Steroid Binding Site Research Articles

Structural determinants of parabens in inhibiting human and rat gonadal 3β-hydroxysteroid dehydrogenase

This study delved into the impacts of 10 parabens on the activity of human and rat gonadal 3β-hydroxysteroid dehydrogenase (3β-HSD) within human KGN cell and rat testicular microsomes, as well as on the secretion of progesterone in KGN cells and the inhibitory potency was compared between human and rats. Intriguingly, the outcomes revealed that ethyl, propyl, butyl, hexyl, heptyl, nonyl, phenyl, and benzyl parabens displayed varying IC50 values for human 3β-HSD2, from 4.15 to 139.96 μM, demonstrating characteristics of mixed inhibitors. Notably, within KGN cells, all examined parabens, excluding nonyl and phenyl parabens, significantly inhibited progesterone secretion at 5–50 μM. In the case of rats, the IC50 values for these parabens on gonadal 3β-HSD1 fluctuated between 7.15 and 110.76 μM, likewise functioning as mixed inhibitors. Through docking analysis, it was proposed that most parabens effectively bind to NAD+ and/or steroid binding site. Moreover, bivariate correlation analysis unveiled an inverse correlation between IC50 values and structural characteristics such as LogP, molecular weight, heavy atom number, and carbon number within the alcohol moiety of parabens. 3D-QSAR elucidated pivotal regions, comprising hydrogen bond donor, hydrogen bond acceptor, and hydrophobic region, with the most potent inhibitor nonyl paraben engaging with all regions, while the weakest inhibitor ethyl paraben interacted with the regions except for the hydrophobic region. In conclusion, this investigation underscored the inhibitory effects imparted by several parabens on both human and rat gonadal 3β-HSD activity, with their inhibitory potency being modulated by aspects of hydrophobicity and carbon chain length.

Tyramine Derivatives as Versatile Pharmacophores With Potent Biological Properties: Sex Hormone-Binding Globulin Inhibition, Colon Cancer Antimigration, and Antimicrobial Activity.

Guided by the idea that the presence of a heterocyclic aromatic core and tyramine moiety, under the umbrella of a single molecular scaffold could bring interesting biological properties, herein we present synthesis, characterization, with two crystal structures reported, and biological evaluation of some tyramine derivates. Cytotoxic and antimigratory potential was addressed by using a colorectal cancer cell line as a model system. Although possessing no cytotoxic effects, two compounds have shown strong antimigratory potential in low doses, with no effect on healthy MRC-5 cells. Evaluation of their antimicrobial activities suggested prominent antimicrobial activity, where Compound 4 outperformed streptomycin against Escherichia coli and Proteus mirabilis. Hormone-dependent types of cancer, such as prostate, ovary, and breast, are highly dependent on human sex hormone-binding globulin (SHBG) blood levels. A molecular docking study has shown that 1 has high affinity to bind and therefore compete with natural steroids for the SHBG steroid-binding site. DNA-binding study have shown that 4 interacts with CT-DNA in a groove-binding mode. In silico ADME/T study revealed that all compounds have suitable physicochemical properties for oral bioavailability and druglikeness, while toxicity tests for 1, 4, and 6 suggested potential for mutagenicity (4, 6), hepatotoxicity (6), and skin sensation (1).

Inhibition of human and rat placental 3β-hydroxysteroid dehydrogenases by bisphenol A analogues depends on their hydrophobicity: In silico docking analysis

Bisphenol A (BPA) and its analogues are widely used industrial chemicals. Placental 3β-hydroxysteroid dehydrogenases (3β-HSDs) catalyse the conversion of pregnenolone to progesterone. However, the potency of BPA analogues in inhibiting 3β-HSDs activity remains unclear. We investigated the inhibitory effect of 10 BPA analogues on 3β-HSDs activity using an in vitro assay and performed the structure-activity relationship and in silico docking analysis. BPH was the most potent inhibitor of human 3β-HSD1, with an IC50 value of 0.95 μM. BPFL, BPG, DABPA, BPAP, BPZ, DMBPA, and BPB also inhibited human 3β-HSD1 activity, albeit with lower potency. BPG was the most potent inhibitor of rat 3β-HSD4, with an IC50 value of 1.14 μM. BPAP, BPFL, BPG, BPH, BPZ, DABPA, and DMBPA are mixed inhibitors of human 3β-HSD1 and they significantly inhibited human JAr cells to secrete progesterone. The LogP values were inversely correlated with the inhibitory effects. Docking analysis showed that most BPA analogues bind to steroid-binding site of both 3β-HSDs. A pharmacophore containing hydrogen bond donor and hydrophobic region was generated for predicting the inhibitory strength of BPA analogues. In conclusion, this study demonstrates that some BPA analogues are potent inhibitors of 3β-HSDs and lipophilicity determines the inhibitory potency.

Structural and functional analysis of a bile salt hydrolase from the bison microbiome

The bile salt hydrolases (BSHs) are significant constituents of animal microbiomes. An evolving appreciation of their roles in health and disease has established them as targets of pharmacological inhibition. These bacterial enzymes belong to the N-terminal nucleophile superfamily and are best known to catalyze the deconjugation of glycine or taurine from bile salts to release bile acid substrates for transformation and or metabolism in the gastrointestinal tract. Here, we identify and describe the BSH from a common member of the Plains bison microbiome, Arthrobacter citreus (BSHAc). Steady-state kinetic analyses demonstrated that BSHAc is a broad-spectrum hydrolase with a preference for glycine-conjugates and deoxycholic acid (DCA). Second-order rate constants (kcat/KM) for BSHAc-catalyzed reactions of relevant bile salts-glyco- and tauro-conjugates of cholic acid and DCA- varied by ∼30-fold and measured between 1.4× 105 and 4.3× 106M-1s-1. Interestingly, a pan-BSH inhibitor named AAA-10 acted as a slow irreversible inhibitor of BSHAc with a rate of inactivation (kinact)of ∼2h-1 and a second order rate constant (kinact/KI) of ∼24M-1s-1 for the process. Structural characterization of BSHAc reacted with AAA-10 showed covalent modification of the N-terminal cysteine nucleophile, providing molecular details for an enzyme-stabilized product formed from this mechanism-based inhibitor's α-fluoromethyl ketone warhead. Structural comparison of the BSHs and BSH:inhibitor complexes highlighted the plasticity of the steroid-binding site, including a flexible loop that is variable across well-studied BSHs.

Food additive salicylates inhibit human and rat placental 3β-hydroxysteroid dehydrogenase: 3D-QSAR and in silico analysis

The use of salicylates as flavoring agents in food and beverages is common, but their potential to disrupt the endocrine system remains unclear. Human placental 3β-hydroxysteroid dehydrogenase 1 (h3β-HSD1) plays a role in progesterone synthesis and is the potential target. This study evaluated the inhibition of 13 salicylates on h3β-HSD1, structure-activity relationship (SAR) and compared with rat placental homolog r3β-HSD4. Salicylates inhibited h3β-HSD1, depending on carbon chain number in the alcohol moiety and the IC50 values for hexyl, ethylhexyl, homomenthyl, and menthyl salicylates were 53.27, 15.78, 2.35, and 2.31 μM, as mixed inhibitors, respectively, while methyl to benzyl salicylates were ineffective at 100 μM. Interestingly, only hexyl salicylate inhibited r3β-HSD4 with IC50 of 31.05 μM. Bivariate analysis revealed a negative correlation between IC50 and hydrophobicity (LogP), molecular weight, heavy atoms, and carbon number in the alcohol moiety against h3β-HSD1. Docking analysis demonstrated that these salicylates bind to cofactor binding sites or between the steroid and cofactor binding sites. Additionally, 3D-QSAR showed distinct binding via hydrogen bond donors and hydrophobic regions. In conclusion, the inhibition of h3β-HSD1 by salicylates appears to be dependent on factors such as LogP, molecular weight, heavy atoms, and carbon-chain length and there is species-dependent inhibition sensitivity.

Resveratrol analogues and metabolites selectively inhibit human and rat 11β-hydroxysteroid dehydrogenase 1 as the therapeutic drugs: structure–activity relationship and molecular dynamics analysis

ABSTRACT Resveratrol is converted to various metabolites by gut microbiota. Human and rat liver 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) are critical for glucocorticoid activation, while 11β-HSD2 in the kidney does the opposite reaction. It is still uncertain whether resveratrol and its analogues selectively inhibit 11β-HSD1. In this study, the inhibitory strength, mode of action, structure–activity relationship (SAR), and docking analysis of resveratrol analogues on human, rat, and mouse 11β-HSD1 and 11β-HSD2 were performed. The inhibitory strength of these chemicals on human 11β-HSD1 was dihydropinosylvin (6.91 μM) > lunularin (45.44 μM) > pinostilbene (46.82 μM) > resveratrol (171.1 μM) > pinosylvin (193.8 μM) > others. The inhibitory strength of inhibiting rat 11β-HSD1 was pinostilbene (9.67 μM) > lunularin (17.39 μM) > dihydropinosylvin (19.83 μM) > dihydroresveratrol (23.07 μM) > dihydroxystilbene (27.84 μM) > others and dihydropinosylvin (85.09 μM) and pinostilbene (>100 μM) inhibited mouse 11β-HSD1. All chemicals did not inhibit human, rat, and mouse 11β-HSD2. It was found that dihydropinosylvin, lunularin, and pinostilbene were competitive inhibitors of human 11β-HSD1 and that pinostilbene, lunularin, dihydropinosylvin, dihydropinosylvin and dihydroxystilbene were mixed inhibitors of rat 11β-HSD1. Docking analysis showed that they bind to the steroid-binding site of human and rat 11β-HSD1. In conclusion, resveratrol and its analogues can selectively inhibit human and rat 11β-HSD1, and mouse 11β-HSD1 is insensitive to the inhibition of resveratrol analogues.

Inhibitory effects of parabens on human and rat 17β-hydroxysteroid dehydrogenase 1: Mechanisms of action and impact on hormone synthesis

Parabens are commonly used preservatives in cosmetics, food, and pharmaceutical products. The objective of this study was to examine the effect of nine parabens on human and rat 17β-hydroxysteroid dehydrogenase 1 (17β-HSD1) in human placental and rat ovarian cytosols, as well as on estradiol synthesis in BeWo cells. The results showed that the IC50 values for these compounds varied from methylparaben with the weakest inhibition (106.42 μM) to hexylparaben with the strongest inhibition (2.05 μM) on human 17β-HSD1. Mode action analysis revealed that these compounds acted as mixed inhibitors. For rats, the IC50 values ranged from the weakest inhibition for methylparaben (no inhibition at 100 μM) to the most potent inhibition for hexylparaben (0.87 μM), and they functioned as mixed inhibitors. Docking analysis indicated that parabens bind to the region bridging the NADPH and steroid binding sites of human 17β-HSD1 and the NADPH binding site of rat 17β-HSD1. Bivariate correlation analysis demonstrated negative correlations between LogP, molecular weight, heavy atoms, and apolar desolvation energy, and the IC50 values of these compounds. In conclusion, this study identified the inhibitory effects of parabens and their binding mechanisms on human and rat 17β-HSD1, as well as their impact on hormone synthesis.

Effects of organochlorine pesticides on human and rat 17β-hydroxysteroid dehydrogenase 1 activity: Structure-activity relationship and in silico docking analysis

The objective of this study was to examine the effect of 11 organochlorine pesticides on human and rat 17β-Hydroxysteroid dehydrogenase 1 (17β-HSD1) in human placental and rat ovarian microsome and on estradiol production in BeWo cells. The results showed that the IC50 values for endosulfan, fenhexamid, chlordecone, and rhothane on human 17β-HSD1 were 21.37, 73.25, 92.80, and 117.69 μM. Kinetic analysis revealed that endosulfan acts as a competitive inhibitor, fenhexamid as a mixed/competitive inhibitor, chlordecone and rhothane as a mixed/uncompetitive inhibitor. In BeWo cells, all insecticides except endosulfan significantly decreased estradiol production at 100 μM. For rats, the IC50 values for dimethomorph, fenhexamid, and chlordecone were 11.98, 36.92, and 109.14 μM. Dimethomorph acts as a mixed inhibitor, while fenhexamid acts as a mixed/competitive inhibitor. Docking analysis revealed that endosulfan and fenhexamid bind to the steroid-binding site of human 17β-HSD1. On the other hand, chlordecone and rhothane binds to a different site other than the steroid and NADPH-binding site. Dimethomorph binds to the steroid/NADPH binding site, and fenhexamid binds to the steroid binding site of rat 17β-HSD1. Bivariate correlation analysis showed a positive correlation between IC50 values and LogP for human 17β-HSD1, while a slight negative correlation was observed between IC50 values and the number of HBA. ADMET analysis provided insights into the toxicokinetics and toxicity of organochlorine pesticides. In conclusion, this study identified the inhibitory effects of 3–4 organochlorine pesticides and binding mechanisms on human and rat 17β-HSD1, as well as their impact on hormone production.

Per- and polyfluoroalkyl substances inhibit human and rat 17β-hydroxysteroid dehydrogenase 1: Quantitative structure-activity relationship and molecular docking analysis

Per- and polyfluoroalkyl (PFAS) substances are enduring industrial materials. 17β-Hydroxysteroid dehydrogenase isoform 1 (17β-HSD1) is an estrogen metabolizing enzyme, which transforms estrone into estradiol in human placenta and rat ovary. Whether PFAS inhibit 17β-HSD1 and what the structure-activity relationship (SAR) remains unexplored. We screened 18 PFAS for inhibiting human and rat 17β-HSD1 in microsomes and studied their SAR and mode of action(MOA). Of the 11 perfluorocarboxylic acids (PFCAs), C8-C14 PFCAs at a concentration of 100 μM substantially inhibited human 17β-HSD1, with order of C11 (half-maximal inhibition concentration, IC50, 8.94 μM) > C10 (10.52 μM) > C12 (14.90 μM) > C13 (30.97 μM) > C9 (43.20 μM) > C14 (44.83 μM) > C8 (73.38 μM) > others. Of the 7 per- and poly-fluorosulfonic acids (PFSAs), the potency was C8S (IC50, 14.93 μM) > C7S (80.70 μM) > C6S (177.80 μM) > others. Of the PFCAs, C8-C14 PFCAs at 100 μM markedly reduced rat 17β-HSD1 activity, with order of C11 (IC50, 9.11 μM) > C12 (14.30 μM) > C10 (18.24 μM) > C13 (25.61 μM) > C9 (67.96 μM) > C8 (204.39 μM) > others. Of the PFSAs, the potency was C8S (IC50, 37.19 μM) > C7S (49.38 μM) > others. In contrast to PFOS (C6S), the partially fluorinated compound 6:2 FTS with an equivalent number of carbon atoms demonstrated no inhibition of human and rat 17β-HSD1 activity at a concentration of 100 μM. The inhibition of human and rat enzymes by PFAS followed a V-shaped trend from C4 to C14, with a nadir at C11. Moreover, human 17β-HSD1 was more sensitive than rat enzyme. PFAS inhibited human and rat 17β-HSD1 in a mixed mode. Docking analysis revealed that they bind to the NADPH and steroid binding site of both 17β-HSD1 enzymes. The 3D quantitative SAR (3D-QSAR) showed that hydrophobic region, hydrogen bond acceptor and donor are key factors in binding to 17β-HSD1 active sites. In conclusion, PFAS exhibit inhibitory effects on human and rat 17β-HSD1 depending on factors such as carbon chain length, degree of fluorination, and the presence of carboxylic acid or sulfonic acid groups, with a notable V-shaped shift observed at C11.

Progesterone activation of β1-containing BK channels involves two binding sites

Progesterone (≥1 µM) is used in recovery of cerebral ischemia, an effect likely contributed to by cerebrovascular dilation. The targets of this progesterone action are unknown. We report that micromolar (µM) progesterone activates mouse cerebrovascular myocyte BK channels; this action is lost in β1-/- mice myocytes and in lipid bilayers containing BK α subunit homomeric channels but sustained on β1/β4-containing heteromers. Progesterone binds to both regulatory subunits, involving two steroid binding sites conserved in β1-β4: high-affinity (sub-µM), which involves Trp87 in β1 loop, and low-affinity (µM) defined by TM1 Tyr32 and TM2 Trp163. Thus progesterone, but not its oxime, bridges TM1-TM2. Mutation of the high-affinity site blunts channel activation by progesterone underscoring a permissive role of the high-affinity site: progesterone binding to this site enables steroid binding at the low-affinity site, which activates the channel. In support of our model, cerebrovascular dilation evoked by μM progesterone is lost by mutating Tyr32 or Trp163 in β1 whereas these mutations do not affect alcohol-induced cerebrovascular constriction. Furthermore, this alcohol action is effectively counteracted both in vitro and in vivo by progesterone but not by its oxime.

Carbon chain length of perfluoroalkylated carboxylic acids determines inhibitory strength on gonadal 3β-hydroxysteroid dehydrogenases in humans, rats, and mice

Perfluoroalkylated carboxylic acids (PFCAs) are a subclass of man-made chemicals that have been widely used in industrial production and consumer products. As a result, PFCAs have been found to accumulate in the environment and bioaccumulate in organisms, leading to potential health and environmental impacts. This study investigated the inhibition of 11 PFCAs on gonadal 3β-hydroxysteroid dehydrogenases in humans, rats, and mice. We observed a V-shaped inhibition pattern against human granulosa (KGN) cell 3β-HSD2 starting from C9 (half-maximal inhibitory concentration, IC50, 100.8 μM) to C11 (8.92 μM), with a V-shaped turn. The same V-shaped inhibition pattern was also observed for PFCAs against rat testicular 3β-HSD1 from C9 (IC50, 50.43 μM) to C11 (6.60 μM). Mouse gonadal 3β-HSD6 was insensitive to the inhibition of PFCAs, with an IC50 of 50.43 μM for C11. All of these PFCAs were mixed inhibitors of gonadal 3β-HSDs. Docking analysis showed that PFCAs bind to the nicotinamide adenine dinucleotide (NAD+)/steroid binding sites of these enzymes and bivariate correlation analysis showed that molecular length determines the inhibitory pattern of PFCAs on these enzymes. In conclusion, the carbon chain length determines the inhibitory strength of PFCAs on human, rat, and mouse gonadal 3β-HSDs, and the inhibitory strength of PFCAs against human and rat 3β-HSD enzymes shows V-shaped turn.

THU578 Concept: Role Of Spiral Steroids (SS) During Pregnancy

Abstract Disclosure: F.I. Chasalow: None. Background: Earlier, my laboratory reported 3 novel endogenous steroids (SS) that cross react with digoxin-specific antibodies (DLM). The SS are readily distinguished from all other steroids by 3 characteristics: [1] each is a phosphocholine diester, [2] each has more than 21 carbon atoms in the steroid fragment and [3] the extra carbons form an E-ring with a cyclic lactone. The discovery included a biosynthetic scheme with known enzymes and no need for a ‘magic’ step. Function: NaK-ATPase has 3 subunits, designated FXYD, α, and β. Affinity for Na+ and K+ ions is regulated by FXYD proteins. Each SS matches a tissue-specific α-subunit. Each α-subunit has 2 different binding sites for steroids. One site binds a SS; the ligand of the other site is unknown. For example, Ionotropin is specific for the α subunit present in cardiac and renal tissue. Kaleotropin is isolated from gonads, but the α subunit which matches each specific ovarian cell type is unknown. Galotropin (G-trop): One of the SS, G-trop, was detected as a DLM in human breast cyst fluids, specifically cysts with high K+ ion levels. Solubility properties distinguished it from previously known cardiac glycosides and, like other DLM, it inhibited NaK-ATPase. G-trop was also detected in milk from cows, sheep and goats. Ions derived from Ionotropin (m/z=341 Da) or Kaleotropin (m/z=369 Da) were not present in milk extracts. On LC-MS, G-trop fragmented to an ion at m/z= 381 Da. Trial and error analysis suggests C25O4H34. No other formula is consistent with m/z=381 Da. G-trop: G-trop wasn’t present in serum from children. At 22-26 weeks of gestation, whether or not a pregnant woman had pre-eclampsia, serum had low levels of G-trop. Maternal G-trop increases during the 3rd trimester, coincidentally with initiation of milk production; simultaneously, K+ ion levels in milk increased. Pre-eclampsia. In a pilot study, 65% of patients (n=20) with pre-eclampsia had elevated levels of Ionotropin precursor and 30% had elevated levels of Kaleotropin precursor. 25% of patients with pre-eclampsia develop life-threatening hypertension or seizures, typical of the expected response to elevated Ionotropin levels. Therapy includes immediate delivery of the infant at 30-36 weeks of gestation. Maternal G-trop synthesis has not yet begun and mother’s milk has low K+ ion levels. Elevated maternal Kaleotropin is not the cause of maternal hypertension, suggesting a specific gestational maternal α-subunit is its target. Kaleotropin from placenta may be the signal to respond to maternal hypokalemia. Necrotizing enterocolitis (NEC):In utero the fetus does not absorb K+ from the GI tract because fetal need for K+ ion is provided via the placenta. Concept: After premature delivery, without G-trop in milk, the infant can’t absorb K+ from the GI tract; the GI tract fails to grow and tight junctions do not form. As a result, infants are at high risk for developing NEC. Presentation Date: Thursday, June 15, 2023

Bisphenol analogues inhibit human and rat 17β-hydroxysteroid dehydrogenase 1: 3D-quantitative structure-activity relationship (3D-QSAR) and in silico docking analysis

Bisphenols, estrogenic endocrine-disrupting chemicals, disrupt at least one of three endocrine pathways (estrogen, androgen, and thyroid). 17β-Hydroxysteroid dehydrogenase 1 (17β-HSD1) is a steroidogenic enzyme that catalyzes the activation of estradiol from estrone in human placenta and rat ovary. However, whether bisphenols inhibit 17β-HSD1 and the mode of action remains unclear. This study we screened 17 bisphenols for inhibiting human 17β-HSD1 in placental microsomes and rat 17β-HSD1 in ovarian microsomes and determined 3D-quantitative structure-activity relationship (3D-QSAR) and mode of action. We observed some bisphenols with substituents were found to significantly inhibit both human and rat 17β-HSD1 with the most potent inhibition on human enzyme by bisphenol H (IC50 = 0.90 μM) when compared to bisphenol A (IC50 = 113.38 μM). Rat enzyme was less sensitive to the inhibition of bisphenols than human enzyme with bisphenol H (IC50 = 32.94 μM) for rat enzyme. We observed an inverse correlation between IC50 and hydrophobicity (expressed as Log P). Docking analysis showed that they bound steroid-binding site of 17β-HSD1. The 3D-QSAR models demonstrated that hydrophobic region, hydrophobic aromatic, ring aromatic, and hydrogen bond acceptor are key factors for the inhibition of steroid synthesis activity of 17β-HSD1.

Inhibition of Resveratrol Analogs on Human and Rat 3β-Hydroxysteroid Dehydrogenases: Structure–Activity Relationship and Docking Analysis

Resveratrol and its analogs are phytochemicals. Human 3β-hydroxysteroid dehydrogenase 1 (3β-HSD1) synthesizes steroid hormones for normal pregnancy or promoting cancer metastasis. Whether they inhibit 3β-HSD1 remains unclear. In this study, the inhibitory potency, mode of action, structure-activity relationship, and docking parameters of resveratrol and its analogs on 3β-HSD1 and rat homolog 3β-HSD4 were analyzed. The inhibitory potency of these chemicals on human 3β-HSD1 was 4,4'-dihydroxystilbene (IC50, 3.68 μM) > pinostilbene (8.07 μM) > pinosylvin (10.60 μM) > lunularin (26.84 μM) > resveratrol (30.20 μM) > dihydroresveratrol (>100 μM) = oxyresveratrol (>100 μM) > dihydropinosylvin (ineffective at 100 μM). Resveratrol analogs and metabolites are mixed or competitive inhibitors of human 3β-HSD1. Resveratrol and 4,4'-dihydroxystilbene inhibited progesterone secretion by human JAr cells at ≥1 μM. Resveratrol (IC50, 32.09 μM) and pinosylvin (34.71 μM) significantly inhibited rat placental 3β-HSD4 activity. Docking analysis shows that resveratrol analogs and metabolites bind the steroid-binding sites of human 3β-HSD1 and rat 3β-HSD4 and interact with the catalytic residues Ser125/Thr125 and Tyr155. The negative correlation of LogP and IC50 values for human 3β-HSD1 indicates that lipophilicity of chemicals plays a critical role in the inhibitory effect of chemicals. In conclusion, 4,4'-dihydroxystilbene, pinostilbene, and pinosylvin effectively inhibit human 3β-HSD1 depending on their lipophilicity, thereby acting as potential therapeutic agents.

Structural insight to elucidate the binding specificity of the anti-cortisol Fab fragment with glucocorticoids

Cortisol is a steroid hormone that is produced by the adrenal gland. It is a primary stress hormone that increases glucose levels in the blood stream. High concentrations of cortisol in the body can be used as a biomarker for acute and chronic stress and related mental and physiological disorders. Therefore, the accurate quantification of cortisol levels in body fluids is essential for clinical diagnosis. In this article, we describe the isolation of recombinant anti-cortisol antibodies with high affinity for cortisol and discover their cross-reactivity with other glucocorticoids. To describe the cortisol binding site and elucidate the structural basis for the binding specificity, the high-resolution crystal structures of the anti-cortisol (17) Fab fragment in the absence of glucocorticoid (2.00 Å) and the presence of cortisol (2.26 Å), corticosterone (1.86 Å), cortisone (1.85 Å) and prednisolone (2.00 Å) were determined. To our knowledge, this is the first determined crystal structure of a cortisol-specific antibody. The recognition of cortisol is driven by hydrophobic interactions and hydrogen bonding at the protein–ligand interface coupled with a conformational transition. Comparison of ligand-free and ligand-bound structures showed that the side chains of residues Tyr58-H and Arg56-H can undergo local conformational changes at the binding site, most likely prior to the binding event via a conformational selection mechanism. Compared to other anti-steroid antibody–antigen complexes, (17) Fab possesses a structurally unique steroid binding site, as the H3 loop from the CDR area has only a minor contribution, but framework residues have a prominent contribution to hapten binding.

The analysis of pesticides and fungicides in the inhibition of human and rat placental 3β-hydroxysteroid dehydrogenase activity: Mode of inhibition and mechanism

3β-Hydroxysteroid dehydrogenase/steroid Δ5,4-isomerase 1 (3β-HSD1) plays a critical role in the biosynthesis of progesterone from pregnenolone in the human placenta to maintain normal pregnancy. Whether they inhibit placental 3β-HSD1 and mode of inhibition remains unclear. In this study, we screened 21 pesticides and fungicides in five classes to inhibit human 3β-HSD1 and compared them to rat homolog 3β-HSD4. 3β-HSD activity was measured by catalyzing pregnenolone to progesterone in the presence of NAD+. Of the 21 chemicals, azoles (difenoconazole), thiocarbamates (thiram and ferbam) and organochlorine (hexachlorophene) significantly inhibited human 3β-HSD1 with half maximal inhibitory concentration (IC50) values of 2.77, 0.24, 0.68, and 17.96 μM, respectively. We also found that difenoconazole, ferbam and hexachlorophene are mixed/competitive inhibitors of 3β-HSD1 while thiram is a mixed/noncompetitive inhibitor. Docking analysis showed that difenoconazole and hexachlorophene bound steroid-binding site. Difenoconazole and hexachlorophene except thiram and ferbam also significantly inhibited rat 3β-HSD4 activity with IC50 of 1.12 and 2.28 µM, respectively. Thiram and ferbam significantly inhibited human 3β-HSD1 possibly by interfering with cysteine residues, while they had no effects on rat 3β-HSD4. In conclusion, some pesticides potently inhibit placental 3β-HSD, leading to the reduction of progesterone formation.

Direct inhibition of human and rat 11β-hydroxysteroid dehydrogenase 2 by per- and polyfluoroalkyl substances: Structure-activity relationship and in silico docking analysis

Per- and polyfluoroalkyl substances (PFAS) are persistent in the environment and may disrupt the endocrine system. Our previous study showed that perfluorooctanoic acid (PFOA, C8) and perfluorooctanesulfonic acid (PFOS, C8S) can inhibit 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) activity leading to an active glucocorticoid accumulation. In this study, we extended investigation for 17 PFAS, including carboxylic and sulfonic acids, with different carbon-chain lengths, to determine their inhibitory potency and structure-activity relationship in human placental and rat renal 11β-HSD2. C8-C14 PFAS at 100 μM significantly inhibited human 11β-HSD2 with a potency as C10 (half-maximal inhibitory concentration, IC50, 9.19 μM) > C11 (15.09 μM) > C12 (18.43 μM) > C9 (20.93 μM) > C13 (124 μM) > C14 (147.3 μM) > other C4-C7 carboxylic acids, and C8S > C7S = C10S > other sulfonic acids. For rat 11β-HSD2, only C9 and C10 and C7S and C8S PFAS exhibited significant inhibitory effects. PFAS are primarily mixed/competitive inhibitors of human 11β-HSD2. Preincubation and simultaneous incubation with the reducing agent dithiothreitol significantly increased human 11β-HSD2 but not rat 11β-HSD2, and preincubation but not simultaneous incubation with dithiothreitol partially reversed C10-mediated inhibition on human 11β-HSD2. Docking analysis showed that all PFAS bound to the steroid-binding site and carbon-chain length determined the potency of inhibition, with the optimal molecular length (12.6 Å) for potent inhibitors PFDA and PFOS, which is comparable to the molecular length (12.7 Å) of the substrate cortisol. The length between 8.9 and 17.2 Å is the probable threshold molecular length to inhibit human 11β-HSD2. In conclusion, the carbon-chain length determines the inhibitory effect of PFAS on human and rat 11β-HSD2, and the inhibitory potency of long-chain PFAS on human and rat 11β-HSD2 showed V-shaped pattern. Long-chain PFAS may partially act on the cysteine residues of human 11β-HSD2.

Direct inhibition of bisphenols on human and rat 11β-hydroxysteroid dehydrogenase 2: Structure-activity relationship and docking analysis

Bisphenols (BPs) as endocrine-disrupting compounds have drawn attention to their health hazards. Whether a BP interferes with glucocorticoid metabolism remains unclear. 11β-Hydroxysteroid dehydrogenase 2 (11β-HSD2) is a key glucocorticoid-metabolizing enzyme that controls fetal glucocorticoid levels across the placental barrier and mineralocorticoid receptor specificity in the kidney. In this study, 11 BPs were tested to inhibit human placental and rat renal 11β-HSD2 and were analyzed for inhibitory potency, mode action, and docking parameters. BPs had inhibitory potency against human 11β-HSD2: BPFL>BPAP>BPZ>BPB>BPC>BPAF>BPA>TDP and the IC10 values were 0.21, 0.55, 1.04, 2.04, 2.43, 2.57, 14.43, and 22.18 μM, respectively. All BPs are mixed inhibitors except BPAP, which is a competitive inhibitor for human 11β-HSD2. Some BPs also inhibited rat renal 11β-HSD2, with BPB (IC50, 27.74 ± 0.95) > BPZ (42.14 ± 0.59) > BPAF (54.87 ± 1.73) > BPA (77.32 ± 1.20) > other BPs (about 100 μM). Docking analysis showed that all BPs bound to the steroid-binding site, interacting with the catalytic residue Tyr232 of both enzymes and the most potent human 11β-HSD2 inhibitor BPFL acts possibly due to its large fluorene ring that has hydrophobic interaction with residues Glu172 and Val270 and π-stacking interaction with catalytic residue Tyr232. The increase in the size of substituted alkanes and halogenated groups in the methane moiety of the bridge of BPs increases its inhibitory potency. Regressions of the lowest binding energy with inhibition constant indicated that there was an inverse regression. These results indicated that BPs significantly inhibited human and rat 11β-HSD2 activity and that there were species-dependent differences.

Designing Selective Drug-like Molecular Glues for the Glucocorticoid Receptor/14-3-3 Protein–Protein Interaction

The ubiquitously expressed glucocorticoid receptor (GR) is a nuclear receptor that controls a broad range of biological processes and is activated by steroidal glucocorticoids such as hydrocortisone or dexamethasone. Glucocorticoids are used to treat a wide variety of conditions, from inflammation to cancer but suffer from a range of side effects that motivate the search for safer GR modulators. GR is also regulated outside the steroid-binding site through protein-protein interactions (PPIs) with 14-3-3 adapter proteins. Manipulation of these PPIs will provide insights into noncanonical GR signaling as well as a new level of control over GR activity. We report the first molecular glues that selectively stabilize the 14-3-3/GR PPI using the related nuclear receptor estrogen receptor α (ERα) as a selectivity target to drive design. These 14-3-3/GR PPI stabilizers can be used to dissect noncanonical GR signaling and enable the development of novel atypical GR modulators.

Inhibition of human and rat placental 3β-hydroxysteroid dehydrogenase/Δ5,4-isomerase activities by insecticides and fungicides: Mode action by docking analysis

Many insecticides and fungicides are endocrine-disrupting compounds, which possibly interfere with the placental endocrine system. In the placenta, 3β-hydroxysteroid dehydrogenase/Δ5,4-isomerase type 1 (HSD3B1) is the major steroidogenic enzyme, which makes progesterone from pregnenolone to support the placental stability. In this study, we screened 12 classes of insecticides and fungicides to inhibit placental HSD3B1 activity and compared them to the rat homolog type 4 (HSD3B4) isoform. Human HSD3B1 activity and rat HSD3B4 activity were measured in the presence of 200 nM pregnenolone and 0.2 mM NAD+ and 100 μM of test chemical. Triclosan, triflumizole, dichlone, and oxine at 100 μM significantly inhibited human HSD3B1 activity with the residual activity being less than 50% of the control. Further study showed that the half-maximal inhibitory concentration (IC50) values of triclosan, triflumizole, dichlone, and oxine were 85.53 ± 9.14, 73.75 ± 3.42, 2.54 ± 0.40, and 102.93 ± 6.10 μM, respectively. In the presence of pregnenolone, triclosan, triflumizole, and dichlone were mixed inhibitors of HSD3B1, while oxine was a noncompetitive inhibitor. In the presence of NAD+, triclosan exhibited competitive inhibition while triflumizole possessed uncompetitive inhibition. Docking analysis showed that triclosan bound NAD+-binding site, while triflumizole, dichlone, and oxine mostly bound steroid-binding site. When the effect of these insecticides on rat placental HSD3B4 activity was screened in the presence of 200 nM pregnenolone, atrazine, triclosan, triflumizole, oxine, cyprodinil, and diphenyltin at 100 μM significantly inhibited rat HSD3B4 activity, with IC50 values of triclosan, triflumizole, oxine, and cyprodinil were 82.99 ± 6.48, 35.45 ± 2.73, 105.59 ± 12.04, and 43.37 ± 3.00 μM, respectively. The mode action analysis showed that triflumizole and cyprodinil were almost competitive inhibitors, while triclosan and oxine were almost noncompetitive inhibitors of rat HSD3B4. Docking analysis showed that triclosan and oxine bound cofactor NAD+ binding residues more than steroid-binding residues of rat HSD3B4 while triflumizole and cyprodinil bound most pregnenolone-interactive residues. In conclusion, some insecticides such as triclosan, triflumizole, and oxine can effectively inhibit both human and rat placental HSD3B activity and they have unique mode action due to the structure difference.