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N-methyl-D-aspartate receptors (NMDARs) play a critical role in synaptic plasticity, and mutations in human genes encoding NMDAR subunits have been described in individuals with various neuropsychiatric disorders. Compounds with a positive allosteric effect are thought to compensate for reduced receptor function.We have used whole-cell patch-clamp electrophysiology on recombinant rat NMDARs and human variants found in individuals with neuropsychiatric disorders, in combination with in silico modelling, to explore the site of action of novel epipregnanolone-based NMDAR modulators.Analysis of the action of 4-(20-oxo-5β-pregnan-3β-yl) butanoic acid (EPA-But) at the NMDAR indicates that the effect of this steroid with a "bent" structure is different from that of cholesterol and oxysterols and shares a disuse-dependent mechanism of NMDAR potentiation with the "planar" steroid 20-oxo-pregn-5-en-3β-yl sulfate (PE-S). The potentiating effects of EPA-But and PE-S are additive. Alanine scan mutagenesis identified residues that reduce the potentiating effect of EPA-But. No correlation was found between the effects of EPA-But and PE-S at mutated receptors that were less sensitive to either steroid. The relative degree of potentiation induced by the two steroids also differed in human NMDARs carrying rare variants of hGluN1 or hGluN2B subunits found in individuals with neuropsychiatric disorders, including intellectual disability, epilepsy, developmental delay, and autism spectrum disorder.Our results show novel sites of action for pregnanolones at the NMDAR and provide an opportunity for the development of new therapeutic neurosteroid-based ligands to treat diseases associated with glutamatergic system hypofunction.

Background and purpose Neurosteroids influence neuronal function and have multiple promising clinical applications. Direct modulation of postsynaptic neurotransmitter receptors by neurosteroids is well characterized, but presynaptic effects remain poorly understood. Here, we report presynaptic glutamate release potentiation by neurosteroids pregnanolone and pregnanolone sulfate and compare their mechanisms of action to phorbol 12,13-dibutyrate (PDBu), a mimic of the second messenger DAG. Experimental approach We use whole-cell patch-clamp electrophysiology and pharmacology in rat hippocampal microisland cultures and total internal reflection fluorescence (TIRF) microscopy in HEK293 cells expressing GFP-tagged vesicle priming protein Munc13-1, to explore the mechanisms of neurosteroid presynaptic modulation. Key results Pregnanolone sulfate and pregnanolone potentiate glutamate release downstream of presynaptic Ca2+ influx, resembling the action of a phorbol ester PDBu. PDBu partially occludes the effect of pregnanolone, but not of pregnanolone sulfate. Calphostin C, an inhibitor that disrupts DAG binding to its targets, reduces the effect PDBu and pregnanolone, but not of pregnanolone sulfate, suggesting that pregnanolone might interact with a well-known DAG/phorbol ester target Munc13-1. However, TIRF microscopy experiments found no evidence of pregnanolone-induced membrane translocation of GFP-tagged Munc13-1, suggesting that pregnanolone may regulate Munc13-1 indirectly or interact with other DAG targets. Conclusion and implications We describe a novel presynaptic effect of neurosteroids pregnanolone and pregnanolone sulfate to potentiate glutamate release downstream of presynaptic Ca2+ influx. The mechanism of action of pregnanolone, but not of pregnanolone sulfate, partly overlaps with that of PDBu. Presynaptic effects of neurosteroids may contribute to their therapeutic potential in the treatment of disorders of the glutamate system.

NMDARs are ligand-gated ion channels that cause an influx of Na+and Ca2+into postsynaptic neurons. The resulting intracellular Ca2+transient triggers synaptic plasticity. When prolonged, it may induce excitotoxicity, but it may also activate negative feedback to control the activity of NMDARs. Here, we report that a transient rise in intracellular Ca2+(Ca2+challenge) increases the sensitivity of NMDARs but not AMPARs/kainate receptors to the endogenous inhibitory neurosteroid 20-oxo-5β-pregnan-3α-yl 3-sulfate and to its synthetic analogs, such as 20-oxo-5β-pregnan-3α-yl 3-hemipimelate (PAhPim). In cultured hippocampal neurons, 30 μmPAhPim had virtually no effect on NMDAR responses; however, following the Ca2+challenge, it inhibited the responses by 62%; similarly, the Ca2+challenge induced a 3.7-fold decrease in the steroid IC50on recombinant GluN1/GluN2B receptors. The increase in the NMDAR sensitivity to PAhPim was dependent on three cysteines (C849, C854, and C871) located in the carboxy-terminal domain of the GluN2B subunit, previously identified to be palmitoylated (Hayashi et al., 2009). Our experiments suggested that the Ca2+challenge induced receptor depalmitoylation, and single-channel analysis revealed that this was accompanied by a 55% reduction in the probability of channel opening. Results ofin silicomodeling indicate that receptor palmitoylation promotes anchoring of the GluN2B subunit carboxy-terminal domain to the plasma membrane and facilitates channel opening. Depalmitoylation-induced changes in the NMDAR pharmacology explain the neuroprotective effect of PAhPim on NMDA-induced excitotoxicity. We propose that palmitoylation-dependent changes in the NMDAR sensitivity to steroids serve as an acute endogenous mechanism that controls NMDAR activity.SIGNIFICANCE STATEMENTThere is considerable interest in negative allosteric modulators of NMDARs that could compensate for receptor overactivation by glutamate orde novogain-of-function mutations in neurodevelopmental disorders. By a combination of electrophysiological, pharmacological, and computational techniques we describe a novel feedback mechanism regulating NMDAR activity. We find that a transient rise in intracellular Ca2+increases NMDAR sensitivity to inhibitory neurosteroids in a process dependent on GluN2B subunit depalmitoylation. These results improve our understanding of the molecular mechanisms of steroid action at the NMDAR and indeed of the basic properties of this important glutamate-gated ion channel and may aid in the development of therapeutics for treating neurologic and psychiatric diseases related to overactivation of NMDARs without affecting normal physiological functions.

N-methyl-D-aspartate receptor (NMDAR) hypofunction has been implicated in several neurodevelopmental disorders. NMDAR function can be augmented by positive allosteric modulators, including endogenous compounds, such as cholesterol and neurosteroid pregnenolone sulfate (PES). Here we report that PES accesses the receptor via the membrane, and its binding site is different from that of cholesterol. Alanine mutagenesis has identified residues that disrupt the steroid potentiating effect at the rat GluN1 (G638; I642) and GluN2B (W559; M562; Y823; M824) subunit. Molecular dynamics simulation indicates that, in the absence of PES, the GluN2B M1 helix residue W559 interacts with the M4 helix residue M824. In the presence of PES, the M1 and M4 helices of agonist-activated receptor rearrange, forming a tighter interaction with the GluN1 M3 helix residues G638 and I642. This stabilizes the open-state position of the GluN1 M3 helices. Together, our data identify a likely binding site for the NMDAR-positive allosteric modulator PES and describe a novel molecular mechanism by which NMDAR activity can be augmented.SIGNIFICANCE STATEMENT There is considerable interest in drugs that enhance NMDAR function and could compensate for receptor hypofunction associated with certain neuropsychiatric disorders. Positive allosteric modulators of NMDARs include an endogenous neurosteroid pregnenolone sulfate (PES), but the binding site of PES on the NMDAR and the molecular mechanism of potentiation are unknown. We use patch-clamp electrophysiology in combination with mutagenesis and in silico modeling to describe the interaction of PES with the NMDAR. Our data indicate that PES binds to the transmembrane domain of the receptor at a discrete group of residues at the GluN2B membrane helices M1 and M4 and the GluN1 helix M3, and that PES potentiates NMDAR function by stabilizing the open-state position of the GluN1 M3 helices.

Evidence from clinical and preclinical studies implicates dysfunction of N-methyl-D-aspartate receptors (NMDARs) in schizophrenia progression and symptoms. We investigated the antipsychotic effect of two neuroactive steroids in an animal model of schizophrenia induced by systemic application of MK-801. The neuroactive steroids differ in their mechanism of action at NMDARs. MS-249 is positive, while PA-Glu is a negative allosteric NMDAR modulator. We hypothesized that the positive NMDA receptor modulator would attenuate deficits caused by MK-801 co-application more effectively than PA-Glu. The rats were tested in a battery of tests assessing spontaneous locomotion, anxiety and cognition. Contrary to our expectations, PA-Glu exhibited a superior antipsychotic effect to MS-249. The performance of MS-249-treated rats in cognitive tests differed depending on the level of stress the rats were exposed to during test sessions. In particular, with the increasing severity of stress exposure, the performance of animals worsened. Our results demonstrate that enhancement of NMDAR function may result in unspecific behavioral responses. Positive NMDAR modulation can influence other neurobiological processes besides memory formation, such as anxiety and response to stress.

International audience; Branched esters of palmitic acid and hydroxy stearic acid are antiinflammatory and antidiabetic lipokines that belong to a family of fatty acid (FA) esters of hydroxy fatty acids (HFAs) called FAHFAs. FAHFAs themselves belong to oligomeric FA esters, known as estolides. Glycerol-bound FAHFAs in triacylglycerols (TAGs), named TAG estolides, serve as metabolite reservoir of FAHFAs mobilized by lipases upon demand. Here, we characterized the involvement of two major metabolic lipases, adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL), in TAG estolide and FAHFA degradation. We synthesized a library of 20 TAG estolide isomers with FAHFAs varying in branching position, chain length, saturation grade, and position on the glycerol backbone and developed an in silico mass spectra library of all predicted catabolic intermediates. We found that ATGL alone or coactivated by comparative gene identification-58 efficiently liberated FAHFAs from TAG estolides with a preference for more compact substrates where the estolide branching point is located near the glycerol ester bond. ATGL was further involved in transesterification and remodeling reactions leading to the formation of TAG estolides with alternative acyl compositions. HSL represented a much more potent estolide bond hydrolase for both TAG estolides and free FAHFAs. FAHFA and TAG estolide accumulation in white adipose tissue of mice lacking HSL argued for a functional role of HSL in estolide catabolism in vivo. Our data show that ATGL and HSL participate in the metabolism of estolides and TAG estolides in distinct manners and are likely to affect the lipokine function of FAHFAs.

International audience; Branched esters of palmitic acid and hydroxystearic acid (PAHSA) are anti-inflammatory and antidiabetic lipokines that connect glucose and lipid metabolism. We aimed to characterize involvement of the 5-PAHSA regioisomer in the adaptive metabolic response of white adipose tissue (WAT) to cold exposure (CE) in mice, exploring the cross talk between glucose utilization and lipid metabolism. CE promoted local production of 5-and 9-PAHSAs in WAT. Metabolic labeling of de novo lipogenesis (DNL) using 2 H 2 O revealed that 5-PAHSA potentiated the effects of CE and stimulated triacylglycerol (TAG)/fatty acid (FA) cycling in WAT through impacting lipogenesis and lipolysis. Adipocyte lipolytic products were altered by 5-PAHSA through selective FA re-esterification. The impaired lipolysis in global adipose triglyceride lipase (ATGL) knockout mice reduced free PAHSA levels and uncovered a metabolite reservoir of TAG-bound PAHSAs (TAG estolides) in WAT. Utilization of 13 C isotope tracers and dynamic metab-olomics documented that 5-PAHSA primes adipocytes for glucose metabolism in a different way from insulin, promoting DNL and impeding TAG synthesis. In summary , our data reveal new cellular and physiological mechanisms underlying the beneficial effects of 5-PAHSA and its relation to insulin action in adipocytes and independently confirm a PAHSA metabolite reservoir linked to ATGL-mediated lipolysis.

The Pregnane X (PXR), Vitamin D (VDR) and Farnesoid X (FXR) nuclear receptors have been shown to be receptors of bile acids controlling their detoxification or synthesis. Chenodeoxycholic (CDCA) and lithocholic (LCA) acids are ligands of FXR and VDR, respectively, whereas 3-keto and acetylated derivates of LCA have been described as ligands for all three receptors. In this study, we hypothesized that oxidation or acetylation at position 3, 7 and 12 of bile acids DCA (deoxycholic acid), LCA, CA (cholic acid), and CDCA by detoxification enzymes or microbiome may have an effect on the interactions with bile acid nuclear receptors. We employed reporter gene assays in HepG2 cells, the TR-FRET assay with recombinant PXR and RT-PCR to study the effects of acetylated and keto bile acids on the nuclear receptors activation and their target gene expression in differentiated hepatic HepaRG cells. We demonstrate that the DCA 3,12-diacetate and CA 3,7,12-triacetate derivatives are ligands of PXR and DCA 3,12-diacetate induces PXR target genes such as CYP3A4, CYP2B6 and ABCB1/MDR1. In conclusion, we found that acetylated DCA and CA are potent ligands of PXR. Whether the acetylated bile acid derivatives are novel endogenous ligands of PXR with detoxification or physiological functions should be further studied in ongoing experiments.

Herein, we report a new class of amide-based inhibitors (1–4) of N-methyl- d -aspartate receptors (NMDARs) that were prepared as analogues of pregnanolone sulfate (PAS) and pregnanolone glutamate (PAG) – the steroidal neuroprotective NMDAR inhibitors. A series of experiments were conducted to evaluate their physicochemical and biological properties: (i) the inhibitory effect of compounds 3 and 4 on NMDARs was significantly improved (IC50 = 1.0 and 1.4 μM, respectively) as compared with endogenous inhibitor – pregnanolone sulfate (IC50 = 24.6 μM) and pregnanolone glutamate (IC50 = 51.7 μM); (ii) physicochemical properties (logP and logD) were calculated; (iii) Caco-2 assay revealed that the permeability properties of compounds 2 and 4 are comparable with pregnanolone glutamate; (iv) compounds 1–4 have minimal or no adverse hepatic effect; (v) compounds 1–4 cross blood-brain-barrier.

Neuroactive steroid 20-oxo-5β-pregnan-3α-yl L-glutamyl 1-ester (PA-Glu), a synthetic analogue of naturally occurring 20-oxo-5β-pregnan-3α-yl sulfate (pregnanolone sulfate, PA-S), inhibits N-methyl-D-aspartate (NMDA) receptors and possesses neuroprotective properties and minimal adverse effects. Herein, we report in vivo effects of new structural modifications of the PA-S molecule: a nonpolar modification of the steroid D-ring (5β-androstan-3α-yl L-glutamyl 1-ester, AND-Glu), attachment of a positively charged group to C3 (20-oxo-5β-pregnan-3α-yl L-argininate dihydrochloride salt, PA-Arg) and their combination (5β-androstan-3α-yl L-argininate dihydrochloride salt, AND-Arg). The first aim of this study was to determine the structure-activity relationship for neuroprotective effects in a model of excitotoxic hippocampal damage in rats, based on its behavioral correlate in Carousel maze. The second aim was to explore side effects of neuroprotective steroids on motor functions, anxiety (elevated plus maze) and locomotor activity (open field) and the effect of their high doses in mice. The neuroprotective properties of PA-Glu and AND-Glu were proven, with the effect of the latter appearing to be more pronounced. In contrast, neuroprotective efficacy failed when positively charged molecules (PA-Arg, AND-Arg) were used. AND-Glu and PA-Glu at the neuroprotective dose (1 mg/kg) did not unfavorably influence motor functions of intact mice. Moreover, anxiolytic effects of AND-Glu and PA-Glu were ascertained. These findings corroborate the value of research of steroidal inhibitors of NMDA receptors as potential neuroprotectants with slight anxiolytic effect and devoid of behavioral adverse effects. Taken together, the results suggest the benefit of the nonpolar D-ring modification, but not of the attachment of a positively charged group to C3.

A broad variety of central nervous system diseases have been associated with glutamate induced excitotoxicity under pathological conditions. The neuroprotective effects of neurosteroids can combat this excitotoxicity. Herein, we have demonstrated the neuroprotective effect of novel steroidal N-methyl-D-aspartate receptor inhibitors against glutamate- or NMDA- induced excitotoxicity. Pretreatment with neurosteroids significantly reduced acute L-glutamic acid or NMDA excitotoxicity mediated by Ca2+ entry and consequent ROS (reactive oxygen species) release and caspase-3 activation. Compounds 6 (IC50 = 5.8 μM), 7 (IC50 = 12.2 μM), 9 (IC50 = 7.8 μM), 13 (IC50 = 1.1 μM) and 16 (IC50 = 8.2 μM) attenuated glutamate-induced Ca2+ entry more effectively than memantine (IC50 = 18.9 μM). Moreover, compound 13 shows comparable effect with MK-801 (IC50 = 1.2 μM) and also afforded significant protection without any adverse effect upon prolonged exposure. This drop in Ca2+ level resulted in corresponding ROS suppression and prevented glutamate-induced caspase-3 activation. Therefore, compound 13 has great potential for development into a therapeutic agent for improving glutamate-related nervous system diseases.

N-Methyl-d-aspartate receptors (NMDARs) display a critical role in various diseases of the central nervous system. The activity of NMDARs can be modulated by neurosteroids. Herein, we report a structure-activity relationship study for perhydrophenanthrene analogues possessing a framework that mimics the steroidal ring system. This study comprises the design, synthesis, and assessment of the biological activity of a library of perhydrophenanthrene 2-sulfates and 2-hemisuccinates (1-10). Their ability to modulate NMDAR-induced currents was tested on recombinant GluN1/GluN2B receptors. Our results demonstrate that such structural optimization leads to compounds that are inhibitors of NMDARs. Notably, compound 9 (IC50 = 15.6 μM) was assessed as a more potent inhibitor of NMDAR-induced currents than the known endogenous neurosteroid, pregnanolone sulfate (IC50 = 24.6 μM).

PostsynapticN-methyl-d-aspartate receptors (NMDARs) phasically activated by presynaptically released glutamate are critical for synaptic transmission and plasticity. However, under pathological conditions, excessive activation of NMDARs by tonically increased ambient glutamate contributes to excitotoxicity associated with various acute and chronic neurological disorders. Here, using heterologously expressed GluN1/GluN2A and GluN1/GluN2B receptors and rat autaptic hippocampal microisland cultures, we show that pregnanolone sulfate inhibits NMDAR currents induced by a prolonged glutamate application with a higher potency than the NMDAR component of EPSCs. For synthetic pregnanolone derivatives substituted with a carboxylic acid moiety at the end of an aliphatic chain of varying length and attached to the steroid skeleton at C3, the difference in potency between tonic and phasic inhibition increased with the length of the residue. The steroid with the longest substituent, pregnanolone hemipimelate, had no effect on phasically activated receptors while inhibiting tonically activated receptors. In behavioral tests, pregnanolone hemipimelate showed neuroprotective activity without psychomimetic symptoms. These results provide insight into the influence of steroids on neuronal function and stress their potential use in the development of novel therapeutics with neuroprotective action.SIGNIFICANCE STATEMENTSynaptic activation ofN-methyl-d-aspartate receptors (NMDARs) plays a key role in synaptic plasticity, but excessive tonic NMDAR activation mediates excitotoxicity associated with many neurological disorders. Therefore, there is much interest in pharmacological agents capable of selectively blocking tonically activated NMDARs while leaving synaptically activated NMDARs intact. Here, we show that an endogenous neurosteroid pregnanolone sulfate is more potent at inhibiting tonically than synaptically activated NMDARs. Further, we report that a novel synthetic analog of pregnanolone sulfate, pregnanolone hemipimelate, inhibits tonic NMDAR currents without inhibiting the NMDAR component of the EPSC and shows neuroprotective activityin vivowithout inducing psychomimetic side effects. These results suggest steroids may have a clinical advantage over other known classes of NMDAR inhibitors.

Herein, we report the synthesis, structure-activity relationship study, and biological evaluation of neurosteroid inhibitors of N-methyl-D-aspartate receptors (NMDARs) receptors that employ an amide structural motif, relative to pregnanolone glutamate (PAG) - a compound with neuroprotective properties. All compounds were found to be more potent NMDAR inhibitors (IC50 values varying from 1.4 to 21.7 μM) than PAG (IC50 = 51.7 μM). Selected compound 6 was evaluated for its NMDAR subtype selectivity and its ability to inhibit AMPAR/GABAR responses. Compound 6 inhibits the NMDARs (8.3 receptors (8.3 ± 2.1 μM) more strongly than it does at the GABAR and AMPARs (17.0 receptors (17.0 ± 0.2 μM and 276.4 ± 178.7 μM, respectively). In addition, compound 6 (10 μM) decreases the frequency of action potentials recorded in cultured hippocampal neurons. Next, compounds 3, 5-7, 9, and 10 were not associated with mitotoxicity, hepatotoxicity nor ROS induction. Lastly, we were able to show that all compounds have improved rat and human plasma stability over PAG.

Neurosteroids are endogenous steroidal compounds that can modulate neuronal receptors. N-Methyl-D-aspartate receptors (NMDARs) are glutamate-gated, calcium-permeable ion channels that are of particular interest, as they participate in synaptic transmission and are implicated in various processes, such as learning, memory, or long-term neuronal potentiation. Positive allosteric modulators that increase the activity of NMDARs may provide a therapeutic aid for patients suffering from neuropsychiatric disorders where NMDAR hypofunction is thought to be involved, such as intellectual disability, autism spectrum disorder, or schizophrenia. We recently described a new class of pregn-5-ene and androst-5-ene 3β-dicarboxylic acid hemiesters (2–24) as potent positive modulators of NMDARs. Considering the recommended guidelines for the early stage development of new, potent compounds, we conducted an in vitro safety assessment and plasma stability screening to evaluate their druglikeness. First, compounds were screened for their hepatotoxicity and mitochondrial toxicity in a HepG2 cell line. Second, toxicity in primary rat postnatal neurons was estimated. Next, the ability of compounds 2–24 to cross a Caco-2 monolayer was also studied. Finally, rat and human plasma stability screening revealed an unforeseen high stability of the C-3 hemiester moiety. In summary, by using potency/efficacy towards NMDARs data along with toxicity profile, Caco-2 permeability and plasma stability, compounds 14 and 15 were selected for further in vivo animal studies.

Here, we report the synthesis of pregn-5-ene and androst-5-ene dicarboxylic acid esters and explore the structure–activity relationship (SAR) for their modulation of N-methyl-d-aspartate receptors (NMDARs). All compounds were positive modulators of recombinant GluN1/GluN2B receptors (EC50 varying from 1.8 to 151.4 μM and Emax varying from 48% to 452%). Moreover, 10 compounds were found to be more potent GluN1/GluN2B receptor modulators than endogenous pregnenolone sulfate (EC50 = 21.7 μM). The SAR study revealed a relationship between the length of the residues at carbon C-3 of the steroid molecule and the positive modulatory effect at GluN1/GluN2B receptors for various D-ring modifications. A selected compound, 20-oxo-pregnenolone hemiadipate, potentiated native NMDARs to a similar extent as GluN1/GluN2A-D receptors and inhibited AMPARs and GABAAR responses. These results provide a unique opportunity for the development of new steroid based drugs with potential use in the treatment of neuropsychiatric di...

N-methyl-D-aspartate receptors (NMDARs), glutamate-gated ion channels, mediate signaling at the majority of excitatory synapses in the nervous system. Recent sequencing data for neurological and psychiatric patients have indicated numerous mutations in genes encoding for NMDAR subunits. Here, we present surface expression, functional, and pharmacological analysis of 11 de novo missense mutations of the human hGluN2B subunit (P553L; V558I; W607C; N615I; V618G; S628F; E657G; G820E; G820A; M824R; L825V) located in the pre-M1, M1, M2, M3, and M4 membrane regions. These variants were identified in patients with intellectual disability, developmental delay, epileptic symptomatology, and autism spectrum disorder. Immunofluorescence microscopy indicated that the ratio of surface-to-total NMDAR expression was reduced for hGluN1/hGluN2B(S628F) receptors and increased for for hGluN1/hGluN2B(G820E) receptors. Electrophysiological recordings revealed that agonist potency was altered in hGluN1/hGluN2B(W607C; N615I; and E657G) receptors and desensitization was increased in hGluN1/hGluN2B(V558I) receptors. The probability of channel opening of hGluN1/hGluN2B (V558I; W607C; V618G; and L825V) receptors was diminished ~10-fold when compared to non-mutated receptors. Finally, the sensitivity of mutant receptors to positive allosteric modulators of the steroid origin showed that glutamate responses induced in hGluN1/hGluN2B(V558I; W607C; V618G; and G820A) receptors were potentiated by 59–96% and 406-685% when recorded in the presence of 20-oxo-pregn-5-en-3β-yl sulfate (PE-S) and androst-5-en-3β-yl hemisuccinate (AND-hSuc), respectively. Surprisingly hGluN1/hGluN2B(L825V) receptors were strongly potentiated, by 197 and 1647%, respectively, by PE-S and AND-hSuc. Synaptic-like responses induced by brief glutamate application were also potentiated and the deactivation decelerated. Further, we have used homology modeling based on the available crystal structures of GluN1/GluN2B NMDA receptor followed by molecular dynamics simulations to try to relate the functional consequences of mutations to structural changes. Overall, these data suggest that de novo missense mutations of the hGluN2B subunit located in membrane domains lead to multiple defects that manifest by the NMDAR loss of function that can be rectified by steroids. Our results provide an opportunity for the development of new therapeutic neurosteroid-based ligands to treat diseases associated with hypofunction of the glutamatergic system.

A unique asymmetric total synthesis of the unnatural enantiomer of pregnanolone, as well as a study of its biological activity at the NMDA receptor, is reported. The asymmetry is introduced by a highly atom-economic organocatalytic Robinson annulation. A new method for the construction of the cyclopentane D-ring consisting of CuI-catalyzed conjugate addition and oxygenation followed by thermal cyclization employing the persistent radical effect was developed. ent-Pregnanolone sulfate is surprisingly only 2.6-fold less active than the natural neurosteroid.

N-Methyl-D-aspartate receptors (NMDARs) are glutamate-gated ion channels that play a crucial role in excitatory synaptic transmission. However, the overactivation of NMDARs can lead to excitotoxic cell damage/death, and as such, they play a role in numerous neuropathological conditions. The activity of NMDARs is known to be influenced by a wide variety of allosteric modulators, including neurosteroids, which in turn makes them promising therapeutic targets. In this study, we describe a new class of neurosteroid analogues which possess structural modifications in the steroid D-ring region. These analogues were tested on recombinant GluN1/GluN2B receptors to evaluate the structure-activity relationship. Our results demonstrate that there is a strong correlation between this new structural feature and the in vitro activity, as all tested compounds were evaluated as more potent inhibitors of NMDA-induced currents (IC50 values varying from 90 nM to 5.4 μM) than the known endogeneous neurosteroid-pregnanolone sulfate (IC50 = 24.6 μM).

Neuroactive steroids modulate receptors for neurotransmitters in the brain and thus might be efficacious in the treatment of various diseases of the central nervous system such as schizophrenia. We have designed and synthetized a novel use-dependent NMDA receptor antagonist 3α5β-pregnanolone glutamate (3α5β-P-Glu). In this study, we evaluate procognitive properties of 3α5β-P-Glu in an animal model of schizophrenia induced by systemic application of MK-801. The procognitive properties were evaluated using active place avoidance on a rotating arena (Carousel maze). We evaluated effects of 3α5β-P-Glu on the avoidance, on locomotor activity, and anxiety. 3α5β-P-Glu alone altered neither spatial learning nor locomotor activity in control animals. In the model animals, 3α5β-P-Glu reversed the MK-801-induced cognitive deficit without reducing hyperlocomotion. The highest dose of 3α5β-P-Glu also showed anxiolytic properties. Taken together, 3α5β-P-Glu may participate in the restoration of normal brain functioning and these results may facilitate the development of new promising drugs improving cognitive functioning in schizophrenia.

BACKGROUND AND PURPOSE NMDA receptors are glutamatergic ionotropic receptors involved in excitatory neurotransmission, synaptic plasticity and excitotoxic cell death. Many allosteric modulators can influence the activity of these receptors positively or negatively, with behavioural consequences. 20-Oxo-5β-pregnan-3α-yl sulphate (pregnanolone sulphate; PA-6) is an endogenous neurosteroid that inhibits NMDA receptors and is neuroprotective. We tested the hypothesis that the interaction of PA-6 with the plasma membrane is critical for its inhibitory effect at NMDA receptors. EXPERIMENTAL APPROACH Electrophysiological recordings and live microscopy were performed on heterologous HEK293 cells expressing GluN1/GluN2B receptors and cultured rat hippocampal neurons. KEY RESULTS Our experiments showed that the kinetics of the steroid inhibition were slow and not typical of drug-receptor interaction in an aqueous solution. In addition, the recovery from steroid inhibition was accelerated by β- and γ-cyclodextrin. Values of IC50 assessed for novel synthetic C3 analogues of PA-6 differed by more than 30-fold and were positively correlated with the lipophilicity of the PA-6 analogues. Finally, the onset of inhibition induced by C3 analogues of PA-6 ranged from use-dependent to use-independent. The onset and offset of cell staining by fluorescent analogues of PA-6 were slower than those of steroid-induced inhibition of current responses mediated by NMDA receptors. CONCLUSION AND IMPLICATIONS We conclude that steroid accumulation in the plasma membrane is the route by which it accesses a binding site on the NMDA receptor. Thus, our results provide a possible structural framework for pharmacologically targeting the transmembrane domains of the receptor.

Glutamate is the main neurotransmitter released at synapses in the central nervous system of vertebrates. Its excitatory role is mediated through activation of specific glutamatergic ionotropic receptors, among which the N-methyl-D-aspartate (NMDA) receptor subtype has attracted considerable attention in recent years. Substantial progress has been made in elucidating the roles these receptors play under physiological and pathological conditions and in our understanding of the functional, structural, and pharmacological properties of NMDA receptors. Many pharmacological compounds have been identified that affect the activity of NMDA receptors, including neurosteroids. This review summarizes our knowledge about molecular mechanisms underlying the neurosteroid action at NMDA receptors as well as about the action of neurosteroids in animal models of human diseases.

We have prepared 20-oxo-5α- and 20-oxo-5β-pregnane-3-carboxylic acids by palladium catalyzed alkoxycarbonylation using triflate and nonaflate as the leaving groups in this substitution reaction. The activity of the synthesized compounds was studied in cultured rat hippocampal neurons under voltage-clamp conditions. The 5β-carboxylic acid derivatives were found to diminish NMDA-induced responses, whereas the 5α-derivative potentiated the response.

A series of keto steroids were reduced with sodium borohydride in the presence of cerium(III) chloride or samarium(III) iodide (Luche reduction). The ratios of axial and equatorial alcohols were determined by HPLC and the results were compared with those obtained by a standard sodium borohydride reduction. The best results were obtained with the 2-keto derivative 1, 7-keto derivatives 5 and 6, and 12-keto derivative 8 where the cerium(III) ion addition resulted in the inversion of the axial/equatorial ratios. The Luche reduction of the 20-keto derivative 11 improved the proportion of the (20S)-alcohol in a mixture of (20S)/(20R) alcohols up to 35% from 5% in a standard sodium borohydride reduction.

A procedure is described for the preparation of A-homo-5-pregnenes via an acid catalyzed rearrangement of cyclopropylcarbinols assisted by microwave irradiation. 3alpha-Hydroxy and 4alpha-hydroxy-A-homo-5-pregnen-20-one, analogues of the neuroactive steroid allopregnanolone, were obtained by means of a regioselective epoxidation of a double bond in the expanded A-ring, using a fructose-derived chiral ketone as catalyst and oxone as oxidant. Although both these compounds were marginally active in inhibiting TBPS binding to GABA(A) receptors, 3beta-hydroxy-A-homo-5-pregnen-20-one was almost as active as allopregnanolone. Reduction of the double bond of the latter compound resulted in a ten fold loss of activity.

The lipophilic nature of allopregnanolone prevents its user-friendly application in human medicine. On inspiration by pReviously prepared allopregnanolone with a 16α -bound tetraethylammonium salt, an attempt was made to produce allopregnanolone analogues with polar groups introduced into position 16α with the goal of increasing water solubility, brain accessibility, and potency of neuroactive steroids. The Michael addition to derivatives of pregn-16-en-20-one was the key reaction step. The link between the steroid skeleton and the new side chain was either a methylene group (when diethyl malonate was added) or an oxygen atom (when a hydroxy derivative was added). [35S]TBPS displacement was used to evaluate the products. Several carbamates (but not their parent alcohols) displaced TBPS from the picrotoxin binding site on GABAA receptors. Although none of them was more potent than the above ammonium salt, which stimulated this study, their nonionic nature should not prevent their passage into the brain. © 2009 American Chemical Society. Fil: Slavíková, Barbora. Academy of Sciences of the Czech Republic; República Checa Fil: Krištofíková, Zdena. Prague Psychiatric Centre; República Checa Fil: Chodounská, Hana. Academy of Sciences of the Czech Republic; República Checa Fil: Buděšínský, Miloš. Academy of Sciences of the Czech Republic; República Checa Fil: Duran, Fernando Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos en Química Orgánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Unidad de Microanálisis y Métodos Físicos en Química Orgánica; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; Argentina Fil: Veleiro, Adriana Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos en Química Orgánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Unidad de Microanálisis y Métodos Físicos en Química Orgánica; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; Argentina Fil: Burton, Gerardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos en Química Orgánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Unidad de Microanálisis y Métodos Físicos en Química Orgánica; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; Argentina Fil: Kasal, Alexander. Academy of Sciences of the Czech Republic; República Checa

Sulfamoyl chloride was used for the preparation of 3α- and 3β-sulfamates of steroids: 20-oxo-5α-pregnan-3α-yl sulfamate (4), 20-oxo-5α-pregnan-3β-yl sulfamate (5), 20-oxo-5β-pregnan-3α-yl sulfamate (7), and 20-oxo-5β-pregnan-3β-yl sulfamate (9) from the corresponding 3α- and 3β-alcohols. Sulfur trioxide–pyridine complex was employed for the preparation of 20-oxo-5α-pregnan-3α-yl pyridinium sulfate (10). The specific steroid binding was detected by the decrease of the specific [35S]-tert-butylbicyclo[2.2.2]phosphorothionate binding after application of the tested compounds.

Steroidal 3-fluoroderivatives were prepared from corresponding tosylates using tetrabutylammonium difluorodimethylphenylsilicate as fluorinating agent. The reaction was tested on all four possible C-3 and C-5 stereoisomers of cholestane and 17-oxoandrostane skeletons. In this reaction only one isomer was always formed with opposite configuration at C-3 to starting tosylate. The reaction is accompanied by elimination which affords a mixture of corresponding olefines.

N-methyl-D-aspartate receptors (NMDARs) mediate synaptic plasticity and their dysfunction is implicated in multiple brain disorders. NMDARs can be allosterically modulated by numerous compounds, including endogenous neurosteroid pregnanolone sulfate. Here, we identify the molecular basis of the use-dependent and voltage-independent inhibitory effect of neurosteroids on NMDAR responses. The site of action is located at the extracellular vestibule of the receptor’s ion channel pore and is accessible after receptor activation. Mutations in the extracellular vestibule in the SYTANLAAF motif disrupt the inhibitory effect of negatively charged steroids. In contrast, positively charged steroids inhibit mutated NMDAR responses in a voltage-dependent manner. These results, in combination with molecular modeling, characterize structure details of the open configuration of the NMDAR channel. Our results provide a unique opportunity for the development of new therapeutic neurosteroid-based ligands to treat diseases associated with dysfunction of the glutamate system.

NMDA receptors are ligand-gated ion channels permeable to calcium and play a critical role in excitatory synaptic transmission, synaptic plasticity, and excitotoxicity. They are heteromeric complexes of NR1 combined with NR2A-D and/or NR3A-B subunits that are activated by glutamate and glycine and whose activity is modulated by allosteric modulators. In this study, patch-clamp recordings from human embryonic kidney 293 cells expressing NR1/NR2 receptors were used to study the molecular mechanism of the endogenous neurosteroid 20-oxo-5beta-pregnan-3alpha-yl sulfate (3alpha5betaS) action at NMDA receptors. 3alpha5betaS was a twofold more potent inhibitor of responses mediated by NR1/NR2C-D receptors than those mediated by NR1/NR2A-B receptors. The structure of the extracellular loop between the third and fourth transmembrane domains of the NR2 subunit was found to be critical for the neurosteroid inhibitory effect. The degree of 3alpha5betaS-induced inhibition of responses to glutamate was voltage independent, with recovery lasting several seconds. In contrast, application of 3alpha5betaS in the absence of agonist had no effect on the subsequent response to glutamate made in the absence of the neurosteroid. A kinetic model was developed to explain the use-dependent action of 3alpha5betaS at NMDA receptors. In accordance with the model, 3alpha5betaS was a less potent inhibitor of NMDA receptor-mediated EPSCs and responses induced by a short application of 1 mm glutamate than of those induced by a long application of glutamate. These results suggest that 3alpha5betaS is a use-dependent but voltage-independent inhibitor of NMDA receptors, with more potent action at tonically than at phasically activated receptors. This may be important in the treatment of excitotoxicity-induced neurodegeneration.

(Diethylamino)sulfur trifluoride (DAST) was used for the preparation of 3α-fluorides (e.g., 3α-fluoro-5α-pregnane-12,20-dione, 3α-fluoro-16α-[(methoxycarbonyl)methyl]-5α-pregnan-20-one, methyl 3α-fluoro-5α-androstane-17β-carboxylate, 3α-fluoro-5β-pregnan-20-one) from the corresponding 3β-alcohols and for the preparation of 3,3-difluorides from 3-ketones (e.g., 3,3-difluoro-5α-pregnan-20-one). Boron trifluoride etherate was used for the conversion of an epoxide into 3α-fluoro-2β-hydroxy-5α-pregnan-20-one. Thein vitrobinding of the 3α-fluorides and the corresponding 3α-alcohols to the GABAAreceptor was established using [3H]muscimol and [35S]-tert-butylbicyclo[2.2.2]phosphorothionate as ligands.

The effect of steroids on NMDA receptors and excitatory postsynaptic transmission was studied in fluorescence-labelled motoneurons in thin spinal cord slices. In outside-out patches, NMDA-induced responses were potentiated by 79% in the presence of 20-oxopregn-5-en-3beta-yl sulfate (PS), while in the presence of 20-oxo-5alpha-pregnan-3alpha-yl sulfate (3alpha5alphaS) and 20-oxo-5beta-pregnan-3alpha-yl sulfate (3alpha5betaS) they were diminished by 57% and 66%, respectively. PS and 3alpha5betaS had no effect on the amplitude of single NMDA receptor channel openings, however, both compounds altered relative distribution of the openings to individual conductance levels. In control cases, the most frequent openings of the NMDA receptor channels were at the 70 pS conductance level, while in the presence of PS or 3alpha5betaS, the most frequent openings were at the 55 pS conductance level. Analysis of the mean current transferred by NMDA receptor channel openings at individual conductance levels indicated that in the presence of PS, the mean current induced by 55 pS conductance openings was significantly increased. In the presence of 3alpha5betaS, the mean currents induced by 55 pS and 70 pS conductance openings were significantly decreased. The amplitude of NMDA receptor-mediated EPSCs was potentiated by 54% in the presence of PS and the deactivation kinetics slowed. Neither the amplitude nor the kinetics of NMDA receptor-mediated EPSCs was significantly changed in the presence of 3alpha5betaS. The results of our experiments indicate that neurosteroids affect NMDA receptors in motoneurons. The effect appears to be influenced by the receptor subunit composition.

a-Hydroxy-20-oxo-5a-pregnan-21-yl hemisuccinate (8) was produced by partial acylation of 3a,21-dihydroxy-5a-pregnan-20-one (14). 3a-Fluoro-5a-pregnan-20-one (9) was prepared by treatment of 3b-hydroxy-5a-pregnan-20-one (11) with DAST and by solvolysis of tosylate 12 with tetrabutylammonium fluoride. A behavioral test on mice was performed using 3 a-hydroxy-5a-pregnan-20-one (1) and compounds 8 and 9. Compound 8 was found to be inactive, while the fluoro derivative 9 selectively reduced aggressive behavior in mice more than the corresponding 3a-hydroxy compound 1; locomotion and other behavioral features were not affected. © 2001 Elsevier Science Inc. All rights reserved.

A simple method to distinguish the 5α- from the 5β-isomers of 3-oxosteroids based on low-frequency 1H NMR spectra was proposed. Additional 1H and 13C NMR characteristics were derived from the comparison of completely assigned spectra of the 5α- and 5β-isomers. The effect of substitution at different positions of steroid skeleton was evaluated on a series of isomeric 3-oxosteroids, prepared for this purpose.

Pregnenolone sulfate (PS), an endogenously occurring neurosteroid, has been shown to modulate the activity of several neurotransmitter-gated channels, including the NMDA receptor (NMDAR). NMDARs are glutamate-gated ion channels involved in excitatory synaptic transmission, synaptic plasticity, and excitotoxicity. In this study, we analyzed the effects of PS on calcium signaling in cultured hippocampal neurons and HEK293 cells expressing NMDAR. The cells were loaded with the Ca2+ sensor Fura-2. In agreement with previous electrophysiological experiments, PS potentiated the increases in intracellular Ca2+ induced by an exogenous application of glutamate; however, PS also increased intracellular Ca2+ in the absence of exogenous NMDA agonist. The agonist-independent effect of PS was induced in all neurons studied and in HEK293 cells expressing GluN1/GluN2A-B receptors in a neurosteroid-specific manner. We conclude that PS is an endogenous NMDA agonist that activates the GluN1/GluN2A-B receptors.

3α-Hydroxy-7a-homo-5α-pregnan-20-one and 3α-hydroxy-7a-homo-5α-pregnane-7,20-dione were prepared from (20R)-3α-(methoxymethoxy)-7-oxo-5α-pregnan-20-yl benzoate. Homo- logation of the B ring was carried out by the Demyanov rearrangement of the corresponding cyanohydrin. Alkaline hydrolysis of the protecting group and oxidation of the formed 20-hydroxy group and deprotection of the 3-hydroxy group were performed either with (20R)-3α-(methoxymethoxy)-7-oxo-7a-homo-5α-pregnan-20-ol derivative or with the product of the Huang Minlon deoxygenation at carbon 7.

(20S)-2α,3α-Dihydroxy-6-oxo-7-oxa-7a-homo-5α-pregnane-20-carboxylic acid (12) as a brassinosteroid ligand for affinity chromatography carriers was synthesized from bisnorcholenic acid. The brassinolide activity in the bean second internode bioassay of the synthesized (20S)-5α-pregnane-20-carboxylic acid derivatives is also described.

3α,7α-Dihydroxy-5α-pregnan-20-one (12) was prepared from (20R)-pregn-5-ene-3β,20-diyl 3-acetate 20-benzoate (1). ∆5-Olefin was oxidized in an allylic position and hydrogenated. Inversion of a configuration at carbon C-3 was carried out by solvolysis in N,N-dimethylformamide of 3β-tosylate in the presence of sodium nitrite. For stereoselective reduction of the 7-oxo group, L-Selectride® was used.

Reduction with diimide was employed in the synthesis of potential antiandrogens - 17β-hydroxy-B-nor-5α-androstan-3-one (26) and its 17α-methyl derivative (25). Other methods of reduction (hydroboration, catalytic hydrogenation) were less effective.

5α-Androstan-17-one and 6β-methoxy-3α,5-cyclo-5α-androstan-17-one were reduced by sodium in deuterium oxide to [16,16,17-2H3]-17β-alcohols. The 17β-tosyloxy group of [16,16,17-2H3]-6β-methoxy-3α,5-cyclo-5α-androstan-17β-ol tosylate was found to be stable under the conditions of the i-steroid rearrangement. The SN2 reaction of the 17β-tosyloxy group with potassium nitrite yielded the corresponding 17α-hydroxy derivative without any loss of deuterium.

11β-Hydroxyandrost-4-ene-3,17-dione (III) was converted into a potential metabolite of epitestosterone - 11β,17α-dihydroxyandrost-4-en-3-one (II) in 5 steps, including the inversion of configuration of a 17β-hydroxy group. This inversion was not feasible in the preparation of the analogues X, XIV, XX, and XXII, where the 17α-hydroxy group was introduced first and only then was the rest of the molecule modified.

It is found that the 5β-carboxylic acid derivatives diminish NMDA- induced responses, whereas the 5α-derivative potentiate the response.

20-Oxo-5β-[9,12,12-(2)H(3)]pregnan-3α-yl-l-glutamyl 1-ester 11 was synthesized as an internal standard for quantification of a neuroprotective NMDA receptor ligand, 20-oxo-5β-pregnan-3α-yl-l-glutamyl 1-ester 18 and its metabolites, in plasma and tissue. 11α-Hydroxy-progesterone (1) was reduced under basic conditions to yield the corresponding 5β-steroid. Protection of the 3- and 20-oxo groups and oxidation of the 11α-hydroxy group was then followed by a deuterium exchange, conducted under basic conditions using deuterated methanol. Next, the carbonyl moiety at C-11 was reduced and the 11α-hydroxyl group removed through utilization of the Barton-McCombie reaction. Subsequent deprotection of the 3- and 20-acetals and stereoselective reduction of the 3-oxo group gave the desired trideuterated pregnanolone (8). This was coupled with protected glutamic acid, which was then deprotected to yield [9,12,12-(2)H(3)]-pregnanolone glutamate (11) with >99% isotopic purity.