Ligand-binding properties and catalytic activity of the purified human 24-hydroxycholesterol 7α-hydroxylase, CYP39A1.

Oxysterols are derivatives of cholesterol and biologically active molecules that are involved in a number of functions, including cholesterol homeostasis, immune response, embryogenic development and pathophysiology of neurodegenerative diseases. Enzymes catalyzing their synthesis and metabolism are of particular interest as potential or evaluated drug targets. Here we report for the first time biochemical analysis of purified human oxysterol 7α-hydroxylase selective for 24-hydroxycholesterol. Binding analyses indicated a tight binding of the oxysterols and estrone. Ligand screening revealed that CYP39A1 binds with high affinity antifungal drugs and prostate cancer drug galeterone (TOK-001). Site-directed mutagenesis of conserved Asn residue in the active site revealed its crucial role for protein folding and heme incorporation. Developed protocol for expression and purification enables further investigation of this hepatic enzyme as off-target in development of specific drugs targeting cytochrome P450 enzymes.

The development and use of a general route to brassinolide, its biosynthetic precursors, metabolites and analogues.

A new method for the construction of steroid side chains through the addition of lithium salts of dithianes to a C-22 aldehyde was developed. An efficient one-pot procedure for the preparation of a suitable C-22 aldehyde from commercial epibrassinolide in three steps in 86% isolated yield was described. Enantioselective hydroxymethylation of isovaleraldehyde and Kulinkovich cyclopropanation of silylated Roche esters were used as key steps for the dithiane syntheses. The method was applied for the preparation of brassinolide, its biosynthetic precursors and metabolites. In addition, a number of brassinosteroids with a double bond in the side chain were prepared as precursors for tritiated derivatives for biosynthetic studies.

Formation of the steroidal C-25 chiral center via the asymmetric alkylation methodology.

A novel approach for the preparation of steroids containing a chiral center at C-25 is reported. The key stereochemistry inducing step was asymmetric alkylation of pseudoephenamine amides of steroidal C-26 acids. The reaction proceeded with high diastereoselectivity (dr > 99 : 1). The developed methodology was successfully applied to the synthesis of (25R)- and (25S)-cholestenoic acids as well as (25R)- and (25S)-26-hydroxy brassinolides.

[Synthesis and biological activity of probable 24-norbrassinolide biosynthetic precursors].

A number of 24-norbrassinolide biosynthetic precursors containing low polar functional groups (3beta3-OH, 3-keto-, delta2- or 2alpha,3alpha-epoxy-) in A-cycle and (22R,23R)-diol in the side chain has been prepared. Studies of these compounds as proliferation regulators in MCF-7 human breast cancer and LnCaP human prostate adenocarcinoma cells showed that most nonpolar (22R,23R)-derivatives effectively suppressed proliferation. Dependence of proliferation on concentration of studied compounds was found in human prostate carcinoma LnCaP cells (IC50 = 13-28 microM at 72 h of incubation in a medium containing 10% FBS; suppression of DNA biosynthesis). A number of compounds induced apoptosis (23-33%); arrested cell cycle in S- and G2/M-phases; and caused partial cells detachment during prolonged incubations.

[Effect of the structure of the brassinosteroid side chain on monooxygenase activity of liver microsomes].

Possible pathways by which brassinosteroids affect the monooxygenase enzymatic system of mammalian liver microsomes, which is involved in the transformation of a broad spectrum of xenobiotics, were studied. The role of the structure of the side chain of brassinosteroids in the regulation of monooxygenase activity was studied using two natural compounds (24-epibrassinolide and 28-homobrassinolide) and two synthetic analogues, (22S, 23S-dihydroxy) stereoisomers. The results of this study show that brassinosteroids can directly influence the functioning of the microsomal enzymatic system. It was found that the degree of this influence depends on the side chain structure. This suggests the possibility of targeted modification of natural compounds to ensure the desired physiological effects.

[Synthesis of secasterol and 24-episecasterol and their toxicity in MCF-7 cells].

A convergent synthesis of biosynthetic precursors of brassinosteroids - secasterol and 24-episecasterol with Δ²-bond in cycle A is described. The key stages in the construction of the side chain of these compounds were Julia olefination of steroid 22-aldehyde followed by asymmetric Sharpless dihydroxylation of the intermediate Δ²²-olefin. Toxicity of synthesized compounds against breast carcinoma MCF-7 cells was studied.

[Synthesis of brassinolide and its biosynthetic precursors using methyl-3-hydroxy-2-methylpropionate].

Formal synthesis of plant hormones that belong to the group of 24alpha-methylbrassinosteroids, including brassinolide and its biosynthetic precursors with one hydroxyl group in their side chain, was performed. Stereochemistry of a methyl group at the C24 atom was provided by the choice of the desired enanthiomer of methyl-3-hydroxy-2-methylpropionate and by the sequence of its conversions into the chiral intermediate that was necessary for the formation of the C23-C28 fragment of the side chain. The (22R,23R)-diol group was introduced by the Sharpless asymmetric dihydroxylation of the intermediate delta22-steroids, the products of consecutive reactions of attachment of a low-molecular sulfone to the steroid C22-aldehyde, acetylation, and reductive desulfurization of the intermediate beta-acetoxysulfones. Reduction of 22-acetoxy-23,25-disulfones was shown to proceed with the preservation of the functional group at atom C22.

[Synthesis of sterols oxygenated in the terminal fragment of their side chains].

Methods of stereoselective synthesis of oxysterols are considered by the examples of (25R)-26-hydroxycholesterol, (24S)-24,25-epoxycholesterol, and (24S)-24-hydroxycholesterol containing functional groups in the terminal fragments of their side chains. Special attention is paid to the problems of construction of chiral centers C24 and C25.

[Oxysterols: genesis and basic functions].

The structural peculiarities of the most widespread oxysterols, the products of oxidative transformations of cholesterol are discussed. The transformations proceed with the participation of enzymatic systems of the body or as a result of various nonenzymatic reactions. The pathways of their formation from cholesterol are also considered. The role of oxysterols in the maintenance of cholesterol homeostasis and in the development of atherosclerosis is reviewed. The possibility of using oxysterols as markers of pathological processes is demonstrated.

[Enzyme immunoassay of (24R)-brassinosteroids].

Brassinosteroids are a new group of phytohormones that are widely distributed in plants and play an important role in the processes of plant growth and development. Physiological concentrations of brassinosteroids in plants are extremely low, and their analysis in organs and tissues is very difficult. This study is devoted to the chemical aspects of elaboration and to bioanalytical parameters of an immunoenzymatic system for quantitative determination of the phytohormones 24-epicastasterone and 24-epibrassinolide.

[A new synthesis of cerebrosterol and its 24-epimer from lithocholic acid].

A new method for the synthesis of both isomers of 24-hydroxycholesterol starting from lithocholic acid is proposed.

[Synthesis of (24S)-hydroxy- and (24S)-24,25-epoxycholesterol analogues, potential agonists of nuclear LXR receptors].

A new approach to the synthesis of a series of isomeric 24-hydroxy- and 24,25-epoxysteroids starting from lithocholic acid was proposed. Sharpless asymmetric hydroxylation of intermediate delta24-olefines was used as a reaction determining the stereochemistry of target compounds. The resulting derivatives are potential agonists of nuclear receptors LXRalpha and LXRbeta and are potentially useful in the structure-function studies.

[Synthesis of C24-functionalized hydroxysterols]. / Sintez C24-funktsionalizirovannykh oksisterinov.

The syntheses of (24S)-24,25-epoxycholesterol, (24S)-hydroxycholesterol, and 24-ketocholesterol are described. The compounds belong to oxysterols, which can be considered to be the modulators of cholesterol metabolism. The asymmetric hydroxylation of desmosterol acetate according to Sharpless was used as the key reaction in the stereoselective introduction of functionality in position 24.

Synthesis of C-5'-alkyl substituted 17-spirofuran 19-norsteroids.

A number of new steroidal 17-spirofuran derivatives of the 19-nor series containing Me, Et or (i)Pr-substituents in the heterocyclic moiety has been prepared, which are expected to have a strong progestagenic activity. The proposed approach made use of the 1-3-dipolar cycloaddition of low-molecular nitrile oxides with steroidal acetylenic alcohols followed by transformation of the isoxazole side chain.