Novel neuroactive steroids as positive allosteric modulators of NMDA receptors: mechanism, site of action, and rescue pharmacology on GRIN variants associated with neurological conditions.

N-methyl-D-aspartate receptors (NMDARs) play vital roles in normal brain functions (i.e., learning, memory, and neuronal development) and various neuropathological conditions, such as epilepsy, autism, Parkinson's disease, Alzheimer's disease, and traumatic brain injury. Endogenous neuroactive steroids such as 24(S)-hydroxycholesterol (24(S)-HC) have been shown to influence NMDAR activity, and positive allosteric modulators (PAMs) derived from 24(S)-hydroxycholesterol scaffold can also enhance NMDAR function. This study describes the structural determinants and mechanism of action for 24(S)-hydroxycholesterol and two novel synthetic analogs (SGE-550 and SGE-301) on NMDAR function. We also show that these agents can mitigate the altered function caused by a set of loss-of-function missense variants in NMDAR GluN subunit-encoding GRIN genes associated with neurological and neuropsychiatric disorders. We anticipate that the evaluation of novel neuroactive steroid NMDAR PAMs may catalyze the development of new treatment strategies for GRIN-related neuropsychiatric conditions.

Metabotropic, but not allosteric, effects of neurosteroids on GABAergic inhibition depend on the phosphorylation of GABAA receptors.

Neuroactive steroids (NASs) are synthesized within the brain and exert profound effects on behavior. These effects are primarily believed to arise from the activities of NASs as positive allosteric modulators (PAMs) of the GABA-type A receptor (GABAAR). NASs also activate a family of G protein-coupled receptors known as membrane progesterone receptors (mPRs). Here, using surface-biotinylation assays and electrophysiology techniques, we examined mPRs' role in mediating the effects of NAS on the efficacy of GABAergic inhibition. Selective mPR activation enhanced phosphorylation of Ser-408 and Ser-409 (Ser-408/9) within the GABAAR ß3 subunit, which depended on the activity of cAMP-dependent protein kinase A (PKA) and protein kinase C (PKC). mPR activation did not directly modify GABAAR activity and had no acute effects on phasic or tonic inhibition. Instead, mPR activation induced a sustained elevation in tonic current, which was blocked by PKA and PKC inhibition. Substitution of Ser-408/9 to alanine residues also prevented the effects of mPR activation on tonic current. Furthermore, this substitution abolished the effects of sustained NAS exposure on tonic inhibition. Interestingly, the allosteric effects of NAS on GABAergic inhibition were independent of Ser-408/9 in the ß3 subunit. Additionally, although allosteric effects of NAS on GABAergic inhibition were sensitive to a recently developed "NAS antagonist," the sustained effects of NAS on tonic inhibition were not. We conclude that metabotropic effects of NAS on GABAergic inhibition are mediated by mPR-dependent modulation of GABAAR phosphorylation. We propose that this mechanism may contribute to the varying behavioral effects of NAS.

Pharmacological characterization of SAGE-718, a novel positive allosteric modulator of N-methyl-d-aspartate receptors.

BACKGROUND AND PURPOSE: Select neuroactive steroids tune neural activity by modulating excitatory and inhibitory neurotransmission, including the endogenous cholesterol metabolite 24(S)-hydroxycholesterol (24(S)-HC), which is an N-methyl-d-aspartate (NMDA) receptor positive allosteric modulator (PAM). NMDA receptor PAMs are potentially an effective pharmacotherapeutic strategy to treat conditions associated with NMDA receptor hypofunction. EXPERIMENTAL APPROACH: Using in vitro and in vivo electrophysiological recording experiments and behavioural approaches, we evaluated the effect of SAGE-718, a novel neuroactive steroid NMDA receptor PAM currently in clinical development for the treatment of cognitive impairment, on NMDA receptor function and endpoints that are altered by NMDA receptor hypoactivity and assessed its safety profile. KEY RESULTS: SAGE-718 potentiated GluN1/GluN2A-D NMDA receptors with equipotency and increased NMDA receptor excitatory postsynaptic potential (EPSP) amplitude without affecting decay kinetics in striatal medium spiny neurons. SAGE-718 increased the rate of unblock of the NMDA receptor open channel blocker ketamine on GluN1/GluN2A in vitro and accelerated the rate of return on the ketamine-evoked increase in gamma frequency band power, as measured with electroencephalogram (EEG), suggesting that PAM activity is driven by increased channel open probability. SAGE-718 ameliorated deficits due to NMDA receptor hypofunction, including social deficits induced by subchronic administration of phencyclidine, and behavioural and electrophysiological deficits from cholesterol and 24(S)-HC depletion caused by 7-dehydrocholesterol reductase inhibition. Finally, SAGE-718 did not produce epileptiform activity in a seizure model or neurodegeneration following chronic dosing. CONCLUSIONS AND IMPLICATIONS: These findings provide strong evidence that SAGE-718 is a neuroactive steroid NMDA receptor PAM with a mechanism that is well suited as a treatment for conditions associated with NMDA receptor hypofunction.

Preventing Phosphorylation of the GABA A R ß3 Subunit Compromises the Behavioral Effects of Neuroactive Steroids.

Neuroactive steroids (NASs) have potent anxiolytic, anticonvulsant, sedative, and hypnotic actions, that reflect in part their efficacy as GABA A R positive allosteric modulators (PAM). In addition to this, NAS exert metabotropic effects on GABAergic inhibition via the activation of membrane progesterone receptors (mPRs), which are G-protein coupled receptors. mPR activation enhances the phosphorylation of residues serine 408 and 409 (S408/9) in the ß3 subunit of GABA A Rs, increasing their accumulation in the plasma membrane leading to a sustained increase in tonic inhibition. To explore the significance of NAS-induced phosphorylation of GABA A Rs, we used mice in which S408/9 in the ß3 subunit have been mutated to alanines, mutations that prevent the metabotropic actions of NASs on GABA A R function while preserving NAS allosteric potentiation of GABAergic current. While the sedative actions of NAS were comparable to WT, their anxiolytic actions were reduced in S408/9A mice. Although the induction of hypnosis by NAS were maintained in the mutant mice the duration of the loss of righting reflex was significantly shortened. Finally, ability of NAS to terminate diazepam pharmacoresistant seizures was abolished in S408/9A mice. In conclusion, our results suggest that S408/9 in the GABA A R ß3 subunit contribute to the anxiolytic and anticonvulsant efficacy of NAS, in addition to their ability to regulate the loss of righting reflex.

SAGE-718: A First-in-Class N-Methyl-d-Aspartate Receptor Positive Allosteric Modulator for the Potential Treatment of Cognitive Impairment.

N-Methyl-d-aspartate receptor (NMDAR) positive allosteric modulators (PAMs) have received increased interest as a powerful mechanism of action to provide relief as therapies for CNS disorders. Sage Therapeutics has previously published the discovery of endogenous neuroactive steroid 24(S)-hydroxycholesterol as an NMDAR PAM. In this article, we detail the discovery of development candidate SAGE-718 (5), a potent and high intrinsic activity NMDAR PAM with an optimized pharmacokinetic profile for oral dosing. Compound 5 has completed phase 1 single ascending dose and multiple ascending dose clinical trials and is currently undergoing phase 2 clinical trials for treatment of cognitive impairment in Huntington's disease.

Preclinical characterization of zuranolone (SAGE-217), a selective neuroactive steroid GABAA receptor positive allosteric modulator.

Zuranolone (SAGE-217) is a novel, synthetic, clinical stage neuroactive steroid GABAA receptor positive allosteric modulator designed with the pharmacokinetic properties to support oral daily dosing. In vitro, zuranolone enhanced GABAA receptor current at nine unique human recombinant receptor subtypes, including representative receptors for both synaptic (γ subunit-containing) and extrasynaptic (δ subunit-containing) configurations. At a representative synaptic subunit configuration, α1ß2γ2, zuranolone potentiated GABA currents synergistically with the benzodiazepine diazepam, consistent with the non-competitive activity and distinct binding sites of the two classes of compounds at synaptic receptors. In a brain slice preparation, zuranolone produced a sustained increase in GABA currents consistent with metabotropic trafficking of GABAA receptors to the cell surface. In vivo, zuranolone exhibited potent activity, indicating its ability to modulate GABAA receptors in the central nervous system after oral dosing by protecting against chemo-convulsant seizures in a mouse model and enhancing electroencephalogram ß-frequency power in rats. Together, these data establish zuranolone as a potent and efficacious neuroactive steroid GABAA receptor positive allosteric modulator with drug-like properties and CNS exposure in preclinical models. Recent clinical data support the therapeutic promise of neuroactive steroid GABAA receptor positive modulators for treating mood disorders; brexanolone is the first therapeutic approved specifically for the treatment of postpartum depression. Zuranolone is currently under clinical investigation for the treatment of major depressive episodes in major depressive disorder, postpartum depression, and bipolar depression.

Neuroactive Steroids Reverse Tonic Inhibitory Deficits in Fragile X Syndrome Mouse Model.

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability. A reduction in neuronal inhibition mediated by γ-aminobutyric acid type A receptors (GABAARs) has been implicated in the pathophysiology of FXS. Neuroactive steroids (NASs) are known allosteric modulators of GABAAR channel function, but recent studies from our laboratory have revealed that NASs also exert persistent metabotropic effects on the efficacy of tonic inhibition by increasing the protein kinase C (PKC)-mediated phosphorylation of the α4 and ß3 subunits which increase the membrane expression and boosts tonic inhibition. We have assessed the GABAergic signaling in the hippocampus of fragile X mental retardation protein (FMRP) knock-out (Fmr1 KO) mouse. The GABAergic tonic current in dentate gyrus granule cells (DGGCs) from 3- to 5-week-old (p21-35) Fmr1 KO mice was significantly reduced compared to WT mice. Additionally, spontaneous inhibitory post synaptic inhibitory current (sIPSC) amplitudes were increased in DGGCs from Fmr1 KO mice. While sIPSCs decay in both genotypes was prolonged by the prototypic benzodiazepine diazepam, those in Frm1-KO mice were selectively potentiated by RO15-4513. Consistent with this altered pharmacology, modifications in the expression levels and phosphorylation of receptor GABAAR subtypes that mediate tonic inhibition were seen in Fmr1 KO mice. Significantly, exposure to NASs induced a sustained elevation in tonic current in Fmr1 KO mice which was prevented with PKC inhibition. Likewise, exposure reduced elevated membrane excitability seen in the mutant mice. Collectively, our results suggest that NAS act to reverse the deficits of tonic inhibition seen in FXS, and thereby reduce aberrant neuronal hyperexcitability seen in this disorder.

Neuroactive Steroid N-Methyl-d-aspartate Receptor Positive Allosteric Modulators: Synthesis, SAR, and Pharmacological Activity.

Neuroactive steroids (NASs) play a pivotal role in maintaining homeostasis is the CNS. We have discovered that one NAS in particular, 24(S)-hydroxycholesterol (24(S)-HC), is a positive allosteric modulator (PAM) of NMDA receptors. Using 24(S)-HC as a chemical starting point, we have identified other NASs that have good in vitro potency and efficacy. Herein, we describe the structure activity relationship and pharmacokinetic optimization of this series that ultimately led to SGE-301 (42). We demonstrate that SGE-301 enhances long-term potentiation (LTP) in rat hippocampal slices and, in a dose-dependent manner, improves cognition in a rat social recognition study.

Nucleoside transporters: from scavengers to novel therapeutic targets.

Hydrophilic purine and pyrimidine nucleosides rely on specialized carrier proteins for their membrane translocation. The recent identification of two gene families encoding equilibrative and concentrative nucleoside transporters in mammals and other organisms has provided the essential breakthrough to a more complete understanding of the biological significance of nucleoside transport. Although nucleoside salvage is a primary function of these proteins, recent data indicate functions beyond metabolic recycling. In brain and spinal cord, for example, nucleoside transporters have the potential to regulate synaptic levels of neuroactive purines such as adenosine and, thereby, indirectly modulate physiological processes through G-protein-coupled purine P1 receptors. As described in this review, recent research indicates novel putative functions for CNS nucleoside transporters in sleep, arousal, drug and alcohol addiction, nociception and analgesia. The therapeutic use of nucleoside analogue drugs and nucleoside transporter inhibitors in viral, neoplastic, cardiovascular and infectious disease is also described.

Endogenous and synthetic neuroactive steroids evoke sustained increases in the efficacy of GABAergic inhibition via a protein kinase C-dependent mechanism.

The neuroactive steroid (NAS) tetrahydrodeoxycorticosterone (THDOC) increases protein kinase C (PKC) mediated phosphorylation of extrasynaptic GABAA receptor (GABAAR) subunits leading to increased surface expression of α4/ß3 subunit-containing extrasynaptic GABAARs, leading to a sustained increase in GABAAR tonic current density. Whether other naturally occurring and synthetic NASs share both an allosteric and metabotropic action on GABAARs is unknown. Here, we examine the allosteric and metabotropic properties of allopregnanolone (ALLO), and synthetic NASs SGE-516 and ganaxolone. ALLO, SGE-516, and ganaxolone all allosterically enhanced prototypical synaptic and extrasynaptic recombinant GABAARs. In dentate gyrus granule cells (DGGCs) all three NASs, when applied acutely, allosterically enhanced tonic and phasic GABAergic currents. In separate experiments, slices were exposed to NASs for 15 min, and then transferred to a steroid naïve recording chamber followed by ≥ 30 min wash before tonic currents were measured. A sustained increase in tonic current was observed following exposure to ALLO, or SGE-516 and was prevented by inhibiting PKC with GF 109203X. No increase in tonic current was observed with exposure to ganaxolone. In agreement with the observations of an increased tonic current, the NASs ALLO and SGE-516 increased the phosphorylation and surface expression of the ß3 subunit-containing GABAARs. Our studies demonstrate that neuroactive steroids have differential abilities to induce sustained increases in the efficacy of tonic inhibition by promoting GABAAR phosphorylation and membrane trafficking dependent on PKC activity.

Anticonvulsant profile of the neuroactive steroid, SGE-516, in animal models.

Despite the availability of multiple antiepileptic drugs (AED), failure to adequately control seizures is a challenge for approximately one third of epilepsy patients, and new therapies with a differentiated mechanism of action are needed. The neuroactive steroid, SGE-516, is a positive allosteric modulator of both gamma- and delta-containing GABAA receptors. This broad GABAA receptor activity differentiates neuroactive steroids like SGE-516 from benzodiazepines, a class of anticonvulsants which have been shown in vitro to selectively target gamma-subunit containing GABAA receptors. As a neuroactive steroid, SGE-516 has pharmacokinetic properties that are intended to allow for chronic oral dosing. We investigated the anticonvulsant activity of SGE-516 across numerous in vitro and in vivo models of seizure activity. SGE-516 dose-dependently reduced neuronal firing rates and epileptiform activity in vitro. In mice, SGE-516 protected against acute seizures in the PTZ-induced chemo-convulsant seizure model and the 6Hz psychomotor seizure model. In addition, SGE-516 demonstrated anticonvulsant activity in the mouse corneal kindling model. These data suggest that SGE-516 may have potential for development as a novel oral AED for the treatment of refractory seizures.

Neuroactive Steroids. 2. 3α-Hydroxy-3ß-methyl-21-(4-cyano-1H-pyrazol-1'-yl)-19-nor-5ß-pregnan-20-one (SAGE-217): A Clinical Next Generation Neuroactive Steroid Positive Allosteric Modulator of the (γ-Aminobutyric Acid)A Receptor.

Certain classes of neuroactive steroids (NASs) are positive allosteric modulators (PAM) of synaptic and extrasynaptic GABAA receptors. Herein, we report new SAR insights in a series of 5ß-nor-19-pregnan-20-one analogues bearing substituted pyrazoles and triazoles at C-21, culminating in the discovery of 3α-hydroxy-3ß-methyl-21-(4-cyano-1H-pyrazol-1'-yl)-19-nor-5ß-pregnan-20-one (SAGE-217, 3), a potent GABAA receptor modulator at both synaptic and extrasynaptic receptor subtypes, with excellent oral DMPK properties. Compound 3 has completed a phase 1 single ascending dose (SAD) and multiple ascending dose (MAD) clinical trial and is currently being studied in parallel phase 2 clinical trials for the treatment of postpartum depression (PPD), major depressive disorder (MDD), and essential tremor (ET).

Adenosine contributes to mu-opioid synaptic inhibition in rat substantia gelatinosa in vitro.

The purpose of this study was to investigate the cellular basis of the synergistic anti-nociceptive interaction between adenosine and opioids reported for spinal cord in vivo. Patch clamp recordings from rat substantia gelatinosa neurons in vitro were used to assess whether adenosine receptor antagonists impact upon mu-opioid receptor (MOR)-mediated inhibition of glutamatergic synaptic transmission. The MOR agonist DAMGO inhibited evoked EPSCs and this inhibition was partly reversed by DPCPX, an A1 receptor (A1R) antagonist. The A2a receptor antagonist, ZM241385 had mixed effects on DAMGO-mediated inhibition, producing either a further inhibition or a reversal of the inhibition. These data show that activation of A1R as a secondary consequence of MOR-activation and putative adenosine release will potentiate opioid synaptic inhibition of nociceptive circuitry.

Neuroactive Steroids. 1. Positive Allosteric Modulators of the (γ-Aminobutyric Acid)A Receptor: Structure-Activity Relationships of Heterocyclic Substitution at C-21.

Neuroactive steroids (NASs) have been shown to impact central nervous system (CNS) function through positive allosteric modulation of the GABA(A) receptor (GABA(A)-R). Herein we report the effects on the activity and pharmacokinetic properties of a series of nor-19 pregnanolone analogues bearing a heterocyclic substituent at C-21. These efforts resulted in the identification of SGE-516, a balanced synaptic/extrasynaptic GABA(A) receptor modulator, and SGE-872, a selective extrasynaptic GABA(A) receptor modulator. Both molecules possess excellent druglike properties, making them advanced leads for oral delivery of GABA(A) receptor modulators.

A cellular mechanism for the antinociceptive effect of a kappa opioid receptor agonist.

This study used concordant behavioral and electrophysiological approaches to examine the actions of the prototypic kappa opioid receptor agonist U69593 in the rostral ventromedial medulla (RVM). In vitro whole-cell voltage clamp recordings indicated that bath application of U69593 produced outward currents in primary cells in the RVM. In secondary cells, which comprised 80% of the population, U69593 produced a concentration-dependent and norbinaltorphimine (norBNI)-reversible inhibition of evoked excitatory postsynaptic currents (EPSCs) in the absence of any postsynaptic effect. U69593 also decreased the frequency, but not the amplitude of spontaneous miniature excitatory postsynaptic currents (mEPSCs) in secondary cells. The inhibition of excitatory inputs to secondary cells would be consonant with disinhibition of primary cells and the production of antinociception. Consistent with this expectation, the activation of kappa opioid receptors in the RVM by microinjection of U69593 produced a dose-dependent increase in paw-withdrawal latency that was antagonized by norBNI. Furthermore, microinjection of norBNI in the RVM antagonized the increases in paw-withdrawal latency and hot-plate latency produced by systemically-administered U69593. In contrast, microinjection of norBNI in the RVM did not antagonize the increase in tail-flick latency produced by systemically-administered U69593. Also, microinjection of U69593 in the RVM did not increase tail-flick latency. The highly test-dependent nature of U69593's effects suggests that the mechanisms by which neurons in the RVM modulate thermal nociceptive responses evoked from the tail and hindpaw are not uniform. Collectively, these data suggest that the RVM is a primary site of action for the antinociceptive actions of kappa opioid receptor agonists and that the mechanism most likely involves a presynaptic inhibition of excitatory inputs to secondary cells. Thus, disinhibition of pain inhibitory neurons in the RVM is likely to be a common mechanism by which opioid receptor agonists produce antinociception, whether by the direct inhibition of inhibitory secondary cells, as in the case of mu opioid receptor agonists, or by a reduction in the excitatory drive to these neurons, as in the case of kappa opioid receptor agonists.

Control of glutamatergic neurotransmission in the rat spinal dorsal horn by the nucleoside transporter ENT1.

Adenosine modulates nociceptive processing in the superficial dorsal horn of the spinal cord. In other tissues, membrane transporters influence profoundly the extracellular levels of adenosine. To investigate the putative role of nucleoside transporters in the regulation of excitatory synaptic transmission in the dorsal horn, we employed immunohistochemistry and whole-cell patch-clamp recording of substantia gelatinosa neurons in slices of rat spinal cord in vitro. The rat equilibrative nucleoside transporter (rENT1) was revealed by antibody staining to be abundant in neonatal and mature dorsal horn, especially within laminae I-III. This was confirmed by immunoblots of dorsal horn homogenate. Nitrobenzylthioinosine (NBMPR), a potent non-transportable inhibitor of rENT1, attenuated synaptically evoked EPSCs onto lamina II neurons in a concentration-dependent manner. Application of an adenosine A1 antagonist 1,3-dipropyl-8-cyclopentylxanthine produced a parallel rightward shift in the NBMPR concentration-effect curve. The effects of NBMPR were partially reversed by adenosine deaminase, which facilitates the metabolic degradation of adenosine. The modulation by NBMPR of evoked EPSCs was mimicked by exogenous adenosine or the selective A1 receptor agonist, 2-chloro-N6-cyclopentyl adenosine. NBMPR reduced the frequency but not the amplitude of spontaneous miniature EPSCs and increased the paired-pulse ratio of evoked currents, an effect that is consistent with presynaptic modulation. These data provide the first direct evidence that nucleoside transporters are able to critically modulate glutamatergic synaptic transmission.