C19 Steroids Research Articles

6611 ACTH deficiency due to TBX19 mutation mimics complete adrenal insufficiency in a newborn

Abstract Disclosure: T. Reinehr: None. P.M. Holterhus: None. S.A. Wudy: None. A. Richter-Unruh: None. C. Spratte: None. C. Roll: None. Isolated ACTH deficiency (IAD) is a rare cause of adrenocortical insufficiency. T-box transcription factor 19 (TBX19) mutations are responsible for more than 60% of neonatal cases of IAD. To the best of our knowledge, we present for the first time a newborn with ACTH deficiency due to a TBX19 mutation with almost steroid-free plasma and urine: At day 2 of life, the female newborn (birth weight 3190g, length 52 cm, 40+1 gestational age) was transferred to our neonatal intensive care unit for severe hypoglycemia (12 mg/dl), which responded to intravenous glucose infusion. Surprisingly, the sodium serum concentrations decreased from 140 mmol/l at day 2 of life to 126 mmol/l at day 7 of life. There were no clinical signs of adrenal insufficiency such as vomiting or hypotonia. Treatment with oral sodium-chloride 5.85% was started. Endocrine diagnostic work-up performed at day 8 of life demonstrated normal values of fT4, HGH and IGFBP-3 ruling out pituitary involvement of these axes, while ACTH serum concentrations were markedly decreased (1.5 pg/ml). Liquid- chromatography-mass spectrometry (LC-MS/MS) revealed nearly absent glucocorticoids (17-hydroxyprogesterone <0.12 nmol/l, 11-deoxycortisol <0.08 nmol/l, cortisol <4.2 nmol/l, and cortisone 1.3 nmol/l (norm value [nv]: 17-138 nmol/l)). Androgens were undetectable (testosterone <0.02 nmol/l, androstenedione <0.08 nmol/l). Mineralocorticoids were severely reduced (11-desoxycorticosterone 0.1 nmol/l (nv: 0.2-1.0 nmol/l), corticosterone 0.6 nmol/l (nv: 1-24 nmol/l), aldosterone 0.1 nmol/l (nv: 0.8-2.3 nmol/l)). In urine spot, gas chromatography-mass spectrometry (GC-MS) demonstrated subnormal excretion of C19 and C21 steroids including fetal zone steroids. Serum and urine measurements were interpreted by the labs as a typical finding similar to StAR defect. The genetic analyses revealed a normal female karyotype (46,XX) and ruled out CYP11A1 and StAR, mutations but demonstrated a homozygous missense mutation in TBX19 (c.172A>G, p.(Thr58Ala)). After 3 months of life under treatment with hydrocortisone (10mg/m² divided in 3 doses) and fludrocortisone (0.05 mg divided in 2 doses), cortisol was normal (320 nmol/l) and DHEAS was undetectable (<3 nmol/l). While androgens were in normal range (testosterone 2.6 nmol/l, androstenedione 0.28 nmol/l), 11-deoxycorticosterone was normal (0.74 nmol/l), corticosterone was undetectable (<0.5 nmol/l) and aldosterone was decreased (0.12 nmol/l nv: 0.8-1.2 nmol/l). At this time point, ACTH was <4 pg/ml, renin 1.6 mIU/l, LH 0.7 mIU/l and FSH 24.5 mIU/ml. This case demonstrated that isolated ACTH deficiency should be considered as a potential differential diagnosis in complete adrenal steroid insufficiency in newborns. Our data suggest that ACTH is not only necessary for cortisol biosynthesis but also for sufficient mineralocorticoid and androgen production by the adrenals in neonates. Presentation: 6/3/2024

9265 Racial/Ethnical Differences in Classic and 11-Oxygenated Androgens

Abstract Disclosure: C. Lee: None. R.J. Auchus: None. J. McDonald: None. J. Rege: None. W.E. Rainey: None. A.F. Turcu: None. Background: Malignancies modulated by sex-steroids, such as prostate and breast cancer, as well as disorders of androgen excess, like polycystic ovary syndrome, display prevalence and/or phenotype differences across races/ethnicities. Such differences have been attributed, in part, to testosterone (T). A set of adrenal-derived 11-oxygenated C19 steroids (11-oxyandrogens) has emerged as relevant to several androgen-dependent pathologies. Classic and 11-oxyandrogens are impacted differently by age and adiposity. Data on race and ethnic variations in 11-oxyandrogen concentrations are lacking. Setting and Participants: Participants in the population-based, multiethnic cohort of the Dallas Heart Study, phase 2. Measurements: T, androstenedione (A4), 11β-hydroxyandrostenedione (11OHA4), 11β-ketoandrostenedione (11KA4), 11-hydroxytosterone (11OHT), and 11-ketotestosterone (11KT) were quantified simultaneously in peripheral blood using liquid chromatography-tandem mass spectrometry. Steroids were compared across races stratified by gender, and adjusted for age and BMI, using linear mixed models in SAS (NC, USA). Results: Of the 1966 participants included, 1137 (57.8%) were women, 49% were non-Hispanic Black, 35% non-Hispanic White, and 14% Hispanic. The mean age of study participants was 51.3 +/- 10.3 years, and mean BMI was 31.0 +/- 7.1 kg/m2. Compared to White individuals of similar age and BMI, 11KT concentrations were higher in both Black women (β = 3.5 +/- 1.2, p = 0.004), and Black men (β = 5.8 +/- 1.9, p = 0.002). The immediate precursor of 11KT, 11KA4, displayed higher concentrations in Black vs. White men (β = 2.9+/- 0.7, p < 0.0001), with similar trends in women (β = 3.0 +/- 1.6, p = 0.056). The weak androgen 11OHT was higher in Black vs. White women (β = 3.0 +/- 0.91, p = 0.0009), but did not differ across races in men. Compared to White individuals of the corresponding gender, age- and BMI-adjusted 11OHA4 concentrations were higher in Hispanic women (β = 10.8 +/- 5.4, p = 0.043), and in Black men (β = 20.9 +/- 4.8, p < 0.0001). The classic androgen T was higher in Black women and men (β = 3.7 +/- 1.4 and β = 41.5 +/- 14.7, respectively, p < 0.01 for both), as well as in Hispanic men (β = 46.6 +/- 21.8, p = 0.03) compared to their White counterparts with similar age and BMI. A4 concentrations were similar across races. Conclusions: The bioactive androgens 11KT and T were higher in Black women and men compared to White individuals with similar age and BMI. Conversely, in Hispanic men, only T was higher compared to White men. In addition to T, 11KT might also contribute to disproportionately high prevalence of androgen-related pathologies in Black individuals. Acknowledgements: This work was supported by grant R01AG08051601 from the National Institute of Aging, and, in part, by grant UL1TR001105 from the National Center for Advancing Translational Science, National Institutes of Health. Presentation: 6/1/2024

Electronic senses and UPLC-Q-TOF/MS combined with chemometrics analyses of Cynanchum species (Baishouwu).

Baishouwu, derived from Cynanchum auriculatum (CA) Royle ex Wight, Cynanchum bungei (CB) Decne., and Cynanchum wilfordii (CW) (Maxim.) Hemsl., is a valuable traditional Chinese medicine. CA is also recognized as a new food resource by China's National Health Commission. Given the considerable variations in flavor and chemical composition among these species and lack of their qualitative assessments, accurately differentiating between the species constituting Baishouwu is essential. To develop a method combining electronic tongue (E-tongue), electronic nose (E-nose), and ultra-performance liquid chromatography-quadrupole-time of flight/mass spectrometry (UPLC-Q-TOF/MS) to differentiate between Baishouwu samples. Fifteen batches of Baishouwu samples were analyzed using E-tongue, E-nose, and UPLC-Q-TOF/MS. Flavor differences and key differential metabolites were determined through principal component analysis and orthogonal partial least squares discriminant analysis. E-tongue results revealed umami, sweetness, and richness as the predominant flavors of Baishouwu, with CA having the highest umami response, CW exhibiting the highest bitterness, and CB the highest sweetness. E-nose sensors showed consistent responses across species, with variations in signal strength; W1W and W2W sensors showed the highest response values. A total of 158 and 41 characteristic variables in the positive and negative ion modes, respectively, were selected as candidate differential metabolites, of which 29 and 14 were confirmed through database comparison. Eight critical differential metabolites, including C21 steroids and acetophenone compounds, were identified. This study presents a strategy for differentiating among the species constituting Baishouwu, providing a basis for broader application and establishing quality standards for these medicinal compounds.

Design, Synthesis, Anti-TMV Activity, and Structure-Activity Relationships of Seco-pregnane C21 Steroids and Their Derivatives.

seco-pregnane C21 steroids exhibit high antiviral activity against the tobacco mosaic virus (TMV). However, the structural modification of seco-pregnane C21 steroids and the structure-activity relationship (SAR) of the modified compounds remain unevaluated. Hence, the present study investigated how variations in the original skeletons of natural seco-pregnane C21 steroids affect their antiviral activity. A series of glaucogenin C and A derivatives were designed and synthesized for the first time, and their anti-TMV activity was evaluated. Bioassay results showed that most of the newly designed derivatives exhibited good to excellent antiviral activity; among these derivatives, 5g, 5j, and 5l with higher antiviral activity than that of ningnanmycin emerged as new antiviral candidates. Reverse transcription-polymerase chain reaction and Western blotting assay revealed reduced levels of TMV coat protein (TMV-CP) gene transcription and TMV-CP protein expression, which confirmed the antiviral activity of these derivatives. These compounds also downregulated the expression of NtHsp70-1 and NtHsp70-061. Computational simulations indicated that 5l displayed strong van der Waals energy and electrostatic with the TMV coat protein, affording a lower binding energy (ΔGbind = -56.2 kcal/mol) compared with Ribavirin (ΔGbind = -47.6 kcal/mol). The SAR of these compounds was also evaluated, which demonstrated for the first time that substitutions at C-3 and double bonds of C-5/C-6 and C-13/C-18 are crucial for maintaining high anti-TMV activity.

Association between Disgust Sensitivity during Pregnancy and Endogenous Steroids: A Longitudinal Study.

The emotion of disgust protects individuals against pathogens, and it has been found to be elevated during pregnancy. Physiological mechanisms discussed in relation to these changes include immune markers and progesterone levels. This study aimed to assess the association between steroids and disgust sensitivity in pregnancy. Using a prospective longitudinal design, we analyzed blood serum steroid concentrations and measured disgust sensitivity via text-based questionnaires in a sample of 179 pregnant women during their first and third trimesters. We found positive correlations between disgust sensitivity and the levels of C19 steroids (including testosterone) and its precursors in the Δ5 pathway (androstenediol, DHEA, and their sulfates) and the Δ4 pathway (androstenedione). Additionally, positive correlations were observed with 5α/β-reduced C19 steroid metabolites in both trimesters. In the first trimester, disgust sensitivity was positively associated with 17-hydroxypregnanolone and with some estrogens. In the third trimester, positive associations were observed with cortisol and immunoprotective Δ5 C19 7α/β-hydroxy-steroids. Our findings show that disgust sensitivity is positively correlated with immunomodulatory steroids, and in the third trimester, with steroids which may be related to potential maternal-anxiety-related symptoms. This study highlights the complex relationship between hormonal changes and disgust sensitivity during pregnancy.

CBC-1 as a Cynanbungeigenin C derivative inhibits the growth of colorectal cancer through targeting Hedgehog pathway component GLI 1

Colorectal cancer (CRC) is one of the most common gastrointestinal cancers that results in death in worldwide. The Hedgehog (HH) signalling pathway regulates the initiation and progression of CRC. Inhibiting the HH pathway has been presented as a potential treatment strategy in recent years. Cynanbungeigenin C (CBC) is a new type of C21 steroid that has been previously reported for the treatment of medulloblastoma. However, its further investigation was limited by its poor water solubility. In this study, six new CBC derivatives were synthesized through the structural modification of CBC, and four of them showed better water solubility than CBC. Moreover, their antiproliferative activities on CRC were evaluated. It was found that CBC-1 presented the best inhibitory effect on three types of CRC cell lines, and this effect was superior to that of CBC. Mechanistically, CBC-1 inhibited the proliferation of CRC cells through regulation of mRNA and proteins of the HH pathway according to qRT–PCR and Western blotting analysis. Furthermore, Cellular Thermal Shift Assay results indicated that CBC-1 regulated this signalling pathway by targeting glioma‑associated oncogene (GLI 1).In addition, cell apoptosis was induced increasingly by transfection with GLI 1 siRNA or treatment with CBC-1 to downregulate GLI 1. Last, the in vivo results demonstrated that CBC-1 significantly reduced tumour size and downregulated GLI 1 in CRC. Therefore, this study suggests that CBC-1, a new GLI 1 inhibitor derived from natural products, may be developed as a potential antitumour candidate for CRC treatment.

Efficient Production of 9,22-Dihydroxy-23,24-bisnorchol-4-ene-3-one from Phytosterols by Modifying Multiple Genes in Mycobacterium fortuitum.

C19 steroids and C22 steroids are vital intermediates for the synthesis of steroid drugs. Compared with C19 steroids, C22 steroids are more suitable for synthesizing progesterone and adrenocortical hormones, albeit less developed. 9,22-dihydroxy-23,24-bisnorchol-4-ene-3-one(9-OHBA), due to its substituents at positions C-9 and C-22, is a beneficial and innovative steroid derivative for synthesizing corticosteroids. We focused on the C22 pathway in Mycobacterium fortuitum ATCC 35855, aiming to develop a productive strain that produces 9-OHBA. We used a mutant strain, MFΔkstD, that knocked out kstds from Mycobacterium fortuitum ATCC 35855 named MFKD in this study as the original strain. Hsd4A and FadA5 are key enzymes in controlling the C19 metabolic pathway of steroids in Mycobacterium fortuitum ATCC 35855. After knocking out hsd4A, MFKDΔhsd4A accumulated 81.47% 9-OHBA compared with 4.13% 9-OHBA in the strain MFKD. The double mutant MFKDΔhsd4AΔfadA5 further improved the selectivity of 9-OHBA to 95.13%, and 9α-hydroxy-4-androstenedione (9-OHAD) decreased to 0.90% from 4.19%. In the end, we obtained 6.81 g/L 9-OHBA from 10 g/L phytosterols with a molar yield of 80.33%, which showed the best performance compared with formerly reported strains.

Microbiota modulates the steroid response to acute immune stress in male mice.

Microbiota plays a role in shaping the HPA-axis response to psychological stressors. To examine the role of microbiota in response to acute immune stressor, we stimulated the adaptive immune system by anti-CD3 antibody injection and investigated the expression of adrenal steroidogenic enzymes and profiling of plasma corticosteroids and their metabolites in specific pathogen-free (SPF) and germ-free (GF) mice. Using UHPLC-MS/MS, we showed that 4 hours after immune challenge the plasma levels of pregnenolone, progesterone, 11-deoxycorticosterone, corticosterone (CORT), 11-dehydroCORT and their 3α/β-, 5α-, and 20α-reduced metabolites were increased in SPF mice, but in their GF counterparts, only CORT was increased. Neither immune stress nor microbiota changed the mRNA and protein levels of enzymes of adrenal steroidogenesis. In contrast, immune stress resulted in downregulated expression of steroidogenic genes (Star, Cyp11a1, Hsd3b1, Hsd3b6) and upregulated expression of genes of the 3α-hydroxysteroid oxidoreductase pathway (Akr1c21, Dhrs9) in the testes of SPF mice. In the liver, immune stress downregulated the expression of genes encoding enzymes with 3β-hydroxysteroid dehydrogenase (HSD) (Hsd3b2, Hsd3b3, Hsd3b4, Hsd3b5), 3α-HSD (Akr1c14), 20α-HSD (Akr1c6, Hsd17b1, Hsd17b2) and 5α-reductase (Srd5a1) activities, except for Dhrs9, which was upregulated. In the colon, microbiota downregulated Cyp11a1 and modulated the response of Hsd11b1 and Hsd11b2 expression to immune stress. These data underline the role of microbiota in shaping the response to immune stressor. Microbiota modulates the stress-induced increase in C21 steroids, including those that are neuroactive that could play a role in alteration of HPA axis response to stress in GF animals.

C11-hydroxy and C11-oxo C19 and C21 Steroids: Pre-Receptor Regulation and Interaction with Androgen and Progesterone Steroid Receptors.

C11-oxy C19 and C11-oxy C21 steroids have been identified as novel steroids but their function remains unclear. This study aimed to investigate the pre-receptor regulation of C11-oxy steroids by 11β-hydroxysteroid dehydrogenase (11βHSD) interconversion and potential agonist and antagonist activity associated with the androgen (AR) and progesterone receptors (PRA and PRB). Steroid conversions were investigated in transiently transfected HEK293 cells expressing 11βHSD1 and 11βHSD2, while CV1 cells were utilised for agonist and antagonist assays. The conversion of C11-hydroxy steroids to C11-oxo steroids by 11βHSD2 occurred more readily than the reverse reaction catalysed by 11βHSD1, while the interconversion of C11-oxy C19 steroids was more efficient than C11-oxy C21 steroids. Furthermore, 11-ketodihydrotestosterone (11KDHT), 11-ketotestosterone (11KT) and 11β-hydroxydihydrotestosterone (11OHDHT) were AR agonists, while only progestogens, 11β-hydroxyprogesterone (11βOHP4), 11β-hydroxydihydroprogesterone (11βOHDHP4), 11α-hydroxyprogesterone (11αOHP4), 11α-hydroxydihydroprogesterone (11αOHDHP4), 11-ketoprogesterone (11KP4), 5α-pregnan-17α-diol-3,11,20-trione (11KPdione) and 21-deoxycortisone (21dE) exhibited antagonist activity. C11-hydroxy C21 steroids, 11βOHP4, 11βOHDHP4 and 11αOHP4 exhibited PRA and PRB agonistic activity, while only C11-oxo steroids, 11KP4 and 11-ketoandrostanediol (11K3αdiol) demonstrated PRB agonism. While no steroids antagonised the PRA, 11OHA4, 11β-hydroxytestosterone (11OHT), 11KT and 11KDHT exhibited PRB antagonism. The regulatory role of 11βHSD isozymes impacting receptor activation is clear-C11-oxo androgens exhibit AR agonist activity; only C11-hydroxy progestogens exhibit PRA and PRB agonist activity. Regulation by the downstream metabolites of active C11-oxy steroids at the receptor level is apparent-C11-hydroxy and C11-oxo metabolites antagonize the AR and PRB, progestogens the former, androgens the latter. The findings highlight the intricate interplay between receptors and active as well as "inactive" C11-oxy steroids, suggesting novel regulatory tiers.

Identification of maturation-inducing steroid in sturgeons via comprehensive analyses of steroids produced during oocyte maturation

Although 17α, 20β-dihydroxy-4-pregnen-3-one (DHP) and 17α, 20β, 21-trihydroxy-4-pregnen-3-one (20β-S) have been identified as maturation-inducing steroids (MIS) in several teleosts, to date, no MISs have been identified in sturgeons. As it remains possible that an unidentified steroid is an MIS in sturgeons, this study aimed to identify a sturgeon MIS via comprehensive analyses and maturation-inducing (MI) assay of C21 steroids. In vivo and in vitro comprehensive analyses of C21 steroids revealed that serum DHP concentrations were rapidly elevated in the oocyte maturation phase and the DHP production level was notably high among C21 steroids. MI assay indicated that the MI activity of DHP, 17α-hydroxyprogesterone (17OHP), a precursor of DHP, 17α, 20α-dihydroxy-4-pregnen-3-one (αDHP), and 20β-S was high among C21 steroids, but the MI activity of these steroids were similar. In the C21 steroids produced in ovarian follicles during oocyte maturation, 17OHP, αDHP, and unidentified compounds had a low production level, and 20β-S was suggested to be metabolized from DHP after oocyte maturation. Against this background, this study concluded that DHP is a steroid that possesses strong MI activity and is highly produced during oocyte maturation. Although this study could not identify an MIS in sturgeons by fractionation of plasma and subsequent bio assay, it was suggested that DHP is a major MIS in sturgeons.

Four new pregnane C21 steroids isolated from the roots of Cynanchum auriculatum

Four new steroids cynansteroid G-I (1-3) and cynansteroid K (4), a new natural product 5,6-deacidizingcaudatin (5), and a known compound glycocaudatin (6), were isolated from the roots of Cynanchum auriculatum. The structures of new compounds were identified by comprehensive spectroscopic analyses, including NMR, HRESI-MS, ECD, UV, and IR spectral data. The cytotoxic activities of all the isolates against two human tumour cell lines (COLO-205 and BGC-823) were screened, unfortunately, which were weaker than positive control.

OR22-02 Sex Differences In 11-Oxyandrogen Correlations With Adipose Tissue Compartments

Abstract Disclosure: A.F. Turcu: None. C. Lee: None. R.J. Auchus: None. Background: Adrenal-derived 11-oxygenated C19 steroids (11-oxyandrogens) are mediators of adrenarche and several disorders of androgen excess. 11β-hydroxyandrostenedione (11OHA4), a major adrenal product, is converted to the bioactive androgen 11-ketotestosterone (11KT) via 11β-hydroxysteroid dehydrogenase type 2 (HSD11B2) and aldo-keto reductase 1C3 (AKR1C3). The adipose tissue expresses both AKR1C3 and the type 1 isoenzyme HSD11B1, which participate in modulating peripheral androgen production. The impact of adipose tissue compartments on circulating 11-oxyandrogens in men and women is not well understood. Setting and Participants: A subset of participants in the population-based, multiethnic cohort of the Dallas Heart Study, phase 2 were included. Measurements: All participants underwent subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) measurements by abdominal magnetic resonance imaging (MRI). Testosterone (T), androstenedione (A4), and four 11-oxyandrogens were quantified simultaneously in peripheral blood using liquid chromatography-tandem mass spectrometry. Associations between adipose tissue compartments and androgens were assessed by Pearson correlation tests, stratified by sex. Results: Of the 498 participants included, 282 (56.6%) were women, 49% were Black, 32% White, 18% Hispanic. The mean age of study participants was 54.3 +/- 10.7 years, and mean BMI was 30.9 +/- 6.9 kg/m2. There was no difference in age and race distribution between men and women. 11OHA4, 11β-hydroxytestosterone (11OHT), and 11KT correlated positively with BMI in women (R2 = 0.15-0.18, p<0.01) but not in men. Conversely, T correlated negatively with BMI only in men (R2 = -0.26, p<.0001). 11OHA4 and 11OHT correlated directly with both SAT and VAT in women (R2 = 0.14-0.18, p<0.05), and a similar correlation was observed between 11OHT and VAT in men. Conversely, T and A4 displayed an inverse correlation with SAT and/or VAT in both sexes. 11KT and 11-ketoandrostenedione (11KA4) displayed no association with adipose tissue compartments. Conclusions: 11OHA4 and 11OHT, but not their respective 11-keto metabolites correlate with all adipose tissue compartments in women, while T correlates inversely with adiposity in men, particularly with SAT. Sexual dimorphism in adipose tissue and impact on androgen modulation might contribute to sex differences in cardiometabolic morbidity. Presentation: Saturday, June 17, 2023

Anti-cervical cancer mechanism of bioactive compounds from Alangium platanifolium based on the ‘compound-target-disease’ network

In this study, we analyzed the chemical compositions of Alangium platanifolium (Sieb. et Zucc.) Harms (AP) using ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) non-targeted plant metabolomics integration MolNetEnhancer strategy. A total of 75 compounds, including flavonoids, alkaloids, terpenes, C21 steroids, among others, were identified by comparing accurate mass-to-charge ratios, MS2 cleavage fragments, retention times, and MolNetenhancer-integrated analytical data, and the cleavage rules of the characteristic compounds were analyzed. A total of 125 potential cervical cancer (CC) therapeutic targets were obtained through Gene Expression Omnibus (GEO) data mining, differential analysis, and database screening. Hub targets were obtained by constructing protein-protein interaction (PPI) networks and CytoNCA topology analysis, including SRC, STAT3, TP53, PIK3R1, MAPK3, and PIK3CA. According to Gene ontology (GO) analysis, AP was primarily against CC by influencing gland development, oxidative stress processes, serine/threonine kinase, and tyrosine kinase activity. Enrichment analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) indicated that the PI3K/AKT and MAPK signaling pathways play a crucial role in AP treatment for CC. The compound-target-pathway (C-T-P) network revealed that quercetin, methylprednisolone, and caudatin may play key roles in the treatment of CC. The results of molecular docking revealed that the core compound could bind significantly to the core target. In this study, the compounds in AP were systematically analyzed qualitatively, and the core components, core targets, and mechanisms of action of AP in the treatment of CC were screened through a combination of network pharmacology tools. Providing a scientific reference for the therapeutic material basis and quality control of AP.

Steroid sulfatase inhibitors and sulfated C19 steroids for proteotoxicity-related diseases: a patent spotlight.

Aging and proteotoxicity go hand in hand. Inhibiting proteotoxicity has been proposed to extend lifespan. This invention describes a new strategy to limit proteotoxicity and to extend the lifespan. Loss of function of sul-2, the Caenorhabditis elegans steroid sulfatase, elevates the pool of sulfated steroid hormones, increases longevityand ameliorates protein aggregation diseases. The present invention provides a group of molecules for use in the prevention of aging-associated proteotoxicity caused by proteinaggregation diseases and/or to increase the lifespan of a eukaryotic organism. These molecules are either steroid sulfatase inhibitors or sulfated C19 steroids, both of which reproduce the phenotype of sul-2 mutants. One particular representative example is STX-64. Potential applications of the claims have been demonstrated in animal models of Parkinson's disease, Huntington's diseaseand Alzheimer's disease.

C21 steroids from the roots of Marsdenia tenacissima

A phytochemical investigation of the dichloromethane soluble fraction of the ethanolic extract obtained from the roots of Marsdenia tenacissima led to the discovery of the sixteen undescribed pregnane C21 steroids (1–16) and isolation of eleven known C21 steroidal analogues (17–27). Their chemical structures were elucidated by one- and two-dimensional nuclear magnetic resonance spectroscopy and, high resolution–electrospray ionization mass spectrometry and their absolute configurations were determined using electronic circular dichroism or single-crystal X-ray diffraction. The in vitro anti-proliferative effects of 1–16 were evaluated against HepG2 (human hepatocellular cancer), A549 (lung cancer), and MCF-7 (human breast cancer) cell lines. Even though some of them showed moderate cytotoxic activities, marsectohexol derivative 12 exhibited significant cytotoxicity against A549 cells with an IC50 value of 5.2 μM.

Screening of anti-functional dyspepsia compounds in Cynanchum auriculatum: A spectrum-effect relationship analysis, and ATP-binding cassette transporters inhibitor evaluation

Ethnopharmacological relevanceFunctional dyspepsia (FD) is a disorder caused by abnormal gut–brain axis regulation and is highly prevalent in China. Cynanchum auriculatum (CA) is often used to treat FD in the ethnic minority areas of Guizhou. Although several CA-based products are currently available in the market, it is unclear which components of CA are efficacious and what their oral absorption mechanism is. Aim of the studyThis study aimed to screen anti-FD components of CA based on the spectrum-effect relationship. In addition, the study evaluated the intestinal absorption mechanism of these components using transporter inhibitors. Materials and methodsThe fingerprinting of compounds from CA extract and plasma after oral administration was conducted using ultra-high-performance liquid chromatography quadrupole-time-of-flight tandem mass spectrometry (UHPLC-Q-TOF-MS). The intestinal contractile parameters were then measured in vitro using the BL-420F Biofunctional Experiment System. Multivariate statistical analysis of the result of spectrum-effect relationship assessment was used to elucidate the correlation between prominent peaks of CA-containing plasma and intestinal contractile activity. The effect of ATP-binding cassette (ABC) transporter inhibitors, such as the P-gp inhibitor verapamil, the MRR inhibitor indomethacin, and the BCRP inhibitor Ko143, on the directional transport of the predicted active ingredients was assessed in vivo. ResultsTwenty chromatographic peaks were identified in the CA extract. Of these, three were C21 steroids, four were organic acids, and one was a coumarin, and acetophenone by comparing with reference compounds. Additionally, it is discovered that there are totally 39 migratory components in CA-containing plasma, which was found to significantly promote the contractility of the isolated duodenum. Moreover, multivariate analysis of the spectrum-effect relationship demonstrated that 16 characteristic peaks (3, 6, 8, 10, 11, 13, 14, 18, 21, m1–m4, m7, m15, and m24) in CA-containing plasma were significantly associated with the anti-FD effect. These compounds included seven prototype compounds, i.e., cynanoneside A, syringic acid, deacylmetaplexigenin, ferulic acid, scopoletin, baishouwubenzophenone, and qingyangshengenin. The inhibition of ABC transporters demonstrated that the inhibitors verapamil and Ko143 significantly increased (P < 0.05) the uptake of scopoletin and qingyangshengenin. Thus, these compounds may be substrates for P-gp and BCRP. ConclusionsThe potential anti-FD components of CA and the effect of ABC transporter inhibitors on these active components were preliminarily clarified. These findings lay a foundation for subsequent in vivo studies.

Pregnane steroids and steroid glycosides with multidrug resistance reversal activity from Marsdenia tenacissima

Twenty compounds comprising four pregnane steroids (2-4 & 20) and 16 pregnane glycosides (1 & 5-19) have been obtained from the ethanol extract of the roots of a Dai ethnological herb, Marsdenia tenacissima. Their structures were characterized on the basis of comprehensive spectroscopic analyses with 17 ones (1-17) being reported for the first time, including the rare cases (2 & 3) of free C21 steroids with 17α-acetyl substitution, compounds 4-7 bearing an unusual 14α-OH, and the first examples with simultaneous 14α-OH/17α-acetyl substitution (7) and glycosylation at C-12 position (10 & 11). An empirical rule for the identification of C-17 configuration, in C21 steroids incorporating the marsdenin constitution structure, was also proposed. All the isolates, along with an array of previously reported analogues in our compound library, were screened for their chemo-reversal ability against P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) in MCF-7/ADR cell line, and six compounds exhibited moderate MDR reversal activity with reversal folds ranging from 1.92 to 4.44.

Chemical constituents from the roots of Dolomiaea souliei

A new benzofuran-type neolignan (1), two new phenylpropanoids (2 - 3), and one new C21 steroid (4) were isolated from the ethyl acetate extract of the roots of Dolomiaea souliei by chromatographic methods, including silica gel, ODS column chromatography, MPLC, and semi-preparative HPLC. Their structures were identified as dolosougenin A (1), (S)-3-isopropylpentyl (E)-3-(4-hydroxy-3-methoxyphenyl) acrylate (2), (S)-3-isopropylpentyl (Z)-3-(4-hydroxy-3-methoxyphenyl) acrylate (3) and dolosoucin A (4) through various spectroscopic techniques including 1D NMR, 2D NMR, IR, UV, HR ESI MS, ORD, and computational ORD methods.

Functional analysis of acyl-CoA dehydrogenases and their application to C22 steroid production

Acyl-CoA dehydrogenase (ChsE) is involved in the steroid side-chain degradation process. However, their function in vivo remains unclear. In this study, three ChsE, ChsE1-ChsE2, ChsE3, and ChsE4-ChsE5, were identified in Mycolicibacterium neoaurum, and their functions in vivo are studied and compared with those from Mycobacterium tuberculosis in vitro. By gene knockout, complementation, and the bioconversion of phytosterols, the function of ChsE was elucidated that ChsE4-ChsE5 could utilize C27, C24, and C22 steroids in vivo. ChsE3 could utilize C27 and C24 steroids in vivo. ChsE1-ChsE2 could utilize C27, C24, and C22 steroids in vivo. What is more, the production strain of a C22 steroid, 3-oxo-4,17-pregadiene-20-carboxylic acid methyl ester (PDCE), is constructed with ChsE overexpression. This study improved the understanding of the steroid bioconversion pathway and proposed a method of the production of a new C22 steroid. KEY POINTS: • Three ChsE paralogs from M. neoaurum are identified and studied. • The function of ChsE is overlapped in vivo. • A C22 steroid (PDCE) producer was constructed with ChsE overexpression.

The effect of etanercept therapy on adrenal steroid metabolism in juvenile idiopathic arthritis: a steroid metabolomics approach

ObjectiveTo evaluate the impact of anti-tumor necrosis factor-alpha (TNFα: etanercept [Etanercept ®]) therapy on adrenal activity in juvenile idiopathic arthritis (JIA) .MethodEleven JIA patients aged 12 ± 6.2 years with a disease duration of 6.3 ± 5.2 years were enrolled. They were treated once weekly with etanercept (0.8 mg/kg) for 3 ± 2.8 years. Urine samples for gas chromatography-mass spectrometry steroid hormone analysis were collected before, and 1 and 3 days after etanercept injection and compared to age- and sex-matched healthy controls.ResultsThe levels of 21 of the 31 metabolites were low before etanercept treatment. Those 21 metabolites included 4 C19 steroids (androgens), 5 C C21 steroid hormone intermediates, 10 cortisol metabolites, and 2 corticosterone metabolites. One day after treatment, only 5 of the 21 metabolite levels remained low. They included 2 C19 metabolites, 2 C21 steroid metabolites and 1 cortisol metabolite β –Cortol (β-Cl). Three days after treatment, the only metabolites levels that continued to be low were 2 C19 metabolite, 2 C21 steroid hormone intermediates and 1 cortisol metabolite α-Cortol (a-Cl), while the remaining 15 metabolites had already normalized after 1 day. Dehydroepiandrosterone-sulfate and 17-hydroxypregnenolone metabolite levels were the last ones to recover. Urinary metabolite ratios reflecting cytochrome P450 CYP21A2 (21-hydroxylase) and 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) enzymatic activitieswere lower in JIA patients than in controls, although significant was not reached.ConclusionAlmost all of the pre-etanercept treatment cortisol urinary metabolite levels were significantly lower than normal, and almost all rose to normal values by 1 day after treatment. The therapeutic effect of anti-TNFα treatment in JIA may be related to its effect on the restoration of adrenal function and cortisol levels.