent potency for rat (Kiâ¯=â¯46â¯nM) and human nNOS (Kiâ¯=â¯48â¯nM), respectively, with 388-fold human eNOS and 135-fold human iNOS selectivity. It also displayed excellent permeability (Peâ¯=â¯17.3â¯×â¯10-6 cm s-1) through a parallel artificial membrane permeability assay, a model for blood-brain permeability. We found that increasing lipophilicity by incorporation of fluorine atoms on the backbone of the inhibitors significantly increased potential blood-brain barrier permeability. In addition to measuring potency, isoform selectivity, and permeability of NOS inhibitors, we also explored structure-activity relationships via structures of key inhibitors complexed to various isoforms of nitric oxide synthases.
Subject(s)
Aminopyridines , Nitric Oxide , Aminopyridines/chemistry , Aminopyridines/pharmacology , Animals , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , Nitric Oxide Synthase , Nitric Oxide Synthase Type I/chemistry , Nitric Oxide Synthase Type I/metabolism , Protein Isoforms , RatsABSTRACT
Melanoma is the most fatal type of skin cancer and is notoriously resistant to chemotherapies. The response of melanoma to current treatments is difficult to predict. To combat these challenges, in this study, we utilize a small peptide to increase drug delivery to melanoma cells. A peptide library array was designed and screened using a peptide array-whole cell binding assay, which identified KK-11 as a novel human melanoma-targeting peptide. The peptide and its D-amino acid substituted analogue (VPWxEPAYQrFL or D-aa KK-11) were synthesized via a solid-phase strategy. Further studies using FITC-labeled KK-11 demonstrated dose-dependent uptake in human melanoma cells. D-aa KK-11 significantly increased the stability of the peptide, with 45.3% remaining detectable after 24 h with human serum incubation. Co-treatment of KK-11 with doxorubicin was found to significantly enhance the cytotoxicity of doxorubicin compared to doxorubicin alone, or sequential KK-11 and doxorubicin treatment. In vivo and ex vivo imaging revealed that D-aa KK-11 distributed to xenografted A375 melanoma tumors as early as 5 min and persisted up to 24 h post tail vein injection. When co-administered, D-aa KK-11 significantly enhanced the anti-tumor activity of a novel nNOS inhibitor (MAC-3-190) in an A375 human melanoma xenograft mouse model compared to MAC-3-190 treatment alone. No apparent systemic toxicities were observed. Taken together, these results suggest that KK-11 may be a promising human melanoma-targeted delivery vector for anti-melanoma cargo.
ABSTRACT
Cyclic α-aryl ß-dicarbonyl derivatives are important scaffolds in medicinal chemistry. Palladium-catalyzed coupling reactions of haloarenes were conducted with diverse five- to seven-membered cyclic ß-dicarbonyl derivatives including barbiturate, pyrazolidine-3,5-dione, and 1,4-diazepane-5,7-dione. The coupling reactions of various para- or meta-substituted aryl halides occurred efficiently when Pd(t-Bu3P)2, Xphos, and Cs2CO3 were used under 1,4-dioxane reflux conditions. Although the couplings of ortho-substituted aryl halides with pyrazolidine-3,5-dione and 1,4-diazepane-5,7-dione were moderate, the coupling with barbiturate was limited. Using the optimized reaction conditions, we synthesized several 5-aryl barbiturates as new scaffolds of CaV1.3 Ca2+ channel inhibitors. Among the synthesized molecules, 14e was the most potent CaV1.3 inhibitor with an IC50 of 1.42 µM.
ABSTRACT
Human ornithine aminotransferase (hOAT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that contains a similar active site to that of γ-aminobutyric acid aminotransferase (GABA-AT). Recently, pharmacological inhibition of hOAT was recognized as a potential therapeutic approach for hepatocellular carcinoma. In this work, we first studied the inactivation mechanisms of hOAT by two well-known GABA-AT inactivators (CPP-115 and OV329). Inspired by the inactivation mechanistic difference between these two aminotransferases, a series of analogues were designed and synthesized, leading to the discovery of analogue 10b as a highly selective and potent hOAT inhibitor. Intact protein mass spectrometry, protein crystallography, and dialysis experiments indicated that 10b was converted to an irreversible tight-binding adduct (34) in the active site of hOAT, as was the unsaturated analogue (11). The comparison of kinetic studies between 10b and 11 suggested that the active intermediate (17b) was only generated in hOAT and not in GABA-AT. Molecular docking studies and pKa computational calculations highlighted the importance of chirality and the endocyclic double bond for inhibitory activity. The turnover mechanism of 10b was supported by mass spectrometric analysis of dissociable products and fluoride ion release experiments. Notably, the stopped-flow experiments were highly consistent with the proposed mechanism, suggesting a relatively slow hydrolysis rate for hOAT. The novel second-deprotonation mechanism of 10b contributes to its high potency and significantly enhanced selectivity for hOAT inhibition.
Subject(s)
4-Aminobutyrate Transaminase , Liver Neoplasms , Carboxylic Acids , Enzyme Inhibitors/chemistry , Humans , Kinetics , Molecular Docking Simulation , Ornithine-Oxo-Acid Transaminase , Phenylacetates , Pyridoxal Phosphate/chemistry , gamma-Aminobutyric AcidABSTRACT
Even though amyotrophic lateral sclerosis (ALS) is a disease of the upper and lower motor neurons, to date none of the compounds in clinical trials have been tested for improving the health of diseased upper motor neurons (UMNs). There is an urgent need to develop preclinical assays that include UMN health as a readout. Since ALS is a complex disease, combinatorial treatment strategies will be required to address the mechanisms perturbed in patients. Here, we describe a novel in vitro platform that takes advantage of an UMN reporter line in which UMNs are genetically labeled with fluorescence and have misfolded SOD1 toxicity. We report that NU-9, an analog of the cyclohexane-1,3-dione family of compounds, improves the health of UMNs with misfolded SOD1 toxicity more effectively than riluzole or edaravone, -the only two FDA-approved ALS drugs to date-. Interestingly, when NU-9 is applied in combination with riluzole or edaravone, there is an additive effect on UMN health, as they extend longer axons and display enhanced branching and arborization, two important characteristics of healthy UMNs in vitro.
Subject(s)
Amyotrophic Lateral Sclerosis , Riluzole , Amyotrophic Lateral Sclerosis/drug therapy , Animals , Edaravone/pharmacology , Humans , Mice , Motor Neurons , Riluzole/pharmacology , Riluzole/therapeutic use , Superoxide DismutaseABSTRACT
Interferon-gamma (IFN-γ) is shown to stimulate melanoma development and progression. However, the underlying mechanism has not been completely defined. Our study aimed to determine the role of neuronal nitric oxide synthase (nNOS)-mediated signaling in IFN-γ-stimulated melanoma progression and the anti-melanoma effects of novel nNOS inhibitors. Our study shows that IFN-γ markedly induced the expression levels of nNOS in melanoma cells associated with increased intracellular nitric oxide (NO) levels. Co-treatment with novel nNOS inhibitors effectively alleviated IFN-γ-activated STAT1/3. Further, reverse phase protein array (RPPA) analysis demonstrated that IFN-γ induced the expression of HIF1α, c-Myc, and programmed death-ligand 1 (PD-L1), in contrast to IFN-α. Blocking the nNOS-mediated signaling pathway using nNOS-selective inhibitors was shown to effectively diminish IFN-γ-induced PD-L1 expression in melanoma cells. Using a human melanoma xenograft mouse model, the in vivo studies revealed that IFN-γ increased tumor growth compared to control, which was inhibited by the co-administration of nNOS inhibitor MAC-3-190. Another nNOS inhibitor, HH044, was shown to effectively inhibit in vivo tumor growth and was associated with reduced PD-L1 expression levels in melanoma xenografts. Our study demonstrates the important role of nNOS-mediated NO signaling in IFN-γ-stimulated melanoma progression. Targeting nNOS using highly selective small molecular inhibitors is a unique and effective strategy to improve melanoma treatment.
Subject(s)
Carcinogenesis/chemically induced , Carcinogenesis/drug effects , Disease Progression , Enzyme Inhibitors/administration & dosage , Interferon-gamma/administration & dosage , Melanoma/drug therapy , Melanoma/metabolism , Nitric Oxide Synthase Type I/antagonists & inhibitors , Signal Transduction/drug effects , Skin Neoplasms/drug therapy , Skin Neoplasms/metabolism , Animals , B7-H1 Antigen/metabolism , Carcinogenesis/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Humans , Interferon-alpha/pharmacology , Melanoma/pathology , Mice , Mice, Nude , Nitric Oxide Synthase Type I/metabolism , STAT1 Transcription Factor/metabolism , STAT3 Transcription Factor/metabolism , Skin Neoplasms/pathology , Treatment Outcome , Tumor Burden/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Hepatocellular carcinoma (HCC) is the second or third leading cause of cancer mortality worldwide (depending on which statistics are used), yet there is no effective treatment. Currently, there are nine FDA-approved drugs for HCC, five monoclonal antibodies and four tyrosine kinase inhibitors. Ornithine aminotransferase (OAT) has been validated as a target in preclinical studies, which demonstrates that it is a potential target to treat HCC. Currently, there are no OAT inactivators in clinical trials for HCC. This Innovation describes evidence to support inhibition of OAT as a novel approach for HCC tumor growth inhibition. After the mechanism of OAT is discussed, the origins of our involvement in OAT inactivation, based on our previous work on mechanism-based inactivation of GABA-AT, are described. Once it was demonstrated that OAT inactivation does lead to HCC tumor growth inhibition, new selective OAT inactivators were designed and their inactivation mechanisms were elucidated. A summary of these mechanistic studies is presented. Inactivators of OAT provide the potential for treatment of HCC, targeting the Wnt/ß-catenin pathway.
ABSTRACT
OBJECTIVE: An attractive target to interfere with epileptic brain hyperexcitability is the enhancement of γ-aminobutyric acidergic (GABAergic) inhibition by inactivation of the GABA-metabolizing enzyme GABA aminotransferase (GABA-AT). GABA-AT inactivators were designed to control seizures by raising brain GABA levels. OV329, a novel drug candidate for the treatment of epilepsy and addiction, has been shown in vitro to be substantially more potent as a GABA-AT inactivator than vigabatrin, an antiseizure drug approved as an add-on therapy for adult patients with refractory complex partial seizures and monotherapy for pediatric patients with infantile spasms. Thus, we hypothesized that OV329 should produce pronounced anticonvulsant effects in two different rat seizure models. METHODS: We therefore examined the effects of OV329 (5, 20, and 40 mg/kg ip) on the seizure threshold of female Wistar Unilever rats, using the timed intravenous pentylenetetrazole (ivPTZ) seizure threshold model as a seizure test particularly sensitive to GABA-potentiating manipulations, and amygdala-kindled rats as a model of difficult-to-treat temporal lobe epilepsy. RESULTS: GABA-AT inactivation by OV329 clearly increased the threshold of both ivPTZ-induced and amygdala-kindled seizures. OV329 further showed a 30-fold greater anticonvulsant potency on ivPTZ-induced myoclonic jerks and clonic seizures compared to vigabatrin investigated previously. Notably, all rats were responsive to OV329 in both seizure models. SIGNIFICANCE: These results reveal an anticonvulsant profile of OV329 that appears to be superior in both potency and efficacy to vigabatrin and highlight OV329 as a highly promising candidate for the treatment of seizures and pharmacoresistant epilepsies.
Subject(s)
Epilepsy , Kindling, Neurologic , Amygdala , Animals , Anticonvulsants/adverse effects , Epilepsy/drug therapy , Female , Humans , Kindling, Neurologic/physiology , Pentylenetetrazole/adverse effects , Rats , Rats, Wistar , Seizures/chemically induced , Seizures/drug therapy , Transaminases/adverse effects , Vigabatrin/adverse effects , gamma-Aminobutyric Acid/pharmacologyABSTRACT
BACKGROUND: Calcium dysregulation has been proposed to play a causative role in the development of Alzheimer's disease pathology. Pregabalin is a compound already approved for human use, marketed as the prescription drug Lyrica. It binds the α2-δ subunit of P/Q-type voltagegated calcium channels, lowering calcium influx and providing effective treatment for epilepsy and neuropathic pain. OBJECTIVE: We hypothesize that increased resting calcium in neuronal processes near amyloid plaques plays a role in the development of neuritic dystrophies and further progression of amyloid pathology. METHODS: 5XFAD mice were treated orally for 12 weeks with pregabalin, then immunoblotting and immunofluorescent imaging were used to quantify neuritic dystrophy and amyloid deposition in pregabalin compared to placebo-treated mice. RESULTS: The treatment did not decrease markers of neuritic dystrophy or amyloid deposition. The image analysis of neuritic dystrophy on a plaque-by-plaque basis showed a small non-significant increase in the relative proportion of LAMP1 to Aß42 in plaques with areas of 50-450 µm2 in the cortex of pregabalin-treated mice. In addition, there was a statistically significant positive correlation between the measured cerebral concentration of pregabalin and the relative levels of BACE1 and Aß in the cortex. This relationship was not observed in the hippocampus, and there was no increase in average Aß levels in pregabalin treated mice compared to placebo. We confirmed previous findings that smaller amyloid plaques are associated with a greater degree of neuritic dystrophy. CONCLUSION: Pregabalin may have an effect on Aß that merits further investigation, but our study does not suggest that pregabalin contributes substantially to amyloid pathology.