RESUMEN
Electron transfer mediated by metalloproteins drives many biological processes. Rubredoxins are a ubiquitous [1Fe-0S] class of electron carriers that play an important role in bacterial adaptation to changing environmental conditions. In Mycobacterium tuberculosis, oxidative and acidic stresses as well as iron starvation induce rubredoxins expression. However, their functions during M. tuberculosis infection are unknown. In the present work, we show that rubredoxin B (RubB) is able to efficiently shuttle electrons from cognate reductases, FprA and FdR to support catalytic activity of cytochrome P450s, CYP124, CYP125, and CYP142, which are important for bacterial viability and pathogenicity. We solved the crystal structure of RubB and characterized the interaction between RubB and CYPs using site-directed mutagenesis. Mutations that not only neutralize single charge but also change the specific residues on the surface of RubB did not dramatically decrease activity of studied CYPs. Together with isothermal calorimetry (ITC) experiments, the obtained results suggest that interactions are transient and not highly specific. The redox potential of RubB is -264 mV vs. Ag/AgCl and the measured extinction coefficients are 9931 M-1cm-1 and 8371 M-1cm-1 at 380 nm and 490 nm, respectively. Characteristic parameters of RubB along with the discovered function might be useful for biotechnological applications. Our findings suggest that a switch from ferredoxins to rubredoxins might be crucial for M. tuberculosis to support CYPs activity during the infection.
Asunto(s)
Mycobacterium tuberculosis/metabolismo , Rubredoxinas/metabolismo , Calorimetría , Dicroismo Circular , Clonación Molecular , Cristalización , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/metabolismo , Transporte de Electrón , Regulación Bacteriana de la Expresión Génica , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Unión Proteica , Conformación Proteica , Rubredoxinas/química , Zinc/química , Zinc/metabolismoRESUMEN
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.
Asunto(s)
Flavonoides/química , Luteolina/química , Esterol 14-Desmetilasa/metabolismo , Humanos , Síndrome Metabólico/metabolismo , Simulación del Acoplamiento Molecular , Resonancia por Plasmón de SuperficieRESUMEN
Spreading of the multidrug-resistant (MDR) strains of the one of the most harmful pathogen Mycobacterium tuberculosis (Mtb) generates the need for new effective drugs. SQ109 showed activity against resistant Mtb and already advanced to Phase II/III clinical trials. Fast SQ109 degradation is attributed to the human liver Cytochrome P450s (CYPs). However, no information is available about interactions of the drug with Mtb CYPs. Here, we show that Mtb CYP124, previously assigned as a methyl-branched lipid monooxygenase, binds and hydroxylates SQ109 in vitro. A 1.25 Å-resolution crystal structure of the CYP124-SQ109 complex unambiguously shows two conformations of the drug, both positioned for hydroxylation of the ω-methyl group in the trans position. The hydroxylated SQ109 presumably forms stabilizing H-bonds with its target, Mycobacterial membrane protein Large 3 (MmpL3). We anticipate that Mtb CYPs could function as analogs of drug-metabolizing human CYPs affecting pharmacokinetics and pharmacodynamics of antitubercular (anti-TB) drugs.
Asunto(s)
Adamantano/análogos & derivados , Antituberculosos/química , Sistema Enzimático del Citocromo P-450/química , Etilenodiaminas/química , Simulación del Acoplamiento Molecular , Mycobacterium tuberculosis/enzimología , Adamantano/química , Adamantano/farmacología , Antituberculosos/farmacología , Sitios de Unión , Sistema Enzimático del Citocromo P-450/metabolismo , Etilenodiaminas/farmacología , Hidroxilación , Unión ProteicaRESUMEN
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.
Asunto(s)
Sistema Enzimático del Citocromo P-450/química , Citocromos b5/química , Sitio Alostérico , Animales , Técnicas Biosensibles , Bovinos , Escherichia coli/metabolismo , Caballos , Humanos , Cinética , Unión Proteica , Mapeo de Interacción de Proteínas , Termodinámica , Xenobióticos/químicaRESUMEN
Cytochrome P450scc (CYP 11A1) catalyzes the conversion of cholesterol (Ch) to pregnenolone, the precursor to steroid hormones. This process proceeds via three sequential monooxygenation reactions: two hydroxylations of Ch first form 22(R)-hydroxycholesterol (HC) and then 20α,22(R)-dihydroxycholesterol (DHC); a lyase reaction then cleaves the C20-C22 bond to form pregnenolone. Recent cryoreduction/annealing studies that employed electron paramagnetic resonance (EPR)/electron nuclear double resonance (ENDOR) spectroscopy [Davydov, R., et al. (2012) J. Am. Chem. Soc. 134, 17149] showed that compound I (Cpd I) is the active intermediate in the first step, hydroxylation of Ch. Herein, we have employed EPR and ENDOR spectroscopy to characterize the intermediates in the second and third steps of the enzymatic process, as conducted by 77 K radiolytic one-electron cryoreduction and subsequent annealing of the ternary oxy-cytochrome P450scc complexes with HC and DHC. This procedure is validated by showing that the cryoreduced ternary complexes of oxy-cytochrome P450scc with HC and DHC are catalytically competent and during annealing generate DHC and pregnenolone, respectively. Cryoreduction of the oxy-P450scc-HC ternary complex trapped at 77K produces the superoxo-ferrous P450scc intermediate along with a minor fraction of ferric hydroperoxo intermediates. The superoxo-ferrous intermediate converts into a ferric-hydroperoxo species after annealing at 145 K. During subsequent annealing at 170-180 K, the ferric-hydroperoxo intermediate converts to the primary product complex with the large solvent kinetic isotope effect that indicates Cpd I is being formed, and (1)H ENDOR measurements of the primary product formed in D2O demonstrate that Cpd I is the active species. They show that the primary product contains Fe(III) coordinated to the 20-O(1)H of DHC with the (1)H derived from substrate, the signature of the Cpd I reaction. Hydroperoxo ferric intermediates are the primary species formed during cryoreduction of the oxy-P450scc-DHC ternary complex, and they decay at 185 K with a strong solvent kinetic isotope effect to form low-spin ferric P450scc. Together, these observations indicated that Cpd I also is the active intermediate in the C20,22 lyase final step. In combination with our previous results, this study thus indicates that Cpd I is the active species in each of the three sequential monooxygenation reactions by which P450scc catalytically converts Ch to pregnenolone.
Asunto(s)
Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Colesterol/metabolismo , Hidroxicolesteroles/metabolismo , Pregnenolona/metabolismo , Animales , Bovinos , Colesterol/química , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/química , Espectroscopía de Resonancia por Spin del Electrón , Humanos , Hidroxicolesteroles/química , Hidroxilación , Oxidación-Reducción , Pregnenolona/químicaRESUMEN
Hepatic conversion to bile acids is a major elimination route for cholesterol in mammals. CYP7A1 catalyzes the first and rate-limiting step in classic bile acid biosynthesis, converting cholesterol to 7α-hydroxycholesterol. To identify the structural determinants that govern the stereospecific hydroxylation of cholesterol, we solved the crystal structure of CYP7A1 in the ligand-free state. The structure-based mutation T104L in the B' helix, corresponding to the nonpolar residue of CYP7B1, was used to obtain crystals of complexes with cholest-4-en-3-one and with cholesterol oxidation product 7-ketocholesterol (7KCh). The structures reveal a motif of residues that promote cholest-4-en-3-one binding parallel to the heme, thus positioning the C7 atom for hydroxylation. Additional regions of the binding cavity (most distant from the access channel) are involved to accommodate the elongated conformation of the aliphatic side chain. Structural complex with 7KCh shows an active site rigidity and provides an explanation for its inhibitory effect. Based on our previously published data, we proposed a model of cholesterol abstraction from the membrane by CYP7A1 for metabolism. CYP7A1 structural data provide a molecular basis for understanding of the diversity of 7α-hydroxylases, on the one hand, and cholesterol-metabolizing enzymes adapted for their specific activity, on the other hand.
Asunto(s)
Colesterol 7-alfa-Hidroxilasa/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Dominio Catalítico , Colesterol 7-alfa-Hidroxilasa/genética , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Humanos , Enlace de Hidrógeno , Hidroxilación , Cetocolesteroles/química , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Secundaria de ProteínaRESUMEN
In humans, the precursor to all steroid hormones, pregnenolone, is synthesized from cholesterol by an enzyme complex comprising adrenodoxin reductase (AdR), adrenodoxin (Adx), and a cytochrome P450 (P450scc or CYP11A1). This complex not only plays a key role in steroidogenesis, but also has long been a model to study electron transfer, multistep catalysis, and C-C bond cleavage performed by monooxygenases. Detailed mechanistic understanding of these processes has been hindered by a lack of structural information. Here we present the crystal structure of the complex of human Adx and CYP11A1--the first of a complex between a eukaryotic CYP and its redox partner. The structures with substrate and a series of reaction intermediates allow us to define the mechanism underlying sequential hydroxylations of the cholesterol and suggest the mechanism of C-C bond cleavage. In the complex the [2Fe-2S] cluster of Adx is positioned 17.4 Å away from the heme iron of CYP11A1. This structure suggests that after an initial protein-protein association driven by electrostatic forces, the complex adopts an optimized geometry between the redox centers. Conservation of the interaction interface suggests that this mechanism is common for all mitochondrial P450s.
Asunto(s)
Adrenodoxina/química , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/química , Mitocondrias/enzimología , Oxigenasas de Función Mixta/metabolismo , Pregnenolona/biosíntesis , Conformación Proteica , Adrenodoxina/metabolismo , Animales , Dominio Catalítico , Bovinos , Colesterol/metabolismo , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Humanos , Modelos Moleculares , Oxidación-Reducción , Unión Proteica , Electricidad EstáticaRESUMEN
We describe a bielectrode system for evaluation of the electrocatalytic activity of cytochrome P450 2E1 (CYP2E1) towards chlorzoxazone. One electrode of the system was employed to immobilize Bactosomes with human CYP2E1, cytochrome P450 reductase (CPR), and cytochrome b5 (cyt b5). The second electrode was used to quantify CYP2E1-produced 6-hydroxychlorzoxazone by its direct electrochemical oxidation, registered using square-wave voltammetry. Using this system, we determined the steady-state kinetic parameters of chlorzoxazone hydroxylation by CYP2E1 of Bactosomes immobilized on the electrode: the maximal reaction rate (Vmax) was 1.64 ± 0.08 min-1, and the Michaelis constant (KM) was 78 ± 9 µM. We studied the electrochemical characteristics of immobilized Bactosomes and have revealed that electron transfer from the electrode occurs both to the flavin prosthetic groups of CPR and the heme iron ions of CYP2E1 and cyt b5. Additionally, it has been demonstrated that CPR has the capacity to activate CYP2E1 electrocatalytic activity towards chlorzoxazone, likely through intermolecular electron transfer from the electrochemically reduced form of CPR to the CYP2E1 heme iron ion.
RESUMEN
Cytochromes P450 (CYP) are a family of membrane proteins involved in the production of endogenous molecules and the metabolism of xenobiotics. It is well-known that the composition of the membrane can influence the activity and orientation of CYP proteins. However, little is known about how membrane composition affects the ligand binding properties of CYP. In this study, we utilized surface plasmon resonance and fluorescence lifetime analysis to examine the impact of membrane micro-environment composition on the interaction between human microsomal CYP51 (CYP51A1) and its inhibitor, luteolin 7,3'-disulphate (LDS). We observed that membranes containing cholesterol or sphingomyelin exhibited the lowest apparent equilibrium dissociation constant for the CYP51A1-LDS complex. Additionally, the tendency for relation between kinetic parameters of the CYP51A1-LDS complex and membrane viscosity and overall charge was observed. These findings suggest that the specific composition of the membrane, particularly the presence of cholesterol and sphingomyelin, plays a vital role in regulating the interaction between CYP enzymes and their ligands.
Asunto(s)
Sistema Enzimático del Citocromo P-450 , Esfingomielinas , Humanos , Sistema Enzimático del Citocromo P-450/metabolismo , Colesterol/metabolismo , Luteolina/farmacologíaRESUMEN
New antitubercular drugs are vital due to the spread of resistant strains. Carbethoxyhexyl imidazole (CHImi) inhibits cytochrome P450 CYP124, which is a steroid-metabolizing enzyme that is important for the survival of Mycobacterium tuberculosis in macrophages. The available crystal structure of the CYP124-CHImi complex reveals two glycerol molecules in the active site. A 1.15â Å resolution crystal structure of the glycerol-free CYP124-CHimi complex reported here shows multiple conformations of CHImi and the CYP124 active site which were previously restricted by glycerol. Complementary molecular dynamics simulations show coherence of the ligand and enzyme conformations. Spectrophotometric titration confirmed the influence of glycerol on CHImi binding: the affinity decreases more than tenfold in glycerol-containing buffer. In addition, it also showed that glycerol has a similar effect on other azole and triazole CYP124 ligands. Together, these data show that glycerol may compromise structural-functional studies and impede rational drug-design campaigns.
Asunto(s)
Sistema Enzimático del Citocromo P-450 , Mycobacterium tuberculosis , Ligandos , Modelos Moleculares , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/farmacología , Antituberculosos , Cristalografía por Rayos XRESUMEN
Cytochrome P450scc (CYP11A1) catalyzes conversion of cholesterol (CH) to pregnenolone, the precursor to all steroid hormones. This process proceeds via three sequential monooxygenation reactions: two stereospecific hydroxylations with formation first of 22R-hydroxycholesterol (22-HC) and then 20α,22R-dihydroxycholesterol (20,22-DHC), followed by C20-C22 bond cleavage. Herein we have employed EPR and ENDOR spectroscopy to characterize the intermediates in the first hydroxylation step by 77 K radiolytic one-electron cryoreduction and subsequent annealing of the ternary oxy-cytochrome P450scc-cholesterol complex. This approach is fully validated by the demonstration that the cryoreduced ternary complex of oxy-P450scc-CH is catalytically competent and hydroxylates cholesterol to form 22-HC with no detectable formation of 20-HC, just as occurs under physiological conditions. Cryoreduction of the ternary complex trapped at 77 K produces predominantly the hydroperoxy-ferriheme P450scc intermediate, along with a minor fraction of peroxo-ferriheme intermediate that converts into a new hydroperoxo-ferriheme species at 145 K. This behavior reveals that the distal pocket of the parent oxy-P450scc-cholesterol complex exhibits an efficient proton delivery network, with an ordered water molecule H-bonded to the distal oxygen of the dioxygen ligand. During annealing of the hydroperoxy-ferric P450scc intermediates at 185 K, they convert to the primary product complex in which CH has been converted to 22-HC. In this process, the hydroperoxy-ferric intermediate decays with a large solvent kinetic isotope effect, as expected when proton delivery to the terminal O leads to formation of Compound I (Cpd I). (1)H ENDOR measurements of the primary product formed in deuterated solvent show that the heme Fe(III) is coordinated to the 22R-O(1)H of 22-HC, where the (1)H is derived from substrate and exchanges to D after annealing at higher temperatures. These observations establish that Cpd I is the agent that hydroxylates CH, rather than the hydroperoxy-ferric heme.
Asunto(s)
Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Colesterol/metabolismo , Compuestos Férricos/metabolismo , Compuestos Ferrosos/metabolismo , Oxígeno/metabolismo , Pregnenolona/metabolismo , Colesterol/química , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/química , Espectroscopía de Resonancia por Spin del Electrón , Compuestos Férricos/química , Compuestos Ferrosos/química , Modelos Moleculares , Oxidación-Reducción , Oxígeno/química , Pregnenolona/químicaRESUMEN
Electron transfer in metalloproteins is a driving force for many biological processes and widely distributed in nature. Rubredoxin B (RubB) from Mycobacterium tuberculosis is a first example among [1Fe-0S] proteins that support catalytic activity of terminal sterol-monooxygenases enabling its application in metabolic engineering. To explore the tolerance of RubB to the specific amino acid changes we evaluated the effect of surface mutations on its electrochemical properties. Based on the RubB fold we also designed the mutant with a putative additional site for protein-protein interactions to further evaluate electron transfer and electrochemical properties. The investigation of redox properties of mutant variants of RubB was done using screen-printed graphite electrodes (SPEs) modified with stable dispersion of multi-walled carbon nanotubes (MWCNTs). The redox potentials (midpoint potentials, E0ê) of mutants did not significantly differ from the wild type protein and vary in the range of -264 to -231 mV vs. Ag/AgCl electrode. However, all mutations affect electron transfer rate between the protein and electrode. Notably, the modulation of the protein-protein interactions was observed for the insertion mutant suggesting the possibility of tailoring of rubredoxin for the selected redox-partner. Overall, RubB is tolerant to the significant modifications in its structure enabling rational engineering of novel redox proteins.
Asunto(s)
Mutación , Mycobacterium tuberculosis/química , Rubredoxinas/química , Técnicas Electroquímicas , Rubredoxinas/genética , Rubredoxinas/metabolismoRESUMEN
Ferredoxins are small iron-sulfur proteins and key players in essential metabolic pathways. Among all types, 3Fe-4S ferredoxins are less studied mostly due to anaerobic requirements. Their complexes with cytochrome P450 redox partners have not been structurally characterized. In the present work, we solved the structures of both 3Fe-4S ferredoxins from M. tuberculosis-Fdx alone and the fusion FdxE-CYP143. Our SPR analysis demonstrated a high-affinity binding of FdxE to CYP143. According to SAXS data, the same complex is present in solution. The structure reveals extended multipoint interactions and the shape/charge complementarity of redox partners. Furthermore, FdxE binding induced conformational changes in CYP143 as evident from the solved CYP143 structure alone. The comparison of FdxE-CYP143 and modeled Fdx-CYP51 complexes further revealed the specificity of ferredoxins. Our results illuminate the diversity of electron transfer complexes for the production of different secondary metabolites.
RESUMEN
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.
Asunto(s)
Esterol 14-Desmetilasa , Aromatasa , Humanos , EsteroidesRESUMEN
Mycobacterium tuberculosis (Mtb) infection is among top ten causes of death worldwide, and the number of drug-resistant strains is increasing. The direct interception of human immune signaling molecules by Mtb remains elusive, limiting drug discovery. Oxysterols and secosteroids regulate both innate and adaptive immune responses. Here we report a functional, structural, and bioinformatics study of Mtb enzymes initiating cholesterol catabolism and demonstrated their interrelation with human immunity. We show that these enzymes metabolize human immune oxysterol messengers. Rv2266 - the most potent among them - can also metabolize vitamin D3 (VD3) derivatives. High-resolution structures show common patterns of sterols binding and reveal a site for oxidative attack during catalysis. Finally, we designed a compound that binds and inhibits three studied proteins. The compound shows activity against Mtb H37Rv residing in macrophages. Our findings contribute to molecular understanding of suppression of immunity and suggest that Mtb has its own transformation system resembling the human phase I drug-metabolizing system.
Asunto(s)
Metabolismo Energético , Interacciones Huésped-Patógeno , Mycobacterium tuberculosis/inmunología , Tuberculosis/inmunología , Tuberculosis/metabolismo , 3-Hidroxiesteroide Deshidrogenasas/química , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Catálisis , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/metabolismo , Activación Enzimática , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunidad , Isoenzimas , Modelos Moleculares , Oxiesteroles/química , Oxiesteroles/metabolismo , Proteínas Recombinantes , Relación Estructura-Actividad , Tuberculosis/microbiologíaRESUMEN
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⯵M. Abiraterone D4A metabolite demonstrates competitive/mixed type CYP21A2 inhibition with an inhibitory constant of 1.8⯱â¯0.8⯵M, as obtained by Dixon plot. These results make it possible to predict the adverse effects of the new perspective candidate compound for antitumor therapy.
Asunto(s)
Androstenos/farmacología , Inhibidores Enzimáticos del Citocromo P-450/farmacología , Esteroide 21-Hidroxilasa/antagonistas & inhibidores , Androstenos/química , Inhibidores Enzimáticos del Citocromo P-450/química , Relación Dosis-Respuesta a Droga , Humanos , Conformación Molecular , Simulación del Acoplamiento Molecular , Esteroide 21-Hidroxilasa/metabolismo , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Androstenos/farmacología , Antibacterianos/farmacocinética , Citocromo P-450 CYP3A/efectos de los fármacos , Eritromicina/farmacocinética , Antineoplásicos/farmacología , Citocromo P-450 CYP3A/metabolismo , Inhibidores del Citocromo P-450 CYP3A/farmacología , Interacciones Farmacológicas , Humanos , Hidroxilación , Simulación del Acoplamiento MolecularRESUMEN
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 =â¯365â¯nm after treatment with sulfuric acid:ethanol (3:1) mixture (λem = 525⯱â¯2â¯nm and λem = 427⯱â¯2â¯nm, respectively). The detection limit for 6ß-hydroxycortisol was estimated to be 0.32⯵M (corresponding to 0.095â¯nmol in 300⯵L sample) (S/Nâ¯=â¯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¯5â¯pmol of product per min per pmol of electroactive enzyme and the Michaelis constant (KM) as 10⯱â¯2⯵M. In our system, ketoconazole inhibited hydroxylase activity of CYP3A4 towards hydrocortisone with the IC50 value of 70⯱â¯5â¯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.
Asunto(s)
Citocromo P-450 CYP3A/química , Enzimas Inmovilizadas/química , Hidrocortisona/análogos & derivados , Hidrocortisona/química , Ácidos Sulfúricos/química , Catálisis , Electrólisis , FluorometríaRESUMEN
A number of isoxazole, 1,2,3-triazole, tetrazole, and 1,2,4-oxadiazole derivatives of [17(20)E]-21-norpregnene comprising 3ß-hydroxy-5-ene and 3-oxo-4-ene fragments were prepared. Among the key steps for the synthesis of isoxazoles, 1,2,3-triazoles, and tetrazoles were (i) 1,3-dipolar cycloaddition of nitrile oxides or azides to acetylenes or nitriles and ii) dehydration of 17ß-hydroxy-17α-methylene-azoles to [17(20)E]-21-norpregnene derivatives. 1,2,4-Oxadiazoles were prepared through the formation of acetimidamides. Potency of the synthesized compounds to inhibit CYP17A1 and to suppress growth of prostate carcinoma cells was investigated. Among the new azole derivatives, four compounds were found possessing high anti-proliferative activity.
Asunto(s)
Antineoplásicos/farmacología , Azoles/farmacología , Norpregnadienos/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Antineoplásicos/síntesis química , Antineoplásicos/uso terapéutico , Azoles/síntesis química , Azoles/química , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Masculino , Estructura Molecular , Norpregnadienos/síntesis química , Norpregnadienos/uso terapéutico , Células PC-3 , Neoplasias de la Próstata/patología , Células Tumorales CultivadasRESUMEN
Trypanosoma cruzi, which causes Chagas disease, is a significant health threat in many countries and affects millions of people. Given the magnitude of this disease, a broader understanding of trypanocidal mechanisms is needed to prevent and treat infection. Natural endoperoxides, such as ergosterol peroxide, have been shown to be toxic to parasites without causing harm to human cells or tissues. Although prior studies have demonstrated the trypanocidal activity of ergosterol peroxide, the cellular and molecular mechanisms remain unknown. The results of this study indicate that a free-radical reaction occurs in T. cruzi following ergosterol peroxide exposure, leading to cell death. Using a combination of biochemical, microscopic and in silico experimental approaches, we have identified, for the first time, the cellular and molecular cytotoxic mechanism of an ergosterol peroxide obtained from Pleurotus ostreatus (Jacq) P. Kumm. f. sp. Florida.