Search Results (31)

Background and Objectives: Neuroinflammation plays a significant role in liver and neurological disorders via its disruption of neurotransmission, which alters cerebral function, resulting in cognitive and motor impairment, fatigue, anxiety, and depression. A key interaction exists between GABAergic neurotransmission and neuroinflammation, whereby excessive GABA_A receptor activation exacerbates cognitive and behavioural impairment. Golexanolone, a novel GABA_A-receptor-modulating steroid antagonist (GAMSA), primarily attenuates GABAergic potentiation via GABA_A-positive steroid allosteric receptor modulators such as allopregnanolone. This review aims to summarize new evidence showing that golexanolone improves peripheral inflammation, neuroinflammation, and neurological alterations in animal models of different neurological pathologies. We provide an overview of the first clinical trial using this novel compound. Results: In rat models of hyperammonemia and minimal hepatic encephalopathy (MHE), peripheral inflammation induces microglia and astrocyte activation and neuroinflammation, altering GABAergic neurotransmission and resulting in cognitive and motor impairment. Golexanolone’s unique dual action reduces peripheral inflammation and glial activation, thus normalizing neurotransmission and cognitive and motor function. Furthermore, a phase II study in cirrhotic patients with MHE shows that golexanolone is well tolerated and improves cognition. Similarly, in a model of primary biliary cholangitis (PBC) involving bile-duct ligation, peripheral inflammation, neuroinflammation, and altered neurotransmission—associated with fatigue, impaired memory, and locomotor gait and motor incoordination—were reversed by the dual action of golexanolone. In the Parkinson’s disease (PD) rat model induced by neurotoxin 6-OHDA, rats exhibited fatigue, anhedonia, impaired memory, and locomotor gait and motor incoordination, which were associated with microglia and astrocyte activation in the substantia nigra and striatum, in addition to tyrosine hydroxylase (TH) loss. Golexanolone reduces microglia and astrocyte activation, partially reduces TH loss, and improves fatigue, anhedonia, memory, locomotor gait, and motor incoordination. Golexanolone also normalizes elevated levels of α-synuclein. Conclusions: These findings suggest that golexanolone has beneficial therapeutic effects for treating fatigue, depression, motor, and cognitive impairment across diverse neuroinflammatory conditions, including synucleinopathies. Full article

To assess the influence of dietary bile acid (BA) on the phenotype associated with hepatic lipid metabolism and its regulation of lipid homeostasis in gibel carp (Carassius auratus gibelio) under high-fat diet (HFD) conditions, five HFDs were designed using soybean oil (SO) as the single lipid source and supplemented with 0, 200, 400, 600, and 800 mg/kg BA (designated as BA0, BA200, BA400, BA600, and BA800, respectively). Juvenile fish (32.37 ± 0.13 g) were fed five BA-added HFDs (12% SO) for 8 weeks. Considerably lower levels of aspartate transaminase, alanine aminotransferase, low-density lipoprotein, triglyceride, and total cholesterol in the serum were observed in gibel carp fed with HFDs with 400–600 mg/kg BA (p < 0.05). The hepatocytes of the BA400 and BA600 groups were intact without abnormal architecture or histopathological changes, compared to other groups. The presence of most genes related to fatty acid biosynthesis decreased significantly with the addition of 400–600 mg/kg BA (p < 0.05), while the gene expressions of hormone-sensitive lipase, adiponectin receptor 2, and peroxisome proliferator-activated receptor α were variably up-regulated, along with the elevation of dietary BA (p < 0.05). Critical genes involved in bile acid and cholesterol synthesis were obviously down-regulated in gibel carp receiving 600–800 mg/kg dietary BA (p < 0.05), despite the sterol 27-hydroxylase (cyp27a1) gene in the BA800 group (p < 0.05). Moreover, hepatopancreas from the BA0 and BA600 groups were isolated for transcriptome sequencing, identifying 7040 differentially expressed genes (DEGs). The enriched KEGG pathways of DEGs mainly included steroid biosynthesis, protein digestion and absorption, etc. Seven randomly selected DEGs were validated using qRT-PCR and were in agreement with the RNA-seq results. Consequently, the appropriate supplementation of dietary BA for juvenile gibel carp is recommended at doses of 400–600 mg/kg in SO-based HFDs, which could contribute to the amelioration of HFD-induced excessive fat deposition in the hepatopancreas of gibel carp by both inhibiting fatty acid intake, biosynthesis, and steroid production and enhancing lipid decomposition. The findings may elucidate the physiological role of exogenous BA in fish and its underlying mechanism, providing references for the reasonable application of BA in aquafeeds and the prevention of HFD-induced metabolic dysfunction in fish. Full article

Multiple sclerosis (MS) is a long-term disease that causes inflammation and damage to the nervous system. This study evaluated steroidomic alterations related to MS in 57 female MS patients during the follicular phase and 17 during the luteal phase, as well as in age- and phase-matched controls. The data showed that (1) unconjugated and conjugated steroids were strongly linked between the blood and CSF. (2) MS patients have lower levels of unconjugated steroids compared to controls. However, unchanged levels of conjugated steroids suggest a possible increase in steroid sulfotransferase functioning. (3) MS patients show altered levels of steroids linked to 11β-hydroxylase (CYP11B1) function. While direct enzyme activity was not measured, disrupted cortisol biosynthesis—potentially linked to reduced functioning of both CYP11B1 and 17α-hydroxylase/17,20-lyase—is associated with more severe cases of MS. (4) Reduced levels of 5α/β-steroids and protective GABAergic 3α-hydroxy-5α/β-steroids in MS patients might be linked to the pathophysiology of MS. (5) A potential increase in AKR1C3 function in MS could contribute to inflammation, as this enzyme catalyzes the synthesis of both steroids and prostaglandins. However, direct measurements of enzyme activity are needed to confirm this hypothesis. (6) Lower pregnenolone levels in MS patients might weaken neuroprotection, while higher pregnenolone sulfate levels could support cognitive function. (7) Lower levels of protective pregnenolone, DHEA, and androstenediol were associated with worse MS, suggesting these steroids may help shield against the disease. Full article

Background: Aging is associated with the development of various disorders, including postural imbalance, which increases the risk of falls and related health complications. This study examines changes in static postural balance after a 4-week intervention involving appropriate exercise and an optimized daily regimen. Additionally, it explores the relationship between these changes and the steroidome. Methods: The study was conducted on a clinically homogeneous group of 41 females around their sixth decade, diagnosed with anxiety-depressive syndrome and treated with selective serotonin reuptake inhibitors (SSRIs). Postural balance was assessed using the dual-scales method (DLLL-DSM), which estimates postural imbalance by evaluating differences in the lower limb load in the standing position. Correlations between initial DLLL-DSM values, age, BMI, and the baseline levels of nine serum steroids, as well as post-intervention changes in five steroids, were analyzed using multivariate regression (OPLS model). Results: A significant reduction in lower limb load differences (-ΔDLLL-DSM), indicating improved postural balance, was observed. The -ΔDLLL-DSM strongly correlated with initial DLLL-DSM values, age, BMI, initial levels of nine serum steroids, and post-intervention changes in five steroids (R = 0.892, p < 0.001). Furthermore, initial DLLL-DSM values negatively correlated with adrenal androgen androstenediol sulfate and various sulfated 5α/β-reduced androgen metabolites (R = 0.323, p < 0.05), suggesting that the activity of steroid sulfotransferase (SULT2A1) and C17-hydroxylase-C17,20-lyase (CYP17A1) at the lyase step is negatively associated with postural imbalance in elderly females. Conclusions: The findings suggest that even severe postural imbalance can be effectively and relatively rapidly improved through an appropriate exercise-based intervention and an optimized daily regimen, provided that initial adrenal activity is not significantly impaired. Additionally, the identified associations between steroid levels and postural balance provide new insights into the hormonal mechanisms regulating balance control in older individuals. Full article

Multiple sclerosis (MS) is a chronic inflammatory neurodegenerative disease of the central nervous system. The manifestation of MS is related to steroid changes during the menstrual cycle and pregnancy. As data focusing on the effect of anti-MS drug treatment on steroidome are scarce, we evaluated steroidomic changes (79 steroids) in 61 female MS patients of reproductive age 39 (29, 47) years (median with quartiles) after treatment with anti-MS drugs on the GC-MS/MS platform and immunoassays (cortisol and estradiol). The changes were assessed using steroid levels and steroid molar ratios (SMRs) that may reflect the activities of steroidogenic enzymes (SMRs). A repeated measures ANOVA, followed by multiple comparisons and OPLS models, were used for statistical analyses. The anti-MS treatment decreased steroid levels in the follicular phase. Anti-CD20 monoclonal antibodies (mAb), such as ofatumumab and ocrelizumab; inhibitors of the sphingosine-1-phosphate receptor (S1PRI); and IFNβ-1a decreased circulating 17-hydroxy-pregnanes and shifted the CYP17A1 functioning from the hydroxylase- toward the lyase step. Decreased conjugated/unconjugated steroid ratios were found after treatment with anti-MS drugs, especially for glatiramer acetate and anti-CD20 mAb. In the luteal phase, IFN-β1a treatment increased steroidogenesis; both IFN-β1a and ocrelizumab increased AKR1D1, and S1PRI increased SRD5A functioning. Anti-CD20 mAb reduced the functioning of enzymes catalyzing the synthesis of immunomodulatory 7α/β and 16α-hydroxy-androgens, which may affect the severity of MS. The above findings may be important concerning the alterations in bioactive steroids, such as cortisol; active androgens and estrogens; and neuroactive, neuroprotective, and immunomodulatory steroids in terms of optimization of anti-MS treatment. Full article

Steroid hormone imbalance is associated with the pathogenesis of preeclampsia. However, affected enzymes of steroid metabolism and gene and protein expression in serum and placenta have not been elucidated yet. We aimed to investigate steroid hormone profiles and precursor-to-product ratios in preeclamptic women compared to women with healthy pregnancy (controls) to identify potentially affected steroid hormones and their metabolizing enzymes. Also, we aimed to investigate whether the mRNA expression of these enzymes is different between the study groups and whether levels of serum mRNA expression reflect postnatal placental protein expression. Serum levels of 14 steroid hormones were measured at eight time points throughout pregnancy in nine preeclamptic women and 36 controls. Serum mRNA expression of selected steroid-metabolizing enzymes was assessed, and their protein expression was analyzed in additional nine preeclamptic women. Mean levels of sex steroid and corticosteroid hormones were significantly altered in preeclamptic women. Precursor-to-product ratios of 5α-reductase, aromatase and 11β-hydroxysteroid dehydrogenase 1 were significantly increased, those of steroid 17α-hydroxylase, 17β-hydroxysteroid-dehydrogenase, steroid 11β-hydroxylase and 11β-hydroxysteroid dehydrogenase 2 were significantly decreased. Serum mRNA expression and placenta protein expression were comparable between the groups. Results contribute to understanding the heterogeneity of preeclampsia and can thus promote future research in personalized medicine. Full article

Hydroxylation reaction is a significant source of structural diversity in natural products (NPs), playing a crucial role in improving the bioactivity, solubility, and stability of natural product molecules. This review summarizes the latest research progress in the field of natural product hydroxylation, focusing on several key hydroxylases involved in the biosynthesis of NPs, including cytochrome P450 monooxygenases, α-ketoglutarate-dependent hydroxylases, and flavin-dependent monooxygenases. These enzymes achieve selective hydroxylation modification of various NPs, such as terpenoids, flavonoids, and steroids, through different catalytic mechanisms. This review systematically summarizes the recent advances on the hydroxylation of NPs, such as amino acids, steroids, terpenoids, lipids, and phenylpropanoids, demonstrating the potential of synthetic biology strategies in constructing artificial biosynthetic pathways and producing hydroxylated natural product derivatives. Through metabolic engineering, enzyme engineering, genetic engineering, and synthetic biology combined with artificial intelligence-assisted technologies, a series of engineered strains have been successfully constructed for the efficient production of hydroxylated NPs and their derivatives, achieving efficient synthesis of hydroxylated NPs. This has provided new avenues for drug development, functional food, and biomaterial production and has also offered new ideas for the industrial production of these compounds. In the future, integrating artificial synthetic pathway design, enzyme directed evolution, dynamic regulation, and artificial intelligence technology is expected to further expand the application of enzyme-catalyzed hydroxylation reactions in the green synthesis of complex NPs, promoting research on natural product hydroxylation to new heights. Full article

Testosterone holds significant medical and economic importance, with the global market for testosterone replacement therapies valued at approximately USD 1.9 billion in 2023. This hormone is essential for the development and maintenance of male sexual characteristics as well as bone and muscle health. It plays a key role in conditions such as hypogonadism, muscle disorders, and andropause. However, the industrial production of testosterone often involves complex chemical processes that result in low yields, high costs, and environmental damage. Microbial biotransformation of steroids presents an eco-friendly alternative to traditional chemical synthesis. A knockout strain of Aspergillus nidulans deficient in steroid 11α-hydroxylase activity was developed, rendering it incapable of hydroxylating androstenedione, progesterone, and testosterone. In these strains, two newly identified CYP450 enzymes, CYP68L1 from A. nidulans and CYP68L8 from Aspergillus ochraceus, were expressed to confirm their roles as steroid 11α-hydroxylases of androstenedione, progesterone, and testosterone. The availability of these 11α-hydroxylases represents significant progress toward achieving efficient single-step steroid fermentation. Furthermore, the A. nidulans knockout strain serves as an effective model for studying the conversion of androstenedione to testosterone upon the expression of the enzyme 17β-hydroxysteroid dehydrogenase, due to its inability to hydroxylate testosterone. Full article

The metabolic activities of microorganisms to modify the chemical structures of organic compounds are an effective tool for the production of high-value steroidal drugs or active pharmaceutical ingredients (APIs). The integration of biotransformation into the synthesis of APIs can greatly reduce the number of reaction steps and achieve higher process efficiency, thus enabling their greener production. The current research efforts are focused on either the optimization of existing processes or identification of new potentially useful bioconversions. This study aimed to assess the catalytic abilities of the filamentous fungus Fusarium culmorum AM 282 to transform B-nor analogues (5(6→7)abeo compounds) of steroid hormones: androstenedione (AD), dehydroepiandrosterone (DHEA) and its acetate. Our previous studies have demonstrated that this strain is an active hydroxylating catalyst for many steroidal compounds with diverse structures. The results presented in this work showed that the hydroxylation of B-nor steroids occurred with the regio- and stereoselectivity typical of this strain in relation to the corresponding natural hormones of the standard 6:6 A/B series. After the transformations of B-nor-DHEA and its acetate, 15α-hydroxy-B-nor-DHEA was obtained as the sole product of the reaction, while the transformation of the AD analogue resulted in a mixture of its 15α- and 6α-hydroxy derivatives. A detailed analysis of the transformation course indicated that all the obtained hydroxy derivatives could be the result of the activity of the same enzyme. The presented results may provide a basis for research aimed at understanding the molecular nature of cytochrome P-450 monooxygenase from F. culmorum AM 282 with its ability for 15α-hydroxylation of steroidal compounds. An analysis of the pharmacokinetic and pharmacodynamic properties of the obtained metabolites with cheminformatics tools suggests their potential biological activity. Full article

High-dose vitamin D supplementation is common in the general population, but unsupervised high-dose supplementation in vitamin D-replete individuals poses a risk of severe toxicity. Susceptibility to vitamin D toxicity shows a significant inter-individual variability that may in part be explained by genetic predispositions (i.e., CYP24A1 polymorphism). The classic manifestation of vitamin D toxicity is hypercalcemia, which may be refractory to conventional therapy. Its causes include the endogenous overaction of 1α-hydroxylase, monogenic alterations affecting vitamin D metabolizing enzymes and exogenous vitamin D intoxication. In this manuscript, we include a literature review of potential pharmacological interventions targeting calcitriol metabolism to treat vitamin D intoxication and present a case of severe, exogenous vitamin D intoxication responding to systemic corticosteroids after the failure of conventional therapy. Systemic glucocorticoids alleviate acute hypercalcemia by inhibiting enteric calcium absorption and increasing the degradation of vitamin D metabolites but may cause adverse effects. Inhibitors of 1α-hydroxylase (keto/fluconazole) and inducers of CYP3A4 (rifampicin) may be considered steroid-sparing alternatives for the treatment of vitamin D intoxication. Full article

Steroids with hydroxylation at C14 are drawing increased attention because of their diverse biological activities and applications. P-450_lun from Cochliobolus lunatus is the first fungal cytochrome P450 reported to have 14α-hydroxylase activity. Studies have shown that P-450_lun catalyzes the hydroxylation of progesterone (PROG) at C14α with low regiospecificity and activity. To improve its regiospecificity and activity for PROG, truncated forms of P-450_lun and its cognate redox partner CPR_lun were functionally co-expressed in Escherichia coli. Then, a semi-rational protein engineering approach was applied to P-450_lun, resulting in a double-site mutant E109A/F297W with enhanced 14α-position selectivity for PROG compared with the wild-type P-450_lun (97% vs. 28%). Protein structure analysis revealed that the F297W substitution can hinder the binding pose for 11β-hydroxylation product formation. Finally, whole-cell catalysis was optimized, and the final titer of 14α-OH-PROG reached 16.0 mg/L. This is the first report where a fungal 14α-hydroxylase was functionally expressed in Escherichia coli. The steroid hydroxylation system obtained in this study can serve as a basis for the synthesis of 14α-hydroxylated PROG and the rapid evolution of eukaryotic cytochrome P-450_lun. Full article

The hydroxylation of steroids in the C7β position is one of the rare reactions that allow the production of value-added precursors in the synthesis of ursodeoxycholic acid and other pharmaceuticals. Recently, we discovered this activity in the ascomycete Curvularia sp. VKM F-3040. In this study, the novel gene of 7-hydroxylase (P450_cur) was identified as being heterologously expressed and functionally characterized in Pichia pastoris. Transcriptome data mining and differential expression analysis revealed that 12 putative genes in Curvularia sp. mycelia significantly increased their expression in response to dehydroepiandrosterone (DHEA). The transcriptional level of the most up-regulated cytochrome P450_cur gene was increased more than 300-fold. A two-gene construct with a candidate P450_cur gene and the gene of its natural redox partner, NADPH-cytochrome P450 reductase (CPR), which is interconnected by a T2A element, was created. Using this construct, recombinant P. pastoris strains co-expressing fungal P450_cur and CPR genes were obtained. The functional activity of the recombinant P450_cur was studied in vivo during the bioconversion of androstane steroids. The fungal 7-monooxygenase predominantly catalyzed the 7β-hydroxylation of androstadienedione (ADD), DHEA, and androstenediol, whereas 1-dehydrotestosterone was hydroxylated by P450_cur mainly at the C7-Hα position. To our knowledge, this is the first report of a recombinant yeast capable of catalyzing the 7α/β-hydroxylation of ADD and DHEA. Full article

Microbial 1(2)-dehydrogenation of 3-ketosteroids is an important basis for the production of many steroid pharmaceuticals and synthons. When using the wild-type strains for whole cell catalysis, the undesirable reduction of the 20-carbonyl group, or 1(2)-hydrogenation, was observed. In this work, the recombinant strains of Mycolicibacterium neoaurum and Mycolicibacterium smegmatis were constructed with blocked endogenous activity of 3-ketosteroid-9α-hydroxylase, 3-ketosteroid-1(2)-dehydrogenase (3-KSD), and expressing 3-KSD encoded by the gene KR76_27125 (kstD2_NS) from Nocardioides simplex VKM Ac-2033D. The in vivo activity of the obtained recombinant strains against phytosterol, 6α-methyl-hydrocortisone, and hydrocortisone was studied. When using M. smegmatis as the host strain, the 1(2)-dehydrogenation activity of the constructed recombinant cells towards hydrocortisone was noticeably higher compared to those on the platform of M. neoaurum. A comparison of the strengths of inducible acetamidase and constitutive hsp60 promoters in M. smegmatis provided comparable results. Hydrocortisone biotransformation by M. smegmatis BD/pMhsp_k expressing kstD2_NS resulted in 95.4% prednisolone yield, and the selectivity preferred that for N. simplex. Mycolicibacteria showed increased hydrocortisone degradation at 35 °C compared to 30 °C. The presence of endogenous steroid catabolism in Mycolicibacterium hosts does not seem to confer an advantage for the functioning of KstD2_NS. The results allow for the evaluation of the prospects for the development of simple technological methods for the selective 1(2)-dehydrogenation of 3-ketosteroids by growing bacterial cells. Full article

This study reports on the synthesis and evaluation of novel compounds replacing the nitrogen-containing heterocyclic ring on the chemical backbone structure of cytochrome P450 17α-hydroxylase/12,20-lyase (CYP17A1) inhibitors with a phenyl bearing a sulfur-based substituent. Initial screening revealed compounds with marked inhibition of CYP17A1 activity. The selectivity of compounds was thereafter determined against cytochrome P450 21-hydroxylase, cytochrome P450 3A4, and cytochrome P450 oxidoreductase. Additionally, the compounds showed weak inhibitory activity against aldo-keto reductase 1C3 (AKR1C3). The compounds’ impact on steroid hormone levels was also assessed, with some notable modulatory effects observed. This work paves the way for developing more potent dual inhibitors specifically targeting CYP17A1 and AKR1C3. Full article

Mycobacterium neoaurum DSM 1381 originated from Mycobacterium neoaurum ATCC 25790 by mutagenesis screening is a strain of degrading phytosterols and accumulating important C22 steroid intermediates, including 22-hydroxy-23, 24-bisnorchola-4-en-3-one (4-HP) and 22-hydroxy-23, 24-bisnorchola-1,4-dien-3-one (HPD). However, the metabolic mechanism of these C22 products in M. neoaurum DSM 1381 remains unknown. Therefore, the whole-genome sequencing and comparative genomics analysis of M. neoaurum DSM 1381 and its parent strain M. neoaurum ATCC 25790 were performed to figure out the mechanism. As a result, 28 nonsynonymous single nucleotide variants (SNVs), 17 coding region Indels, and eight non-coding region Indels were found between the genomes of the two strains. When the wild-type 3-ketosteroid-9α-hydroxylase subunit A1 (KshA1) and β-hydroxyacyl-CoA dehydrogenase (Hsd4A) were overexpressed in M. neoaurum DSM 1381, the steroids were transformed into the 4-androstene-3, 17- dione (AD) and 1,4-androstadiene-3,17-dione (ADD) instead of C22 intermediates. This result indicated that 173N of KshA1 and 171K of Hsd4A are indispensable to maintaining their activity, respectively. Amino acid sequence alignment analysis show that both N173D in KshA1 and K171E in Hsd4A are conservative sites. The 3D models of these two enzymes were predicted by SWISS-MODEL and AlphaFold2 to understand the inactivation of the two key enzymes. These results indicate that K171E in Hsd4A may destroy the inaction between the NAD+ with the NH3+ and N173D in KshA1 and may disrupt the binding of the catalytic domain to the substrate. A C22 steroid intermediates–accumulating mechanism in M. neoaurum DSM 1381 is proposed, in which the K171E in Hsd4A leads to the enzyme’s inactivation, which intercepts the C19 sub-pathways and accelerates the C22 sub-pathways, and the N173D in KshA1 leads to the enzyme’s inactivation, which blocks the degradation of C22 intermediates. In conclusion, this study explained the reasons for the accumulation of C22 intermediates in M. neoaurum DSM 1381 by exploring the inactivation mechanism of the two key enzymes. Full article