Strong Inhibitory Effect, Low Cytotoxicity and High Plasma Stability of Steroidal Inhibitors of N-Methyl-D-Aspartate Receptors With C-3 Amide Structural Motif

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

Allosteric Modulation of Muscarinic Receptors by Cholesterol, Neurosteroids and Neuroactive Steroids

Muscarinic acetylcholine receptors are membrane receptors involved in many physiological processes. Malfunction of muscarinic signaling is a cause of various internal diseases, as well as psychiatric and neurologic conditions. Cholesterol, neurosteroids, neuroactive steroids, and steroid hormones are molecules of steroid origin that, besides having well-known genomic effects, also modulate membrane proteins including muscarinic acetylcholine receptors. Here, we review current knowledge on the allosteric modulation of muscarinic receptors by these steroids. We give a perspective on the research on the non-genomic effects of steroidal compounds on muscarinic receptors and drug development, with an aim to ultimately exploit such knowledge

Stereoselective Reduction of Steroidal 4‑Ene-3-ketones in the Presence of Biomass-Derived Ionic Liquids Leading to Biologically Important 5β-Steroids

The stereoselective reduction of the steroidal 4-ene-3-ketone moiety (enone) affords the 5β-steroid backbone that is a key structural element of biologically important neuroactive steroids. Neurosteroids have been currently studied as novel and potent central nervous system drug-like compounds for the treatment of, e.g., postpartum depression. As a green methodology, we studied the palladium-catalyzed hydrogenation of steroidal 4-ene-3-ketones in the presence of ionic liquids derived from natural carboxylic acids. The hydrogenation proceeds with improved 5β-selectivity in the presence of tetrabutylammonium carboxylates as additives compared to the exclusive use of an organic solvent. Under optimal conditions, using tetrabutylammonium d-mandelate, the reduction of testosterone led to 5β-dihydrotestosterone in high yield and stereoselectivity and no byproduct formation was observed. Moreover, the catalyst could be recycled. The presence of additional substituents on the steroid backbone showed a significant effect on the 5β-selectivity

The Neuroactive Steroid Pregnanolone Glutamate: Anticonvulsant Effect, Metabolites and Its Effect on Neurosteroid Levels in Developing Rat Brains

: Pregnanolone glutamate (PA-G) is a neuroactive steroid that has been previously demonstrated to be a potent neuroprotective compound in several biological models in vivo. Our in vitro experiments identified PA-G as an inhibitor of N-methyl-D-aspartate receptors and a potentiator of γ-aminobutyric acid receptors (GABAARs). In this study, we addressed the hypothesis that combined GABAAR potentiation and NMDAR antagonism could afford a potent anticonvulsant effect. Our results demonstrated the strong age-related anticonvulsive effect of PA-G in a model of pentylenetetrazol-induced seizures. PA-G significantly decreased seizure severity in 12-day-old animals, but only after the highest dose in 25-day-old animals. Interestingly, the anticonvulsant effect of PA-G differed both qualitatively and quantitatively from that of zuranolone, an investigational neurosteroid acting as a potent positive allosteric modulator of GABAARs. Next, we identified 17-hydroxy-pregnanolone (17-OH-PA) as a major metabolite of PA-G in 12-day-old animals. Finally, the administration of PA-G demonstrated direct modulation of unexpected neurosteroid levels, namely pregnenolone and dehydroepiandrosterone sulfate. These results suggest that compound PA-G might be a pro-drug of 17-OH-PA, a neurosteroid with a promising neuroprotective effect with an unknown mechanism of action that may represent an attractive target for studying perinatal neural diseases

Pitfalls of NMDA Receptor Modulation by Neuroactive Steroids. The Effect of Positive and Negative Modulation of NMDA Receptors in an Animal Model of Schizophrenia

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

C‑3 Steroidal Hemiesters as Inhibitors of 17β-Hydroxysteroid Dehydrogenase Type 10

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 (IC50 = 6.95 ± 0.35 μM) and 23 (IC50 = 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

3β-Isoobeticholic acid efficiently activates the farnesoid X receptor (FXR) due to its epimerization to 3α-epimer by hepatic metabolism

Bile acids (BAs) are important signaling molecules acting via the farnesoid X nuclear receptor (FXR) and the membrane G protein-coupled bile acid receptor 1 (GPBAR1). Besides deconjugation of BAs, the oxidoreductive enzymes of colonic bacteria and hepatocytes enable the conversion of BAs into their epimers or dehydrogenated forms. Obeticholic acid (OCA) is the first-in-class BA-derived FXR agonist approved for the treatment of primary biliary cholangitis. Herein, a library of OCA derivatives, including 7-keto, 6-ethylidene derivatives and 3β-epimers, was synthetized and investigated in terms of interactions with FXR and GPBAR1 in transaction assays and evaluated for FXR target genes expression in human hepatocytes and C57BL/6 mice. The derivatives were further subjected to cell-free analysis employing in silico molecular docking and a TR-FRET assay. The conversion of the 3βhydroxy epimer and its pharmacokinetics in mice were studied using LC-MS. We found that only the 3β-hydroxy epimer of OCA (3β-isoOCA) possesses significant activity to FXR in hepatic cells and mice. However, in a cell-free assay, 3β-isoOCA had about 9-times lower affinity to FXR than did OCA. We observed that 3β-isoOCA readily epimerizes to OCA in hepatocytes and murine liver. This conversion was significantly inhibited by the hydroxy-Δ5-steroid dehydrogenase inhibitor trilostane. In addition, we found that 3,7-dehydroobeticholic acid is a potent GPBAR1 agonist. We conclude that 3β-isoOCA significantly activates FXR due to its epimerization to the more active OCA by hepatic metabolism. Other modifications as well as epimerization on the C3/C7 positions and the introduction of 6-ethylidene in the CDCA scaffold abrogate FXR agonism and alleviate GPBAR1 activation