Discovery of a small molecule modulator of the Kv1.1/Kvβ1 channel complex that reduces neuronal excitability and in vitro epileptiform activity

Nonsynaptic mechanisms exert a powerful influence on seizure threshold. It is well-established that nonsynaptic epileptiform activity can be induced in hippocampal slices by reducing extracellular Ca2+ concentration. We show here that nonsynaptic epileptiform activity can be readily induced in vitro in normal (2 mM) Ca2+ levels. Those conditions sufficient for nonsynaptic epileptogenesis in the CA1 region were determined by pharmacologically mimicking the effects of Ca2+ reduction in normal Ca2+ levels. Increasing neuronal excitability, by removing extracellular Mg2+ and increasing extracellular K+ (6–15 mM), induced epileptiform activity that was suppressed by postsynaptic receptor antagonists [d-(−)-2-amino-5-phosphonopentanoic acid, picrotoxin, and 6,7-dinitroquinoxaline-2,3-dione] and was therefore synaptic in nature. Similarly, epileptiform activity induced when neuronal excitability was increased in the presence of KCaantagonists (verruculogen, charybdotoxin, norepinephrine, tetraethylammonium salt, and Ba2+) was found to be synaptic in nature. Decreases in osmolarity also failed to induce nonsynaptic epileptiform activity in the CA1 region. However, increasing neuronal excitability (by removing extracellular Mg2+ and increasing extracellular K+) in the presence of Cd2+, a nonselective Ca2+channel antagonist, or veratridine, a persistent sodium conductance enhancer, induced spontaneous nonsynaptic epileptiform activity in vitro. Both novel models were characterized using intracellular and ion-selective electrodes. The results of this study suggest that reducing extracellular Ca2+ facilitates bursting by increasing neuronal excitability and inhibiting Ca2+ influx, which might, in turn, enhance a persistent sodium conductance. Furthermore, these data show that nonsynaptic mechanisms can contribute to epileptiform activity in normal Ca2+ levels.

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Diadenosine-Polyphosphate Analogue AppCH2ppA Suppresses Seizures by Enhancing Adenosine Signaling in the Cortex

Cerebral Cortex ◽

10.1093/cercor/bhy257 ◽

2018 ◽

Vol 29 (9) ◽

pp. 3778-3795

Author(s):

Alexandre Pons-Bennaceur ◽

Vera Tsintsadze ◽

Thi-thien Bui ◽

Timur Tsintsadze ◽

Marat Minlebaev ◽

...

Keyword(s):

Human Population ◽

Temporal Summation ◽

Neuronal Excitability ◽

Epileptiform Activity ◽

Treatment Strategies ◽

Anticonvulsant Treatment ◽

A1 Receptor ◽

Adenosine Signaling

Abstract Epilepsy is a multifactorial disorder associated with neuronal hyperexcitability that affects more than 1% of the human population. It has long been known that adenosine can reduce seizure generation in animal models of epilepsies. However, in addition to various side effects, the instability of adenosine has precluded its use as an anticonvulsant treatment. Here we report that a stable analogue of diadenosine-tetraphosphate: AppCH2ppA effectively suppresses spontaneous epileptiform activity in vitro and in vivo in a Tuberous Sclerosis Complex (TSC) mouse model (Tsc1+/−), and in postsurgery cortical samples from TSC human patients. These effects are mediated by enhanced adenosine signaling in the cortex post local neuronal adenosine release. The released adenosine induces A1 receptor-dependent activation of potassium channels thereby reducing neuronal excitability, temporal summation, and hypersynchronicity. AppCH2ppA does not cause any disturbances of the main vital autonomous functions of Tsc1+/− mice in vivo. Therefore, we propose this compound to be a potent new candidate for adenosine-related treatment strategies to suppress intractable epilepsies.

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Calcium-Activated Afterhyperpolarizations Regulate Synchronization and Timing of Epileptiform Bursts in Hippocampal CA3 Pyramidal Neurons

Journal of Neurophysiology ◽

10.1152/jn.00434.2006 ◽

2006 ◽

Vol 96 (6) ◽

pp. 3028-3041 ◽

Cited By ~ 32

Author(s):

David Fernández de Sevilla ◽

Julieta Garduño ◽

Emilio Galván ◽

Washington Buño

Keyword(s):

Neuronal Excitability ◽

Pyramidal Neurons ◽

Epileptiform Activity ◽

Network Activity ◽

Cellular Mechanisms ◽

Burst Frequency ◽

Potassium Conductances ◽

Hippocampal Ca3 ◽

Ca3 Pyramidal Neurons

Calcium-activated potassium conductances regulate neuronal excitability, but their role in epileptogenesis remains elusive. We investigated in rat CA3 pyramidal neurons the contribution of the Ca2+-activated K+-mediated afterhyperpolarizations (AHPs) in the genesis and regulation of epileptiform activity induced in vitro by 4-aminopyridine (4-AP) in Mg2+-free Ringer. Recurring spike bursts terminated by prolonged AHPs were generated. Burst synchronization between CA3 pyramidal neurons in paired recordings typified this interictal-like activity. A downregulation of the medium afterhyperpolarization (mAHP) paralleled the emergence of the interictal-like activity. When the mAHP was reduced or enhanced by apamin and EBIO bursts induced by 4-AP were increased or blocked, respectively. Inhibition of the slow afterhyperpolarization (sAHP) with carbachol, t-ACPD, or isoproterenol increased bursting frequency and disrupted burst regularity and synchronization between pyramidal neuron pairs. In contrast, enhancing the sAHP by intracellular dialysis with KMeSO4 reduced burst frequency. Block of GABAA–B inhibitions did not modify the abnormal activity. We describe novel cellular mechanisms where 1) the inhibition of the mAHP plays an essential role in the genesis and regulation of the bursting activity by reducing negative feedback, 2) the sAHP sets the interburst interval by decreasing excitability, and 3) bursting was synchronized by excitatory synaptic interactions that increased in advance and during bursts and decreased throughout the subsequent sAHP. These cellular mechanisms are active in the CA3 region, where epileptiform activity is initiated, and cooperatively regulate the timing of the synchronized rhythmic interictal-like network activity.

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CA-170 – A Potent Small-Molecule PD-L1 Inhibitor or Not?

Molecules ◽

10.3390/molecules24152804 ◽

2019 ◽

Vol 24 (15) ◽

pp. 2804 ◽

Cited By ~ 27

Author(s):

Bogdan Musielak ◽

Justyna Kocik ◽

Lukasz Skalniak ◽

Katarzyna Magiera-Mularz ◽

Dominik Sala ◽

...

Keyword(s):

Small Molecule ◽

Binding Assay ◽

Production Costs ◽

In Vitro Methods ◽

Therapeutic Results ◽

Direct Binding ◽

High Production ◽

Small Molecule Modulator ◽

Positive Controls

CA-170 is currently the only small-molecule modulator in clinical trials targeting PD-L1 and VISTA proteins – important negative checkpoint regulators of immune activation. The reported therapeutic results to some extent mimic those of FDA-approved monoclonal antibodies overcoming the limitations of the high production costs and adverse effects of the latter. However, no conclusive biophysical evidence proving the binding to hPD-L1 has ever been presented. Using well-known in vitro methods: NMR binding assay, HTRF and cell-based activation assays, we clearly show that there is no direct binding between CA-170 and PD-L1. To strengthen our reasoning, we performed control experiments on AUNP-12 – a 29-mer peptide, which is a precursor of CA-170. Positive controls consisted of the well-documented small-molecule PD-L1 inhibitors: BMS-1166 and peptide-57.

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Antiepileptic effects of quinine in the pentylenetetrazole model of seizure

European Psychiatry ◽

10.1016/s0924-9338(11)72991-1 ◽

2011 ◽

Vol 26 (S2) ◽

pp. 1286-1286

Author(s):

B. Torabinejad ◽

M. Nassiri-Asl ◽

F. Zamansoltani

Keyword(s):

Neuronal Excitability ◽

Epileptiform Activity ◽

Tween 80 ◽

Clonic Seizure ◽

Positive Control ◽

The Body ◽

Control Group ◽

Gap Junction Channels ◽

Connexin 36

IntroductionQuinine, is an anti-malarial drug that specifically blocks connexin 36 at gap junction channels.ObjectiveQuinine has suppressed ictal epileptiform activity in vitro without decreasing neuronal excitability.AimWe considered the possible anticonvulsant effects of quinine in the pentylenetetrazole (PTZ) model of seizure.MethodsIn five groups, the mice were given quinine at the doses of 20, 30, 40, 50, or 60 mg/kg 30 min before the administration of PTZ (90 mg/kg). Two groups were injected with diazepam, the positive control (0.5, 1 mg/kg) and one group, the control group, was injected with saline + Tween 80 before the administration of PTZ. The onset of a general clonus was used as the endpoint. The general clonus was characterized by forelimb clonus followed by full clonus of the body.ResultsIn the PTZ model, quinine at the dose of 60 mg/kg increased the latency of seizure. However, quinine at 40-60 mg/kg decreased the duration of seizure, dose dependently.ConclusionThe present study provides evidence for anticonvulsant activity of quinine in the generalized clonic seizure of PTZ model. As a result of these finding, we suggest that gap junctions represent an appropriate target for the development of drugs aimed at decreasing epileptiform synchronization and preventing epileptogenesis.

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CA-170 – a potent small-molecule PD-L1 inhibitor or not?

10.1101/662668 ◽

2019 ◽

Cited By ~ 1

Author(s):

Bogdan Musielak ◽

Justyna Kocik ◽

Lukasz Skalniak ◽

Katarzyna Magiera-Mularz ◽

Dominik Sala ◽

...

Keyword(s):

Clinical Trials ◽

Small Molecule ◽

Binding Assay ◽

Production Costs ◽

Therapeutic Results ◽

Direct Binding ◽

High Production ◽

Small Molecule Modulator ◽

Positive Controls

AbstractCA-170is currently the only small-molecule modulator in clinical trials targeting PD-L1 and VISTA proteins – important negative checkpoint regulators of immune activation. The reported therapeutic results to some extent mimic those of FDA-approved monoclonal antibodies overcoming the limitations of the high production costs and adverse effects of the latter. However, no conclusive biophysical evidence proving the binding to hPD-L1 has ever been presented. Using well-knownin vitromethods: NMR binding assay, HTRF and cell-based activation assays, we clearly show that there is no direct binding betweenCA-170and PD-L1. To strengthen our reasoning, we performed control experiments onAUNP-12– a 29-mer peptide, which is a precursor ofCA-170. Positive controls consisted of the well-documented small-molecule PD-L1 inhibitors:BMS-1166and peptide p57.

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