Mechanisms of NMDA Receptor Inhibition by Sepimostat-Comparison with Nafamostat and Diarylamidine Compounds.

N-methyl-D-aspartate (NMDA) receptors are inhibited by many amidine and guanidine compounds. In this work, we studied the mechanisms of their inhibition by sepimostat-an amidine-containing serine protease inhibitor with neuroprotective properties. Sepimostat inhibited native NMDA receptors in rat hippocampal CA1 pyramidal neurons with IC50 of 3.5 ± 0.3 µM at -80 mV holding voltage. It demonstrated complex voltage dependence with voltage-independent and voltage-dependent components, suggesting the presence of shallow and deep binding sites. At -80 mV holding voltage, the voltage-dependent component dominates, and we observed pronounced tail currents and overshoots evidencing a "foot-in-the-door" open channel block. At depolarized voltages, the voltage-independent inhibition by sepimostat was significantly attenuated by the increase of agonist concentration. However, the voltage-independent inhibition was non-competitive. We further compared the mechanisms of the action of sepimostat with those of structurally-related amidine and guanidine compounds-nafamostat, gabexate, furamidine, pentamidine, diminazene, and DAPI-investigated previously. The action of all these compounds can be described by the two-component mechanism. All compounds demonstrated similar affinity to the shallow site, which is responsible for the voltage-independent inhibition, with binding constants in the range of 3-30 µM. In contrast, affinities to the deep site differed dramatically, with nafamostat, furamidine, and pentamidine being much more active.

Febrile Seizures Cause a Rapid Depletion of Calcium-Permeable AMPA Receptors at the Synapses of Principal Neurons in the Entorhinal Cortex and Hippocampus of the Rat.

Febrile seizures (FSs) are a relatively common early-life condition that can cause CNS developmental disorders, but the specific mechanisms of action of FS are poorly understood. In this work, we used hyperthermia-induced FS in 10-day-old rats. We demonstrated that the efficiency of glutamatergic synaptic transmission decreased rapidly after FS by recording local field potentials. This effect was transient, and after two days there were no differences between control and post-FS groups. During early ontogeny, the proportion of calcium-permeable (CP)-AMPA receptors in the synapses of the principal cortical and hippocampal neurons is high. Therefore, rapid internalization of CP-AMPA receptors may be one of the mechanisms underlying this phenomenon. Using the whole-cell patch-clamp method and the selective CP-AMPA receptor blocker IEM-1460, we tested whether the proportion of CP-AMPA receptors changed. We have demonstrated that FS rapidly reduces synaptic CP-AMPA receptors in both the hippocampus and the entorhinal cortex. This process was accompanied by a sharp decrease in the calcium permeability of the membrane of principal neurons, which we revealed in experiments with kainate-induced cobalt uptake. Our experiments show that FSs cause rapid changes in the function of the glutamatergic system, which may have compensatory effects that prevent excessive excitotoxicity and neuronal death.

Mechanisms of acid-sensing ion channels inhibition by nafamostat, sepimostat and diminazene.

Acid-sensing ion channels (ASICs) are blocked by many cationic compounds. Mechanisms of action, which may include pore block, modulation of activation and desensitization, need systematic analysis to allow predictable design of new potent and selective drugs. In this work, we studied the action of the serine protease inhibitors nafamostat, sepimostat, gabexate and camostat, on native ASICs in rat giant striatal interneurons and recombinant ASIC1a and ASIC2a channels, and compared it to that of well-known small molecule ASIC blocker diminazene. All these compounds have positively charged amidine and/or guanidine groups in their structure. Nafamostat, sepimostat and diminazene inhibited pH 6.5-induced currents in rat striatal interneurons at -80 mV holding voltage with IC50 values of 0.78 ± 0.12 µM, 2.4 ± 0.3 µM and 0.40 ± 0.09 µM, respectively, whereas camostat and gabexate were practically ineffective. The inhibition by nafamostat, sepimostat and diminazene was voltage-dependent evidencing binding in the channel pore. They were not trapped in the closed channels, suggesting "foot-in-the-door" mechanism of action. The inhibitory activity of nafamostat, sepimostat and diminazene was similar in experiments on native ASICs and recombinant ASIC1a channels, while all of them were drastically less active against ASIC2a channels. According to our molecular modeling, three active compounds bind in the channel pore between Glu 433 and Ala 444 in a similar way. In view of the relative safety of nafamostat for clinical use in humans, it can be considered as a potential candidate for the treatment of pathophysiological conditions linked to ASICs disfunction, including inflammatory pain and ischemic stroke.

Biphenyl scaffold for the design of NMDA-receptor negative modulators: molecular modeling, synthesis, and biological activity.

NMDA (N-methyl-d-aspartate) receptor antagonists are promising tools for the treatment of a wide variety of central nervous system impairments including major depressive disorder. We present here the activity optimization process of a biphenyl-based NMDA negative allosteric modulator (NAM) guided by free energy calculations, which led to a 100 times activity improvement (IC50 = 50 nM) compared to a hit compound identified in virtual screening. Preliminary calculation results suggest a low affinity for the human ether-a-go-go-related gene ion channel (hERG), a high affinity for which was earlier one of the main obstacles for the development of first-generation NMDA-receptor negative allosteric modulators. The docking study and the molecular dynamics calculations suggest a completely different binding mode (ifenprodil-like) compared to another biaryl-based NMDA NAM EVT-101.

Mechanisms of NMDA receptor inhibition by nafamostat, gabexate and furamidine.

N-methyl-D-aspartate (NMDA) receptors are affected by many pharmaceuticals. In this work, we studied the action of the serine protease inhibitors nafamostat, gabexate and camostat, and an antiprotozoal compound, furamidine, on native NMDA receptors in rat hippocampal pyramidal neurons. Nafamostat, furamidine and gabexate inhibited these receptors with IC50 values of 0.20 ± 0.04, 0.64 ± 0.13 and 16 ± 3 µM, respectively, whereas camostat was ineffective. Nafamostat and furamidine showed voltage-dependent inhibition, while gabexate showed practically voltage-independent inhibition. Nafamostat and furamidine demonstrated tail currents, implying a 'foot-in-the-door' mechanism of action; gabexate did not demonstrate any signs of 'foot-in-the-door' or trapping channel block. Gabexate action was also not competitive, suggesting allosteric inhibition of NMDA receptors. Furamidine and nafamostat are structurally similar to the previously studied diminazene and all three demonstrated a 'foot-in-the-door' mechanism. They have a rather rigid, elongated structures and cannot fold into more compact forms. By contrast, the gabexate molecule can fold, but its folded structure differs drastically from that of typical NMDA receptor blockers, in agreement with its voltage-independent inhibition. These findings provide a better understanding of the structural determinants of NMDA receptor antagonism, while also supporting the potential clinical repurposing of these drugs as neuroprotectors for glaucoma and other neurodegenerative diseases.

Mechanisms of NMDA receptor inhibition by diarylamidine compounds.

Pentamidine, diminazene and 4',6-diamidino-2-phenylindole (DAPI) are antiprotozoal diarylamidine compounds. In the present work, we have studied their action on native N-methyl-D-aspartate (NMDA) receptors in rat hippocampal pyramidal neurons. All three compounds inhibited NMDA receptors at -80 mV holding voltage with IC50 of 0.41 ± 0.08, 13 ± 3 and 3.1 ± 0.6 µM, respectively. The inhibition by pentamidine was strongly voltage-dependent, while that of DAPI was practically voltage-independent. Inhibition by diminazene had both voltage-dependent and voltage-independent components. Diminazene and DAPI demonstrated tail currents and overshoots suggesting "foot-in-the-door" mechanism of action. In contrast, pentamidine was partially trapped in the closed NMDA receptor channels. Such difference in the mechanism of action can be explained by the difference in the 3D structure of compounds. In the pentamidine molecule, two benzamidine groups are connected with a flexible linker, which allows the molecule to fold up and fit in the cavity of a closed NMDA receptor channel. Diminazene and DAPI, in contrast, have an extended form and could not be trapped.

Psychotropic Drugs for the Management of Chronic Pain and Itch.

Clinical observations have shown that patients with chronic neuropathic pain or itch exhibit symptoms of increased anxiety, depression and cognitive impairment. Such patients need corrective therapy with antidepressants, antipsychotics or anticonvulsants. It is known that some psychotropic drugs are also effective for the treatment of neuropathic pain and pruritus syndromes due to interaction with the secondary molecular targets. Our own clinical studies have identified antipruritic and/or analgesic efficacy of the following compounds: tianeptine (atypical tricyclic antidepressant), citalopram (selective serotonin reuptake inhibitor), mianserin (tetracyclic antidepressant), carbamazepine (anticonvulsant), trazodone (serotonin antagonist and reuptake inhibitor), and chlorprothixene (antipsychotic). Venlafaxine (serotonin-norepinephrine reuptake inhibitor) is known to have an analgesic effect too. The mechanism of such effect of these drugs is not fully understood. Herein we review and correlate the literature data on analgesic/antipruritic activity with pharmacological profile of these compounds.

Multiple modes of action of hydrophobic amines and their guanidine analogues on ASIC1a.

Hydrophobic monoamines containing only a hydrophobic/aromatic moiety and protonated amino group are a recently described class of acid-sensing ion channel (ASIC) modulators. Intensive studies have revealed a number of active compounds including endogenous amines and pharmacological agents and shown that these compounds potentiate and inhibit ASICs depending on their specific structure and on subunit composition of the target channel. The action of monoamines also depends on the application protocol, membrane voltage, conditioning and activating pH, suggesting complex mechanism(s) of the ligand-receptor interaction. Without understanding of these mechanisms analysis of structure-function relationships and predictive search for new potent and selective drugs are hardly possible. To this end, we investigated the modes of action for a representative series of amine and guanidine derivatives of adamantane and phenylcyclohexyl. The study was performed on transfected Chinese hamster ovary (CHO) cells and rat hippocampal interneurons using whole-cell patch clamp recording. We found that complex picture of monoamine action can be rationalized assuming four modes of action: (1) voltage-dependent pore block, (2) acidic shift of activation, (3) alkaline shift of activation and (4) acidic shift of steady-state desensitization. Structure-activity relationships are discussed in the light of this framework. The experiments on native heteromeric ASICs have shown that some of these mechanisms are shared between them and recombinant ASIC1a, implying that our results could also be relevant for amine action in physiological and pathological conditions.

Complex action of tyramine, tryptamine and histamine on native and recombinant ASICs.

Proton-gated channels of the ASIC family are widely distributed in the mammalian brain, and, according to the recent data, participate in synaptic transmission. However, ASIC-mediated currents are small, and special efforts are required to detect them. This prompts the search for endogenous ASIC ligands, which can activate or potentiate these channels. A recent finding of the potentiating action of histamine on recombinant homomeric ASIC1a has directed attention to amine-containing compounds. In the present study, we have analyzed the action of histamine, tyramine, and tryptamine on native and recombinant ASICs. None of the compounds caused potentiation of native ASICs in hippocampal interneurons. Furthermore, when applied simultaneously with channel activation, they produced voltage-dependent inhibition. Experiments on recombinant ASIC1a and ASIC2a allowed for an interpretation of these findings. Histamine and tyramine were found to be inactive on the ASIC2a, while tryptamine demonstrated weak inhibition. However, they induce both voltage-dependent inhibition of open channels and voltage-independent potentiation of closed/desensitized channels on the ASIC1a. We suggest that the presence of an ASIC2a subunit in heteromeric native ASICs prevents potentiation but not inhibition. As a result, the inhibitory action of histamine, which is masked by a strong potentiating effect on the ASIC1a homomers, becomes pronounced in experiments with native ASICs.

Inhibition of the NMDA and AMPA receptor channels by antidepressants and antipsychotics.

It is known that some antidepressants and antipsychotics directly inhibit NMDA-type ionotropic glutamate receptors. In this study we systematically studied action of seven drugs (Fluoxetine, Citalopram, Desipramine, Amitriptyline, Atomoxetine, Chlorpromazine, and Clozapine) on NMDA receptors and Ca2+-permeable and -impermeable AMPA receptors in rat brain neurons by whole-cell patch-clamp technique. Except for weak effect of fluoxetine, all drugs were virtually inactive against Ca2+-impermeable AMPA receptors. Fluoxetine and desipramine significantly inhibited Ca2+-permeable AMPA receptors (IC50=43±7 and 105±12µM, respectively). Desipramine, atomoxetine and chlorpromazine inhibited NMDA receptors in clinically relevant low micromolar concentrations, while citalopram had only weak effect. All tested medicines have been clustered into two groups by their action on NMDA receptors: desipramine, amitriptyline, chlorpromazine, and atomoxetine display voltage- and magnesium-dependent open channel blocking mechanism. Action of fluoxetine and clozapine was found to be voltage- and magnesium-independent. All voltage-dependent compounds could be trapped in closed NMDA receptor channels. Possible contribution of NMDA receptor inhibition by certain antidepressants and antipsychotics to their analgesic effects in neuropathic pain is discussed.

Inhibition of calcium-permeable and calcium-impermeable AMPA receptors by perampanel in rat brain neurons.

Perampanel is an antiepileptic drug that is used to treat partial-onset seizures and generalized tonic-clonic seizures. It is a highly selective AMPA receptor allosteric antagonist. However, published data on perampanel activity vary in different studies. In the present work we studied the inhibition of native calcium-permeable and calcium-impermeable AMPA receptors in rat brain neurons by perampanel using whole-cell patch clamp technique. We found that inhibitory activity and kinetics of perampanel action do not differ between calcium-permeable AMPA receptors of rat giant striatum interneurons and calcium-impermeable receptors of hippocampal CA1 pyramidal neurons (the IC50 value about 60nM). Also, perampanel caused the same inhibition of steady-state currents induced by kainate and glutamate. From the other side perampanel-induced inhibition was markedly reduced in the presence of cyclothiazide (IC50 value increased to 1.2±0.2µM). We demonstrated that perampanel competes with GYKI-52466 for binding site.

Non-classical mechanism of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor channel block by fluoxetine.

Antidepressants have many targets in the central nervous system. A growing body of data demonstrates the influence of antidepressants on glutamatergic neurotransmission. In the present work, we studied the inhibition of native Ca(2+)-permeable and Ca(2+)-impermeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in rat brain neurons by fluoxetine. The Ca(2+)-impermeable AMPA receptors in CA1 hippocampal pyramidal neurons were weakly affected. The IC50 value for the inhibition of Ca(2+)-permeable AMPA receptors in giant striatal interneurons was 43 ± 7 µM. The inhibition of Ca(2+)-permeable AMPA receptors was voltage dependent, suggesting deep binding in the pore. However, the use dependence of fluoxetine action differed markedly from that of classical AMPA receptor open-channel blockers. Moreover, fluoxetine did not compete with other channel blockers. In contrast to fluoxetine, its membrane-impermeant quaternary analog demonstrated all of the features of channel inhibition typical for open-channel blockers. It is suggested that fluoxetine reaches the binding site through a hydrophobic access pathway. Such a mechanism of block is described for ligands of sodium and calcium channels, but was never found in AMPA receptors. Molecular modeling suggests binding of fluoxetine in the subunit interface; analogous binding was proposed for local anesthetics in closed sodium channels and for benzothiazepines in calcium channels.

Neuroprotective activity of creatylglycine ethyl ester fumarate.

BACKGROUND: We have recently shown neuroprotective activity of the creatine amides in the focal cerebral ischemia in rats on the 280 mg/kg administration. In the present study, neuroprotective properties of creatylglycine ethyl ester fumarate (CrGEt) in rats with focal cerebral ischemia were explored in a wide dosage range (30-280 mg/kg, intravenous and intragastric). METHODS: Focal cerebral ischemia was induced by the middle cerebral artery occlusion (MCAO). RESULTS: The CrGEt administration 30 minutes before and at the last 5 minutes of MCAO dose dependently attenuated cerebral ischemic damage on 35%-65%, reduced neurobehavioral deficits, led to high neuronal survival in ischemic rat brains. The neuroprotective activity of CrGEt was mediated by its following abilities: (1) normalize the energy metabolism in the ischemic brains, maintaining adenosine triphosphate levels, and reducing lactate concentration; (2) inhibit the ischemia-reperfusion-related oxidative stress as evidenced by the increased activity of superoxide dismutase and the reduced levels of malondialdehyde. CrGEt served as a substrate for creatine kinase and a partial agonist of N-methyl-D-aspartate receptors; this partly explains mechanism of its neuroprotective action. CONCLUSIONS: In view of the previously mentioned results, CrGEt holds a promise as a compound for treatment of ischemic brain disorders.

Monoamine NMDA receptor channel blockers inhibit and potentiate native and recombinant proton-gated ion channels.

Acid-sensing ion channels (ASICs) are widely distributed in the peripheral and central nervous system. Although they are involved in many physiological functions, the actual processes that activate ASICs remain unclear. This is particularly true for brain ASICs, which produce only a transient response to a fast drop in pH and cannot mediate sustained current. Therefore, the search for ASIC inhibitors and, especially, potentiators/activators is important. We report that NMDA receptor channel blockers with a comparatively simple structure (9-aminoacridine, memantine, IEM-2117 and IEM-1921) potentiate and/or inhibit ASICs in submillimolar concentrations. The experiments were performed using the patch clamp technique on native ASICs from rat hippocampal interneurons and recombinant ASICs of different subunit compositions expressed in CHO cells. Native ASICs were potentiated by IEM-1921 and IEM-2117, and inhibited by memantine and 9-aminoacridine. Homomeric ASIC1a were inhibited by memantine, IEM-2117 and 9-aminoacridine while IEM-1921 was ineffective. In contrast, homomeric ASIC2a were potentiated by IEM-2117, memantine and IEM-1921, whereas 9-aminoacridine was inactive. The compounds caused a complex effect on ASIC3. 9-aminoacridine and IEM-1921 potentiated the steady-state response of ASIC3 and inhibited the peak component. IEM-2117 not only potentiated ASIC3-mediated currents caused by acidification but also evoked steady-state currents at neutral pH. Our results demonstrate that, depending on the subunit composition, ASICs can be activated or inhibited by simple compounds that possess only amino group and aromatic/hydrophobic moieties. This opens up the possibility to search for new ASIC modulators among a number of endogenous ligands.

Argiotoxin in the closed AMPA receptor channel: experimental and modeling study.

Binding of argiotoxin in the closed state of Ca(2+)-permeable AMPA receptor channels was studied using electrophysiological and molecular modeling approaches. Experimental study unambiguously revealed that argiotoxin is trapped in the closed AMPA receptor channels after agonist dissociation. Docking of the argiotoxin to the channel model based on recently published X-ray structure demonstrated that the drug can be effectively accommodated in the cavity of the closed channel only if the terminal moiety of the molecule penetrates in the narrow portion of the pore below the selectivity filter. Combining these results, we conclude that the selectivity filter of the AMPA receptor channels is not sterically occluded in the closed state.

Voltage-dependent and -independent block of α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor channels.

Polyamine-containing toxins and synthetic dicationic derivatives of adamantane and phenylcyclohexyl selectively antagonize Ca(2+)-permeable α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor channels. These compounds demonstrate voltage-dependent open-channel block and are trapped by closed channels. In this study, we describe an alternative mechanism of non-competitive AMPA receptor inhibition caused by 9-aminoacridine and some of its derivatives. These compounds exhibit similar potency against Ca(2+)-permeable and Ca(2+)-impermeable AMPA receptors. The inhibition is largely voltage-independent, binding and unbinding do not require presence of agonist. We conclude that 9-aminoacridine binds to a shallow site in the AMPA receptor, which is located above the activation gate. A comparison of three-dimensional structures of the antagonists suggests that the 'V-like' shape of the hydrophobic headgroup favors voltage-dependent binding to the deep site in the channel pore, whereas the compounds possessing flat aromatic headgroups preferably bind to the shallow site. The characterization of the novel mechanism of AMPA receptor channel antagonism opens a way to develop a new family of pharmacological agents, which can be of scientific and practical importance.

Blockade of NMDA receptor channels by 9-aminoacridine and its derivatives.

9-Aminoacridine is known as "foot-in-the-door" NMDA receptor channel blocker because its binding prevents channel closure. Structural determinants of this mechanism of block were studied using a series of 9-aminoacridine derivatives. Experiments were performed on native NMDA receptors of hippocampal pyramidal neurons, isolated from rat brain slices. The use-dependence of block and kinetics of recovery from block were used to characterize mechanism of block produced by the compounds. Modifications, which preserve the flat structure of the tricyclic 9-aminoacridine moiety, affect blocking activity and kinetics but not the foot-in-the-door mechanism. On the contrary, disruption of the flat structure changes the mechanism of block to trapping. It is concluded that flat aromatic structure is one of the critical determinants of the action mechanism of 9-aminoacridine.

Mechanisms of non-steroid anti-inflammatory drugs action on ASICs expressed in hippocampal interneurons.

The inhibitory action of non-steroid anti-inflammatory drugs was investigated on acid-sensing ionic channels (ASIC) in isolated hippocampal interneurons and on recombinant ASICs expressed in Chinese hamster ovary (CHO) cells. Diclofenac and ibuprofen inhibited proton-induced currents in hippocampal interneurons (IC(50) were 622 +/- 34 muM and 3.42 +/- 0.50 mM, respectively). This non-competitive effect was fast and fully reversible for both drugs. Aspirin and salicylic acid at 500 muM were ineffective. Diclofenac and ibuprofen decreased the amplitude of proton-evoked currents and slowed the rates of current decay with a good correlation between these effects. Simultaneous application of acid solution and diclofenac was required for its inhibitory effect. Unlike amiloride, the action of diclofenac was voltage-independent and no competition between two drugs was found. Analysis of the action of diclofenac and ibuprofen on activation and desensitization of ASICs showed that diclofenac but not ibuprofen shifted the steady-state desensitization curve to more alkaline pH values. The reason for this shift was slowing down the recovery from desensitization of ASICs. Thus, diclofenac may serve as a neuroprotective agent during pathological conditions associated with acidification.