Steroid Ring Research Articles

Structure elucidation and spectroscopic analysis of photodegradants of the anti-rhinitis drug fluticasone furoate

Fluticasone furoate is a novel glucocorticoid receptor agonist marketed as a treatment for seasonal and perennial allergic rhinitis. Forced degradation of fluticasone furoate under conditions of light led to a number of degradation products, the structures of which were elucidated using mass spectrometry and a range of one and two-dimensional NMR experiments. Three structures were derived, including two which involved a rearrangement of the steroid ring A to give cross-linked degradation products. The results demonstrate the applicability of a previously observed mechanism of photodegradation to fluticasone furoate.

Effects of bile acids on rat hepatic microsomal type I 11β-hydroxysteroid dehydrogenase

The aim of this study was to investigate the effect of various bile acids on hepatic type I 11β-hydroxysteroid dehydrogenase (11β-HSD1) activity in vitro. The rat liver microsome fraction was prepared and 11β-HSD1 activity was assayed using cortisol and corticosterone as substrates for the enzyme reaction. The substrate and various concentrations of bile acids were added to the assay mixture. After incubation, the products were extracted and analyzed using high-performance liquid chromatography. All bile acids tested except deoxycholic acid and 7-keto bile acids inhibited the 11β-HSD1 enzyme reaction to some degree. Ursodeoxycholic acid inhibited the activity less than cholic, chenodeoxycholic, and lithocholic acids. Deoxycholic acid and 7-keto bile acids did not inhibit, but enhanced the enzyme activity. Inhibitions of dehydrogenation by corticosterone were weaker than those by cortisol. Kinetic analysis revealed that the inhibition of 11β-HSD1 was competitive. The inhibition of 11β-HSD1 by bile acids depended on the three-dimensional structural difference in the steroid rings and the presence of the 7α-hydroxy molecule of the bile acids was important for the inhibition of rat hepatic 11β-HSD1 enzyme activity. These results suggest that bile acid administration might modulate 11β-HSD1 enzyme activity.

Mycobacterial Cytochrome P450 125 (Cyp125) Catalyzes the Terminal Hydroxylation of C27 Steroids

Cyp125 (Rv3545c), a cytochrome P450, is encoded as part of the cholesterol degradation gene cluster conserved among members of the Mycobacterium tuberculosis complex. This enzyme has been implicated in mycobacterial pathogenesis, and a homologue initiates cholesterol catabolism in the soil actinomycete Rhodococcus jostii RHA1. In Mycobacterium bovis BCG, cyp125 was up-regulated 7.1-fold with growth on cholesterol. A cyp125 deletion mutant of BCG did not grow on cholesterol and accumulated 4-cholesten-3-one when incubated in the presence of cholesterol. Wild-type BCG grew on this metabolite. By contrast, a parallel cyp125 deletion mutation of M. tuberculosis H37Rv did not affect growth on cholesterol. Purified Cyp125 from M. tuberculosis, heterologously produced in R. jostii RHA1, bound cholesterol and 4-cholesten-3-one with apparent dissociation constants of 0.20 +/- 0.02 microM and 0.27 +/- 0.05 microm, respectively. When reconstituted with KshB, the cognate reductase of the ketosteroid 9alpha-hydroxylase, Cyp125 catalyzed the hydroxylation of these steroids. MS and NMR analyses revealed that hydroxylation occurred at carbon 26 of the steroid side chain, allowing unambiguous classification of Cyp125 as a steroid C26-hydroxylase. This study establishes the catalytic function of Cyp125 and, in identifying an important difference in the catabolic potential of M. bovis and M. tuberculosis, suggests that Cyp125 may have an additional function in pathogenesis.

Natural heterocycles as antifertility agents

Natural products have shown an unflinching promise as antifertility agents. Interestingly, most of them are heterocyclic in nature. The extraordinary capability of these compounds to mimic the activity of estrogenic compounds imparts them a remarkable characteristic of being able to regulate female fertility. Nitrogen and oxygen heterocycles form the majority of these compounds of which, in majority of cases, the pharmacologically active part of the molecule is non-aromatic, thus non planar, just as is the steroid ring system. This fact may be responsible for their steroid mimicking activity. This review presents the active compounds identified and pharmacologically evaluated till date.

Cholesterol oxidase: biochemistry and structural features

Cholesterol oxidases are bifunctional flavoenzymes that catalyze the oxidation of steroid substrates which have a hydroxyl group at the 3beta position of the steroid ring system. The enzyme is found, in a wide range of bacterial species, in two forms: one with the FAD cofactor bound noncovalently to the enzyme; and one with the cofactor linked covalently to the protein. Here we discuss, compare and contrast the salient biochemical properties of the two forms of the enzyme. Specifically, the structural features are discussed that affect the redox potentials of the flavin cofactor, the chemical mechanism of substrate dehydrogenation by active-center amino acid residues, the kinetic parameters of both types of enzymes and the reactivity of reduced enzymes with molecular dioxygen. The presence of a molecular tunnel that is proposed to serve in the access of dioxygen to the active site and mechanisms of its control by a 'gate' formed by amino acid residues are highlighted.

CYP7B1: One Cytochrome P450, Two Human Genetic Diseases, and Multiple Physiological Functions

The CYP7B1 cytochrome P450 enzyme hydroxylates carbons 6 and 7 of the B ring of oxysterols and steroids. Hydroxylation reduces the biological activity of these substrates and facilitates their conversion to end products that are readily excreted from the body. CYP7B1 is expressed in the liver, reproductive tract, and brain and performs different physiological functions in each tissue. Hepatic CYP7B1 activity is crucial for the inactivation of oxysterols and their subsequent conversion into bile salts. Loss of CYP7B1 activity is associated with liver failure in children. In the reproductive tract, the enzyme metabolizes androgens that antagonize estrogen action; mice without CYP7B1 have abnormal prostates and ovaries. The role of CYP7B1 in brain is under investigation; recent studies show that spastic paraplegia type 5, a progressive neuropathy, is caused by loss-of-function mutations in the human gene.

Physical properties of the lipid bilayer membrane made of cortical and nuclear bovine lens lipids: EPR spin-labeling studies

The physical properties of membranes derived from the total lipids extracted from the lens cortex and nucleus of a 2-year-old cow were investigated using EPR spin-labeling methods. Conventional EPR spectra and saturation–recovery curves show that spin labels detect a single homogenous environment in membranes made from cortical lipids. Properties of these membranes are very similar to those reported by us for membranes made of the total lipid extract of 6-month-old calf lenses (J. Widomska, M. Raguz, J. Dillon, E. R. Gaillard, W. K. Subczynski, Biochim. Biophys. Acta 1768 (2007) 1454–1465). However, in membranes made from nuclear lipids, two domains were detected by the EPR discrimination by oxygen transport method using the cholesterol analogue spin label and were assigned to the bulk phospholipid–cholesterol domain (PCD) and the immiscible cholesterol crystalline domain (CCD), respectively. Profiles of the order parameter, hydrophobicity, and the oxygen transport parameter are practically identical in the bulk PCD when measured for either the cortical or nuclear lipid membranes. In both membranes, lipids in the bulk PCD are strongly immobilized at all depths. Hydrophobicity and oxygen transport parameter profiles have a rectangular shape with an abrupt change between the C9 and C10 positions, which is approximately where the steroid ring structure of cholesterol reaches into the membrane. The permeability coefficient for oxygen, estimated at 35 °C, across the bulk PCD in both membranes is slightly lower than across the water layer of the same thickness. However, the evaluated upper limit of the permeability coefficient for oxygen across the CCD (34.4 cm/s) is significantly lower than across the water layer of the same thickness (85.9 cm/s), indicating that the CCD can significantly reduce oxygen transport in the lens nucleus.

The X-ray Structure of RU486 Bound to the Progesterone Receptor in a Destabilized Agonistic Conformation

Here we describe the 1.95 A structure of the clinically used antiprogestin RU486 (mifepristone) in complex with the progesterone receptor (PR). The structure was obtained by taking a crystal of the PR ligand binding domain containing the agonist norethindrone and soaking it in a solution containing the antagonist RU486 for extended times. Clear ligand exchange could be observed in one copy of the PR ligand binding domain dimer in the crystal. RU486 binds while PR is in an agonistic conformation without displacing helix 12. Although this is probably because of the constraints of the crystal lattice, it demonstrates that helix 12 displacement is not a prerequisite for RU486 binding. Interestingly, B-factor analysis clearly shows that helix 12 becomes more flexible after RU486 binding, suggesting that RU486, being a model antagonist, does not induce one fixed conformation of helix 12 but changes its positional equilibrium. This conclusion is confirmed by comparing the structures of RU486 bound to PR and RU486 bound to the glucocorticoid receptor.

Foetal ‘flat’ bile acids reappear during human liver regeneration after surgery

Changes in bile acid (BA) pool, such as the reappearance of typically foetal-type molecular species with a 'flat' structure at the steroid ring, occur during hepatocarcinogenesis, both in humans and rodents. Moreover flat-BAs also appear during rat liver regeneration. These changes can be detected in urine. The aim of the present study was to investigate whether flat-BAs also reappear during human liver regeneration, and whether this change correlates with the magnitude of liver resection. Patients undergoing partial hepatectomy were divided in two groups: major hepatectomy group (> 50% of hepatic tissue resection, n = 17) and minor hepatectomy group (< 50%, n = 13). BAs were extracted from serum and urine (collected over 24 h) and analysed by gas chromatography-mass spectrometry. Samples were obtained before surgery (day 0) and on the third and seventh days after hepatectomy. In serum, total BAs significantly increased on day seven after hepatectomy, but only a moderate increase in flat-BA concentrations was observed. By contrast, urinary excretion of total as well as flat-BAs significantly increased at day three and day seven after hepatectomy. Moreover, the amount of flat-BAs excreted in urine during the first week after partial hepatectomy correlated with the magnitude of the resection. Urinary BA output increases and flat-BAs reappear in urine during human liver regeneration. These results suggest that determination of BAs in urine may be an interesting parameter obtained by non-invasive techniques whose actual clinical value during human liver regeneration warrants further evaluation.

Structural determinants of monohydroxylated bile acids to activate β1 subunit-containing BK channels

Lithocholate (LC) (10-300 microM) in physiological solution is sensed by vascular myocyte large conductance, calcium- and voltage-gated potassium (BK) channel beta(1) accessory subunits, leading to channel activation and arterial dilation. However, the structural features in steroid and target that determine LC action are unknown. We tested LC and close analogs on BK channel (pore-forming cbv1+beta(1) subunits) activity using the product of the number of functional ion channels in the membrane patch (N) and the open channel probability (Po). LC (5beta-cholanic acid-3alpha-ol), 5alpha-cholanic acid-3alpha-ol, and 5beta-cholanic acid-3beta-ol increased NPo (EC(50) approximately 45 microM). At maximal increase in NPo, LC increased NPo by 180%, whereas 5alpha-cholanic acid-3alpha-ol and 5beta-cholanic acid-3beta-ol raised NPo by 40%. Thus, the alpha-hydroxyl and the cis A-B ring junction are both required for robust channel potentiation. Lacking both features, 5alpha-cholanic acid-3beta-ol and 5-cholenic acid-3beta-ol were inactive. Three-dimensional structures show that only LC displays a bean shape with clear-cut convex and concave hemispheres; 5alpha-cholanic acid-3alpha-ol and 5beta-cholanic acid-3beta-ol partially matched LC shape, and 5alpha-cholanic acid-3beta-ol and 5-cholenic acid-3beta-ol did not. Increasing polarity in steroid rings (5beta-cholanic acid-3alpha-sulfate) or reducing polarity in lateral chain (5beta-cholanic acid 3alpha-ol methyl ester) rendered poorly active compounds, consistent with steroid insertion between beta(1) and bilayer lipids, with the steroid-charged tail near the aqueous phase. Molecular dynamics identified two regions in beta(1) transmembrane domain 2 that meet unique requirements for bonding with the LC concave hemisphere, where the steroid functional groups are located.

Evolution of the bile salt nuclear receptor FXR in vertebrates*s⃞

Bile salts, the major end metabolites of cholesterol, vary significantly in structure across vertebrate species, suggesting that nuclear receptors binding these molecules may show adaptive evolutionary changes. We compared across species the bile salt specificity of the major transcriptional regulator of bile salt synthesis, the farnesoid X receptor (FXR). We found that FXRs have changed specificity for primary bile salts across species by altering the shape and size of the ligand binding pocket. In particular, the ligand binding pockets of sea lamprey (Petromyzon marinus) and zebrafish (Danio rerio) FXRs, as predicted by homology models, are flat and ideal for binding planar, evolutionarily early bile alcohols. In contrast, human FXR has a curved binding pocket best suited for the bent steroid ring configuration typical of evolutionarily more recent bile acids. We also found that the putative FXR from the sea squirt Ciona intestinalis, a chordate invertebrate, was completely insensitive to activation by bile salts but was activated by sulfated pregnane steroids, suggesting that the endogenous ligands of this receptor may be steroidal in nature. Our observations present an integrated picture of the coevolution of bile salt structure and of the binding pocket of their target nuclear receptor FXR.

Testosterone-inducible Regulator Is a Kinase That Drives Steroid Sensing and Metabolism in Comamonas testosteroni

The mechanism of gene regulation by steroids in bacteria is still a mystery. We use steroid-inducible 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase (3alpha-HSD/CR) as a reporter system to study steroid signaling in Comamonas testosteroni. In previous investigations we cloned and characterized the 3alpha-HSD/CR-encoding gene, hsdA. In addition, we identified two negative regulator genes (repA and repB) in the vicinity of hsdA, the protein products which repress hsdA expression on the level of transcription and translation, respectively. Recently, a positive regulator of hsdA expression, TeiR (testosterone-inducible regulator), was found by transposon mutagenesis, but the mode of its action remained obscure. In the present work we produced a TeiR-green fluorescent fusion protein and showed that TeiR is a membrane protein with asymmetrical localization at one of the cell poles of C. testosteroni. Knock-out mutants of the teiR gene revealed that TeiR provides swimming and twitching motility of C. testosteroni to the steroid substrate source. TeiR also mediated an induced expression of 3alpha-HSD/CR which was paralleled by an enhanced catabolism of testosterone. We also found that TeiR responds to a variety of different steroids other than testosterone. Biochemical analysis with several deletion mutants of the teiR gene revealed TeiR to consist of three different functional domains, an N-terminal domain important for membrane association, a central steroid binding site, and a C-terminal part mediating TeiR function. Finally, we could demonstrate that TeiR works as a kinase in the steroid signaling chain in C. testosteroni. Overall, we provide evidence that TeiR mediates steroid sensing and metabolism in C. testosteroni via its steroid binding and kinase activity.

Characterization of Phytoecdysteroid Glycosides in Meadowfoam (Limnanthes alba) Seed Meal by Positive and Negative Ion LC-MS/MS

Meadowfoam ( Limnanthes alba) is an oilseed crop grown in western Oregon. The seed meal has potential value as a biopesticide due to glucosinolate degradation products and phytoecdysteroids, a group of polyhydroxylated triterpenoids with potent activities as arthropod molting hormones. Liquid chromatography in combination with tandem mass spectrometry operated in the precursor ion mode revealed the presence of four ecdysteroid glycosides in meadowfoam seed meal. The carbohydrate sequence and the identity of the ecdysteroid aglycones, ponasterone A and 20-hydroxyecdysone, were determined by product ion scanning. Ecdysteroids were detected in the negative ion mode as [M + formate] (-) ions, which yielded [M - H] (-) and alpha-cleavage fragments with retention of hydroxyl groups in MS/MS experiments (not seen in the positive ion mode), allowing the determination of the number of hydroxyl groups in the side chain and in the steroid ring system. MS/MS of glycoside ions ([MH] (+) or [M + formate] (-)) provided carbohydrate sequence information.

Identification of a potent synthetic FXR agonist with an unexpected mode of binding and activation

The farnesoid X receptor (FXR), a member of the nuclear hormone receptor family, plays important roles in the regulation of bile acid and cholesterol homeostasis, glucose metabolism, and insulin sensitivity. There is intense interest in understanding the mechanisms of FXR regulation and in developing pharmaceutically suitable synthetic FXR ligands that might be used to treat metabolic syndrome. We report here the identification of a potent FXR agonist (MFA-1) and the elucidation of the structure of this ligand in ternary complex with the human receptor and a coactivator peptide fragment using x-ray crystallography at 1.9-A resolution. The steroid ring system of MFA-1 binds with its D ring-facing helix 12 (AF-2) in a manner reminiscent of hormone binding to classical steroid hormone receptors and the reverse of the pose adopted by naturally occurring bile acids when bound to FXR. This binding mode appears to be driven by the presence of a carboxylate on MFA-1 that is situated to make a salt-bridge interaction with an arginine residue in the FXR-binding pocket that is normally used to neutralize bound bile acids. Receptor activation by MFA-1 differs from that by bile acids in that it relies on direct interactions between the ligand and residues in helices 11 and 12 and only indirectly involves a protonated histidine that is part of the activation trigger. The structure of the FXR:MFA-1 complex differs significantly from that of the complex with a structurally distinct agonist, fexaramine, highlighting the inherent plasticity of the receptor.

Effect of the structure and substituents on the proton affinity of immunoactive azasteroid molecules

The effect of the structure and substitution on the proton affinity of immunoactive 8-azasteroid molecules was studied by semi-empirical methods of the quantum chemistry. It was shown that the proton affinity of these molecules is determined by the carbonyl oxygen atoms. The methoxy substitutents on the aromatic ring of the steroid have a weak effect on the proton affinity of the molecules in the ground state, whereas the direction of the effect for excited molecules depends on the orbital nature of the excited state. The substitution of the NH group or oxygen in the 16-position causes a substantial increase in the proton-acceptor power. The reasons for these changes are discussed.

Neurosteroid Analogues. 14. Alternative Ring System Scaffolds: GABA Modulatory and Anesthetic Actions of Cyclopenta[b]phenanthrenes and Cyclopenta[b]anthracenes

Although the structural features of binding sites for neuroactive steroids on gamma-aminobutryic acid type A (GABA A) receptors are still largely unknown, structure-activity studies have established a pharmacophore for potent enhancement of GABA A receptor function by neuroactive steroids. This pharmacophore emphasizes the importance of the position and stereochemistry of hydrogen-bonding groups on the steroid. However, the importance of the steroid ring system in mediating hydrophobic interactions with the GABA A receptor is unclear. We have taken the cyclopenta[ b]phenanthrene (tetracyclic compounds with a nonlinear ring system different from that of steroids) and cyclopenta[ b]anthracene (tetracyclic molecules with a linear 6-6-6-5 carbocyclic ring system) ring systems and properly substituted them to satisfy the pharmacophore requirements of the critical hydrogen-bond donor and acceptor groups found in neuroactive steroids. We have found these cyclopenta[ b]phenanthrene and cyclopenta[ b]anthracene analogues to have potent activity at the GABA A receptor, rivaling that of the most potent steroid modulators. Single-channel analysis of electrophysiological data indicates that similarly substituted analogues in the different ring systems affect the kinetic components of macroscopic currents in different ways. Mutations to the hydrogen bonding amino acids at the putative steroid binding site (alpha1Q241L mutation and alpha1N407A/Y410F double mutation) produce similar effects on macroscopic current amplitude by the different ring system analogues suggesting that the different kinetic effects are explained by the precise interactions of each analogue with the same binding site(s).

Changes of the Membrane Lipid Organization Characterized by Means of a New Cholesterol-Pyrene Probe

We synthesized 3 β-hydroxy-pregn-5-ene-21-(1-methylpyrenyl)-20-methylidene (Py-met-chol), consisting of cholesterol steroid rings connected to a pyrene group via a linker without polar atoms. This compound has interesting spectroscopic properties when probing membranes: 1), The pyrene has hypochromic properties resulting from probe self-association processes in membranes. Using liposomes of various lipid compositions, we determined the association constants of the probe ( K): K DOPC ≫ K POPC ≫ K DMPC > K DMPC/15 mol % Chol > K DMPC/30 mol % Chol . This indicates a better probe solvation in saturated than in unsaturated lipids, and this effect is enhanced as the cholesterol concentration increases. 2), The pyrene fluorophore is characterized by monomer ( I 1– I 5) and excimer ( I E) emission bands. In model membranes, I 1/ I 3 and I E/ I 3 ratios revealed a correlation between the polarity of the lipid core of the membrane and the amount of cholesterol. 3), Using this probe, we monitored the first steps of the signaling pathway of the mouse δ-opioid receptor, a G-protein-coupled receptor. The thickness of the membrane around this receptor is known to change after agonist binding. Fluorescence spectra of living Chinese hamster ovary cells overexpressing mouse δ-opioid receptor specifically revealed the agonist binding. These results indicate that Py-met-chol may be useful for screening ligands of this family of receptors.

Palladium‐Catalyzed Potassium Enoxyborate Alkylation of Enantiopure Hajos—Parrish Indenone to Construct Rearranged Steroid Ring Systems.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

Structure–antileukemic activity relationship study of B- and D-ring modified and nonmodified steroidal esters of 4-methyl-3-N,N-bis(2-chloroethyl)amino benzoic acid: a comparative study

This study was designed as a rational continuation of our research regarding the functional requirements essential for the antileukemic activity of compounds comprising an alkylating moiety and a modified steroid. The steroidal esteric derivatives of 4-methyl-3-N,N-bis(2-chloroethyl)amino benzoic acid were tested on leukemias P388 and L1210 in vivo and in normal human lymphocytes in vitro. Among them the B-lactamic steroidal esters proved more potent antileukemic agents than the 7-oxidized and those with a simple B-ring, but not more effective inducers of DNA damage and cell cycle arrest in vitro. We speculate that these results indicate a different mechanism of action induced by the lactamized B steroidal ring, in comparison to the 7-keto or the D-lactamic groups, which involves the interaction of the -NHCO- moiety with cellularcomponents essential for tumor growth. 4-Methyl-3-N,N-bis(2-chloroethyl)amino benzoic acid proved a more proper module for the B-lactams than chlorambucil and phenyl acetic acid's nitrogen mustard probably because the esteric bond is less cleaved by the esterases, resulting in an increased concentration of the drug in the vinicity of the target site essential for an antineoplasmatic response.

Do Ligand Binding and Solvent Exclusion Alter the Electrostatic Character within the Oxyanion Hole of an Enzymatic Active Site?

We report the site-specific incorporation of a thiocyanate vibrational probe into the active-site oxyanion hole of ketosteroid isomerase (KSI) to test the effect of hydrophobic steroid binding and solvent exclusion on the local electrostatic environment at this position. While binding of an uncharged ground state steroid analogue shifts the observed −CN vibrational frequency by +0.4 cm-1 relative to unliganded KSI, binding of an intermediate steroid analogue containing localized negative charge results in a +2.8 cm-1 shift. On the basis of a Stark tuning rate of 0.7 cm-1/(MV/cm), this shift indicates a fivefold larger change in the projection of the local electric field along the −CN bond in the presence of the charged ligand. Binding of a single ring phenolate with oxyanion charge localization equivalent to the intermediate steroid analogue but lacking distal hydrocarbon rings results in an identical −CN peak shift. We conclude that solvent exclusion and replacement by hydrophobic steroid rings negligibl...