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Endogenous neurosteroids (NS) and their synthetic analogs, neuroactive steroids (NAS), are potentially useful drug-like compounds affecting the pathophysiology of miscellaneous central nervous system disorders (e.g. Alzheimer´s disease, epilepsy, depression, etc.). Additionally, NS have been shown to promote neuron viability and neurite outgrowth upon injury. The molecular, structural and physicochemical basis of the NS effect on neurons is so far not fully understood, and the development of new, biologically relevant assays is essential for their comparative analysis and for assessment of their mechanism of action. Here, we report the development of a novel, plate-based, high-content in vitro assay for screening of NS and newly synthesized, 5β-reduced NAS for the promotion of postnatal neuron survival and neurite growth using fluorescent, postnatal mixed cortical neuron cultures isolated from thy1-YFP transgenic mice. The screen allows a detailed time course analysis of different parameters, such as the number of neurons or neurite lengths of 7-day, in vitro neuron cultures. Using the screen, we identify a new NAS, compound 42, that promotes the survival and growth of postnatal neurons significantly better than several endogenous NS (dehydroepiandrosterone, progesterone, and allopregnanolone). Interestingly, we demonstrate that compound 42 also promotes the proliferation of glia (in particular oligodendrocytes) and that the glial function is critical for its neuron growth support. Computational analysis of the biological data and calculated physicochemical properties of tested NS and NAS demonstrated that their biological activity is proportional to their lipophilicity. Together, the screen proves useful for the selection of neuron-active NAS and the comparative evaluation of their biologically relevant structural and physicochemical features., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

17β-HSD10 is a mitochondrial enzyme that catalyzes the steroidal oxidation of a hydroxy group to a keto group and, thus, is involved in maintaining steroid homeostasis. The druggability of 17β-HSD10 is related to potential treatment for neurodegenerative diseases, for example, Alzheimer's disease or cancer. Herein, steroidal derivatives with an acidic hemiester substituent at position C-3 on the skeleton were designed, synthesized, and evaluated by using pure recombinant 17β-HSD10 converting 17β-estradiol to estrone. Compounds 22 (IC 50 = 6.95 ± 0.35 μM) and 23 (IC 50 = 5.59 ± 0.25 μM) were identified as the most potent inhibitors from the series. Compound 23 inhibited 17β-HSD10 activity regardless of the substrate. It was found not cytotoxic toward the HEK-293 cell line and able to inhibit 17β-HSD10 activity also in the cellular environment. Together, these findings support steroidal compounds as promising candidates for further development as 17β-HSD10 inhibitors., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

The family of ATP-gated purinergic P2X receptors comprises seven bunits (P2X1-7) that are unevenly distributed in the central and peripheral nervous systems as well as other organs. Endogenous modulators of P2X receptors are phospholipids, steroids and neurosteroids. Here, we analyzed whether bile acids, which are natural products derived from cholesterol, affect P2X receptor activity. We examined the effects of primary and secondary bile acids and newly synthesized derivatives of lithocholic acid on agonist-induced responses in HEK293T cells expressing rat P2X2, P2X4 and P2X7 receptors. Electrophysiology revealed that low micromolar concentrations of lithocholic acid and its structural analog 4-dafachronic acid strongly inhibit ATP-stimulated P2X2 but potentiate P2X4 responses, whereas primary bile acids and other secondary bile acids exhibit no or reduced effects only at higher concentrations. Agonist-stimulated P2X7 responses are significantly potentiated by lithocholic acid at moderate concentrations. Structural modifications of lithocholic acid at positions C-3, C-5 or C-17 abolish both inhibitory and potentiation effects to varying degrees, and the 3α-hydroxy group contributes to the ability of the molecule to switch between potentiation and inhibition. Lithocholic acid allosterically modulates P2X2 and P2X4 receptor sensitivity to ATP, reduces the rate of P2X4 receptor desensitization and antagonizes the effect of ivermectin on P2X4 receptor deactivation. Alanine-scanning mutagenesis of the upper halve of P2X4 transmembrane domain-1 revealed that residues Phe48, Val43 and Tyr42 are important for potentiating effect of lithocholic acid, indicating that modulatory sites for lithocholic acid and ivermectin partly overlap. Lithocholic acid also inhibits ATP-evoked currents in pituitary gonadotrophs expressing native P2X2, and potentiates ATP currents in nonidentified pituitary cells expressing P2X4 receptors. These results indicate that lithocholic acid is a bioactive steroid that may help to further unveil the importance of the P2X2, and P2X4 receptors in many physiological processes., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

P2X receptors (P2XRs) are ATP-gated cationic channels that are allosterically modulated by numerous compounds, including steroids and neurosteroids. These compounds may both inhibit and potentiate the activity of P2XRs, but sex steroids such as 17β-estradiol or progesterone are reported to be inactive. Here, we tested a hypothesis that testosterone, another sex hormone, modulates activity of P2XRs. We examined actions of native testosterone and a series of testosterone derivatives on the gating of recombinant P2X2R, P2X4R and P2X7R and native channels expressed in pituitary cells and hypothalamic neurons. The 17β-ester derivatives of testosterone rapidly and positively modulate the 1 µM ATP-evoked currents in P2X2R- and P2X4R-expressing cells, but not agonist-evoked currents in P2X7R-expressing cells. In general, most of the tested testosterone derivatives are more potent modulators than endogenous testosterone. The comparison of chemical structures and whole-cell recordings revealed that their interactions with P2XRs depend on the lipophilicity and length of the alkyl chain at position C-17. Pre-treatment with testosterone butyrate or valerate increases the sensitivity of P2X2R and P2X4R to ATP by several fold, reduces the rate of P2X4R desensitization, accelerates resensitization, and enhances ethidium uptake by P2X4R. Native channels are also potentiated by testosterone derivatives, while endogenously expressed GABA receptors type A are inhibited. The effect of ivermectin, a P2X4R-specific allosteric modulator, on deactivation is antagonized by testosterone derivatives in a concentration-dependent manner. Together, our results provide evidence for potentiation of particular subtypes of P2XRs by testosterone derivatives and suggest a potential role of ivermectin binding site for steroid-induced modulation. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/., (© 2019 International Society for Neurochemistry.)

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 Ca 2+ entry and consequent ROS (reactive oxygen species) release and caspase-3 activation. Compounds 6 (IC 50 = 5.8 μM), 7 (IC 50 = 12.2 μM), 9 (IC 50 = 7.8 μM), 13 (IC 50 = 1.1 μM) and 16 (IC 50 = 8.2 μM) attenuated glutamate-induced Ca 2+ entry more effectively than memantine (IC 50 = 18.9 μM). Moreover, compound 13 shows comparable effect with MK-801 (IC 50 = 1.2 μM) and also afforded significant protection without any adverse effect upon prolonged exposure. This drop in Ca 2+ 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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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 (IC 50 values varying from 1.4 to 21.7 μM) than PAG (IC 50 = 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.

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 (EC 50 varying from 1.8 to 151.4 μM and E max varying from 48% to 452%). Moreover, 10 compounds were found to be more potent GluN1/GluN2B receptor modulators than endogenous pregnenolone sulfate (EC 50 = 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 GABA A R responses. These results provide a unique opportunity for the development of new steroid based drugs with potential use in the treatment of neuropsychiatric disorders involving hypofunction of NMDARs.

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 (IC 50 =1.0 and 1.4μM, respectively) as compared with endogenous inhibitor - pregnanolone sulfate (IC 50 =24.6μM) and pregnanolone glutamate (IC 50 =51.7μM); (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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

White adipose tissue (WAT) is a complex organ with both metabolic and endocrine functions. Dysregulation of all of these functions of WAT, together with low-grade inflammation of the tissue in obese individuals, contributes to the development of insulin resistance and type 2 diabetes. n-3 polyunsaturated fatty acids (PUFAs) of marine origin play an important role in the resolution of inflammation and exert beneficial metabolic effects. Using experiments in mice and overweight/obese patients with type 2 diabetes, we elucidated the structures of novel members of fatty acid esters of hydroxy fatty acids-lipokines derived from docosahexaenoic acid (DHA) and linoleic acid, which were present in serum and WAT after n-3 PUFA supplementation. These compounds contained DHA esterified to 9- and 13-hydroxyoctadecadienoic acid (HLA) or 14-hydroxydocosahexaenoic acid (HDHA), termed 9-DHAHLA, 13-DHAHLA, and 14-DHAHDHA, and were synthesized by adipocytes at concentrations comparable to those of protectins and resolvins derived from DHA in WAT. 13-DHAHLA exerted anti-inflammatory and proresolving properties while reducing macrophage activation by lipopolysaccharides and enhancing the phagocytosis of zymosan particles. Our results document the existence of novel lipid mediators, which are involved in the beneficial anti-inflammatory effects attributed to n-3 PUFAs, in both mice and humans., (© 2016 by the American Diabetes Association.)

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).

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).

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 IC(50) 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., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

Androstane brassinosteroid analogues with alpha-azido acid ester groups in position 17beta were synthesized from 2alpha,3alpha,17beta-trihydroxy-5alpha-androstan-6-one after the protection of the 2alpha,3alpha-diols upon treatment with the corresponding alpha-azido acid and the subsequent deprotection of the diol grouping. Six new castasterone analogues were prepared. The biological activities were evaluated in two bioassays: a rice lamina inclination test and bean second internode bioassays. The activities of the new analogues differ in these two bioassays., (Copyright 2010 Elsevier Inc. All rights reserved.)

Three types of 5alpha-androstane and ergostane analogues of brassinolide, containing a fluorine atom in either the 3alpha or the 5alpha positions or in 3alpha and 5alpha positions, were prepared using standard operations (reaction of 3beta-alcohols with (diethylamino)sulfur trifluoride, cleavage of epoxide with HF in py or BF 3.Et 2O). The 5alpha-fluorine was found to affect chemical reactivity (e.g., electrophilic addition to the Delta (2)-double bond) as well as physical properties (e.g., NMR, chromatographic behavior) of the products. Cytotoxicity of the products was studied using human normal and cancer cell lines with 28-homocastasterone as positive control and their brassinolide type activity was established using the bean second-internode test with 24-epibrassinolide as standard. The equivalence of F and OH groups was observed in some of the active compounds. The anticancer and the brassinolide-type activity do not correlate with each other: ergostane derivatives were most active in the former test while androstane derivatives were best in the latter.