Human Lanosterol 14-Alpha Demethylase (CYP51A1) Is a Putative Target for Natural Flavonoid Luteolin 7,3'-Disulfate.

Widespread pathologies such as atherosclerosis, metabolic syndrome and cancer are associated with dysregulation of sterol biosynthesis and metabolism. Cholesterol modulates the signaling pathways of neoplastic transformation and tumor progression. Lanosterol 14-alpha demethylase (cytochrome P450(51), CYP51A1) catalyzes one of the key steps in cholesterol biosynthesis. The fairly low somatic mutation frequency of CYP51A1, its druggability, as well as the possibility of interfering with cholesterol metabolism in cancer cells collectively suggest the clinical importance of CYP51A1. Here, we show that the natural flavonoid, luteolin 7,3'-disulfate, inhibits CYP51A1 activity. We also screened baicalein and luteolin, known to have antitumor activities and low toxicity, for their ability to interact with CYP51A1. The Kd values were estimated using both a surface plasmon resonance optical biosensor and spectral titration assays. Unexpectedly, in the enzymatic activity assays, only the water-soluble form of luteolin-luteolin 7,3'-disulfate-showed the ability to potently inhibit CYP51A1. Based on molecular docking, luteolin 7,3'-disulfate binding suggests blocking of the substrate access channel. However, an alternative site on the proximal surface where the redox partner binds cannot be excluded. Overall, flavonoids have the potential to inhibit the activity of human CYP51A1 and should be further explored for their cholesterol-lowering and anti-cancer activity.

Transformations, NMR studies and biological testing of some 17ß-isoxazolyl steroids and their heterocyclic ring cleavage derivatives.

The synthesis and NMR structure analysis of a group of oxygenated steroids containing isoxazole, dihydrofuran, tetrahydrofuran rings or enamino carbonyl fragment in the side chain have been fulfilled. The prepared compounds were tested toward several enzymes (human cytochrome P450s CYP17, CYP19, CYP51 and CYP51 of pathogenic fungus Candida glabrata) as their potential inhibitors. A number steroids show a high level affinity (micro- and submicromole) for the enzyme-ligand complexes of the tested compounds with human CYP51, CYP19 and CYP51 of C. glabrata.

In vitro interactions of abiraterone, erythromycin, and CYP3A4: implications for drug-drug interactions.

Potential drug-drug interactions of the antitumor drug abiraterone and the macrolide antibiotic erythromycin were studied at the stage of cytochrome P450 3A4 (CYP3A4) biotransformation. Using differential spectroscopy, we have shown that abiraterone is a type II ligand of CYP3A4. The dependence of CYP3A4 spectral changes on the concentration of abiraterone is sigmoidal, which indicates cooperative interactions of CYP3A4 with abiraterone; these interactions were confirmed by molecular docking. The dissociation constant (Kd ) and Hill coefficient (h) values for the CYP3A4-abiraterone complex were calculated as 3.8 ± 0.1 µM and 2.3 ± 0.2, respectively. An electrochemical enzymatic system based on CYP3A4 immobilized on a screen-printed electrode was used to show that abiraterone acts as a competitive inhibitor toward erythromycin N-demethylase activity of CYP3A4 (apparent Ki  = 8.1 ± 1.2 µM), while erythromycin and its products of enzymatic metabolism do not affect abiraterone N-oxidation by CYP3A4. In conclusion, the inhibition properties of abiraterone toward CYP3A4-dependent N-demethylation of erythromycin and the biologically inert behavior of erythromycin toward abiraterone hydroxylation were demonstrated.

Estimation of the inhibiting impact of abiraterone D4A metabolite on human steroid 21-monooxygenase (CYP21A2).

Abiraterone D4A metabolite, the product of 3ß-hydroxysteroid dehydrogenase activity toward abiraterone, may serve as a potential antitumor agent for the treatment of prostate cancer. The main adverse effect of abiraterone is the disruption of corticosteroid biosynthesis, and the more pharmacologically active abiraterone D4A metabolite may have the same issues. We therefore estimated the inhibiting impact of the abiraterone D4A metabolite on one of the key corticosteroidogenic enzymes - human steroid 21-monooxygenase (CYP21A2). Molecular docking of D4A into the active site of CYP21A2 has been predicted to be similar to abiraterone binding with the enzyme. Abiraterone D4A metabolite, similar to abiraterone, induces type II spectral changes of CYP21A2. The spectral dissociation constant for the abiraterone D4A metabolite-CYP21A2 complex was calculated as 3.4 ±â€¯0.5 µM. Abiraterone D4A metabolite demonstrates competitive/mixed type CYP21A2 inhibition with an inhibitory constant of 1.8 ±â€¯0.8 µM, as obtained by Dixon plot. These results make it possible to predict the adverse effects of the new perspective candidate compound for antitumor therapy.

A large-scale comparative analysis of affinity, thermodynamics and functional characteristics of interactions of twelve cytochrome P450 isoforms and their redox partners.

The aim of the present work was to establish the thermodynamic and functional differences in the protein-protein interactions between the components of the P450-dependent mitochondrial (mit) and microsomal (mic) monooxygenase systems using 12 different isoforms of cytochromes P450 and two redox partners, NADPH-dependent cytochrome P450 reductase (CPR) and adrenodoxin (Adx). Comparative analysis of the affinity, thermodynamics, enzymatic activity and the ability for one-electron reduction has been carried out. The study of protein-protein interactions to determine the equilibrium dissociation constants (Kd) was performed using surface plasmon resonance (SPR) biosensor Biacore 3000. We demonstrated that CPR and Adx interacted with both, micCYPs and mitCYPs, with different affinities (Kd values ranged from 0.01 to 2 µM). All complexes of microsomal (micCYP) and mitochondrial (mitCYP) cytochrome P450 with redox partners can be divided into three groups depending on the prevalent role of either enthalpy or entropy contribution. About 90% of CYP/redox partner complexes were entropy-driven, while the contribution of enthalpy and entropy differed significantly in case of mitCYP/Adx complexes. The CYP11A1/Adx complex was enthalpy-driven, while CYP11B1/Adx and CYP11B2/Adx complexes were entropy-driven. Thermodynamic discrimination of mitCYPs/Adx complexes is likely associated with the different functional impact of CYP11A1 and CYP11B. The exception was the enthalpy-entropy-driven (mixed type) CYP21A2/Adx complex. CPR and Adx were able to transfer the first electron to micCYPs while mitCYPs demonstrated high specificity to Adx. Productive catalysis for mitCYPs observed only in the presence of Adx/AdR pair, while in case of steroidogenic micCYPs (CYP17A1, CYP19A1, and CYP21A2) it was found either in the presence of a CPR or an Adx/AdR pair. From the evolutionary point of view, the type 1 electron transport system (mitCYPs, Adx and NADPH-dependent adrenodoxin reductase (AdR)) increased the specialization of protein-protein interactions (PPI) significantly, which was accompanied by an increase in the specificity of electron transfer. In contrast, the evolution of the type 2 electron transport system (micCYPs and CPR) led to an increase in versatility of PPI as demonstrated for steroidogenic microsomal cytochrome P450s. Our data enhance the current understanding of molecular recognition and summarize qualitative and thermodynamic characteristics of protein-protein interactions in the P450-dependent mitochondrial and microsomal monooxygenase systems.

Assessment of electrocatalytic hydroxylase activity of cytochrome P450 3A4 (CYP3A4) by means of derivatization of 6ß-hydroxycortisol by sulfuric acid for fluorimetric assay.

We used rapid one-step derivatization of 6ß-hydroxylated hydrocortisone by sulfuric acid for fluorimetric determination of CYP3A4-dependent hydroxylase reaction in the electrochemical system. We have shown that CYP3A4 substrate - hydrocortisone - and its 6ß-hydroxylated product have different emission wavelengths at an excitation λex = 365 nm after treatment with sulfuric acid:ethanol (3:1) mixture (λem = 525 ±â€¯2 nm and λem = 427 ±â€¯2 nm, respectively). The detection limit for 6ß-hydroxycortisol was estimated to be 0.32 µM (corresponding to 0.095 nmol in 300 µL sample) (S/N = 3). Using the fluorimetric method of 6ß-hydroxycortisol detection following the electrolysis of hydrocortisone with CYP3A4 immobilized on a screen-printed graphite electrode modified by didodecyldimethylammonium bromide we have calculated the steady-state kinetic parameters of CYP3A4 for hydrocortisone: the maximal rate of the reaction (Vmax) as 89 ±â€¯5 pmol of product per min per pmol of electroactive enzyme and the Michaelis constant (KM) as 10 ±â€¯2 µM. In our system, ketoconazole inhibited hydroxylase activity of CYP3A4 towards hydrocortisone with the IC50 value of 70 ±â€¯5 nM. The approach proposed for determination of the CYP3A4 electrocatalytic activity can be used for throughput screening of different modulators of this cytochrome P450 isozyme during drug development.

Interaction of 17α-hydroxylase, 17(20)-lyase (CYP17A1) inhibitors - abiraterone and galeterone - with human sterol 14α-demethylase (CYP51A1).

Abiraterone and galeterone induce type I differential spectral changes in human sterol 14α-demethylase (cytochrome P450 51A1, CYP51A1) with the sigmoidal shape of the binding curve. After approximation of the data by Hill model, the half-saturation concentrations (K0.5) were estimated as 22 ±â€¯1 µM and 16 ±â€¯1 µM and the Hill coefficients as 2.4 ±â€¯0.2 and 1.97 ±â€¯0.23 for abiraterone and galeterone, respectively. We analyzed the catalytic activity of CYP51A1 towards abiraterone and galeterone using an electrochemical system based on recombinant CYP51A1 immobilized on the screen-printed graphite electrode (SPE) modified by didodecyldimethylammonium bromide (DDAB) film. The study revealed the amperometric response of CYP51A1 upon addition of abiraterone, which may indicate the substrate properties of abiraterone towards CYP51A1. Galeterone caused negligible amperometric response of CYP51A1. Mass-spectrometric analysis of the products of CYP51A1-dependent electrocatalytic reaction at a controlled potential towards abiraterone and galeterone revealed products with m/z of 366.3 and 405.2, respectively, indicating monohydroxylation of abiraterone and galeterone. We have observed the sigmoidal character of the dependence of the catalytic current on abiraterone concentration. Analysis of molecular docking data demonstrated the ability of abiraterone and galeterone to bind to the active site of CYP51A1, but abiraterone occupies the position closer to the heme.

Thermodynamics of interactions between mammalian cytochromes P450 and b5.

Cytochromes P450 (CYPs) play an important role in the metabolism of xenobiotics and various endogenous substrates. Being a crucial component of the microsomal monooxygenase system, CYPs are involved in numerous protein-protein interactions. However, mechanisms underlying molecular interactions between components of the monooxygenase system still need better characterization. In this study thermodynamic parameters of paired interactions between mammalian CYPs and cytochromes b5 (CYB5) have been evaluated using a Surface Plasmon Resonance (SPR) based biosensor Biacore 3000. Analysis of 18 pairs of CYB5-CYP complexes formed by nine different isoforms of mammalian CYPs and two isoforms of human CYB5 has shown that thermodynamically these complexes can be subdivided into enthalpy-driven and entropy-driven groups. Formation of the enthalpy-driven complexes was observed in the case of microsomal CYPs allosterically regulated by CYB5 (CYB5A-CYP3A4, CYB5A-CYP3A5, CYB5A-CYP17A1). The entropy-driven complexes were formed when CYB5 had no effect on the CYP activity (CYB5A-CYP51A1, CYB5A-CYP1B1, CYB5B-CYP11A1). Results of this study suggest that such interactions determining protein clustering are indirectly linked to the monooxygenase functioning. Positive ΔH values typical for such interactions may be associated with displacement of the solvation shells of proteins upon clustering. CYB5-CYP complex formation accompanied by allosteric regulation of CYP activity by CYB5 is enthalpy-dependent.

Computer-aided design of aptamers for cytochrome p450.

Aptamers are short single-stranded DNA or RNA oligonucleotides that can bind to their targets with high affinity and specificity. Usually, they are experimentally selected using the SELEX method. Here, we describe an approach toward the in silico selection of aptamers for proteins. This approach involves three steps: finding a potential binding site, designing the recognition and structural parts of the aptamers and evaluating the experimental affinity. Using this approach, a set of 15-mer aptamers for cytochrome P450 51A1 was designed using docking and molecular dynamics simulation. An experimental evaluation of the synthesized aptamers using SPR biosensor showed that these aptamers interact with cytochrome P450 51A1 with Kd values in the range of 10(-6)-10(-7) M.