RESUMEN
Cytochrome P450 (P450, CYP) 11A1 is the classical cholesterol side chain cleavage enzyme (P450scc) that removes six carbons of the side chain, the first and rate-limiting step in the synthesis of all mammalian steroids. The reaction is a 3-step, 6-electron oxidation that proceeds via formation of 22R-hydroxy (OH) and 20R,22R-(OH)2 cholesterol, yielding pregnenolone. We expressed human P450 11A1 in bacteria, purified the enzyme in the absence of nonionic detergents, and assayed pregnenolone formation by HPLC-mass spectrometry of the dansyl hydrazone. The reaction was inhibited by the nonionic detergent Tween 20, and several lipids did not enhance enzymatic activity. The 22R-OH and 20R,22R-(OH)2 cholesterol intermediates were bound to P450 11A1 relatively tightly, as judged by steady-state optical titrations and koff rates. The electron donor adrenodoxin had little effect on binding; the substrate cholesterol showed a â¼5-fold stimulatory effect on the binding of adrenodoxin to P450 11A1. Presteady-state single-turnover kinetic analysis was consistent with a highly processive reaction with rates of intermediate oxidation steps far exceeding dissociation rates for products and substrates. The presteady-state kinetic analysis revealed a second di-OH cholesterol product, separable by HPLC, in addition to 20R,22R-(OH)2 cholesterol, which we characterized as a rotamer that was also converted to pregnenolone at a similar rate. The first oxidation step (at C-22) is the slowest, limiting the overall rate of cleavage. d3-Cholesterol showed no kinetic deuterium isotope effect on C-22, indicating that C-H bond cleavage is not rate-limiting in the first hydroxylation step.
Asunto(s)
Enzima de Desdoblamiento de la Cadena Lateral del Colesterol , Colesterol , Pregnenolona , Humanos , Adrenodoxina/metabolismo , Colesterol/química , Colesterol/metabolismo , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/química , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/aislamiento & purificación , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Cinética , Pregnenolona/química , Pregnenolona/metabolismo , Unión Proteica , Oxidación-Reducción , Estructura MolecularRESUMEN
The multifunctional cytochrome P450 17A1 (CYP17A1) plays a crucial role in human steroid hormone synthesis (UniProtKBâP05093). It first carries out standard monooxygenase chemistry, converting pregnenolone (PREG) and progesterone (PROG) into 17OH-PREG and 17OH-PROG, utilizing a "Compound I" to initiate hydrogen abstraction and radical recombination in the classic "oxygen rebound" mechanism. Additionally, these hydroxylated products also serve as substrates in a second oxidative cycle which cleaves the 17-20 carbon-carbon bond to form dehydroepiandrosterone and androstenedione, which are key precursors in the generation of powerful androgens and estrogens. Interestingly, in humans, with 17OH-PREG, this so-called lyase reaction is more efficient than with 17OH-PROG, based on Kcat/Km values. In the present work, the asparagine residue at 202 position was replaced by serine, an alteration which can affect substrate orientation and control substrate preference for the lyase reaction. First, we report studies of solvent isotope effects for the N202S CYP17A1 mutant in the presence of 17OH-PREG and 17OH-PROG, which suggest that the ferric peroxo species is the predominant catalytically active intermediate in the lyase step. This conclusion is further supported by employing a combination of cryoradiolysis and resonance Raman techniques to successfully trap and structurally characterize the key reaction intermediates, including the peroxo, the hydroperoxo, and the crucial peroxo-hemiketal intermediate. Collectively, these studies show that the mutation causes active site structural changes that alter the H-bonding interactions with the key Fe-O-O fragment and the degree of protonation of the reactive ferric peroxo intermediate, thereby impacting lyase efficiency.
Asunto(s)
Asparagina , Esteroide 17-alfa-Hidroxilasa , Androstenodiona , Dominio Catalítico , Humanos , Pregnenolona/química , Progesterona/química , Esteroide 17-alfa-Hidroxilasa/químicaRESUMEN
The GABAA receptor is inhibited by the endogenous sulfated steroids pregnenolone sulfate (PS) and dehydroepiandrosterone sulfate (DHEAS). It has been proposed in previous work that these steroids act by enhancing desensitization of the receptor. Here, we have investigated the modulatory effects of the steroids on the human α1ß3γ2L GABAA receptor. Using electrophysiology and quantitative model-based data analysis, we show that exposure to the steroid promotes occupancy of a nonconducting state that retains high affinity to the transmitter but whose properties differ from those of the classic, transmitter-induced desensitized state. From the analysis of the inhibitory actions of two combined steroids, we infer that PS and DHEAS act through shared or overlapping binding sites. SIGNIFICANCE STATEMENT: Previous work has proposed that sulfated neurosteroids inhibit the GABAA receptor by enhancing the rate of entry into the desensitized state. This study shows that the inhibitory steroids pregnenolone sulfate and dehydroepiandrosterone sulfate act through a common interaction site by stabilizing a distinct nonconducting state.
Asunto(s)
Sulfato de Deshidroepiandrosterona/farmacología , Antagonistas del GABA/farmacología , Pregnenolona/farmacología , Receptores de GABA-A/metabolismo , Animales , Sulfato de Deshidroepiandrosterona/química , Relación Dosis-Respuesta a Droga , Femenino , Antagonistas del GABA/química , Humanos , Neuroesteroides/química , Neuroesteroides/farmacología , Pregnenolona/química , Estabilidad Proteica , Receptores de GABA-A/química , Xenopus laevisRESUMEN
CYP17A1 is an essential human steroidogenic enzyme, which catalyzes two sequential reactions leading to the formation of androstenedione from progesterone and dehydroepiandrosterone from pregnenolone. The second reaction is the C17-C20 bond scission, which is strongly dependent on the presence of cytochrome b5 and displays a heretofore unexplained more pronounced acceleration when 17OH-progesteone (17OH-PROG) is a substrate. The origin of the stimulating effect of cytochrome b5 on C-C bond scission catalyzed by CYP17A1 is still debated as mostly due to either the acceleration of the electron transfer to the P450 oxy complex or allosteric effects of cytochrome b5 favoring active site conformations that promote lyase activity. Using resonance Raman spectroscopy, we compared the effect of Mn-substituted cytochrome b5 (Mn-Cytb5) on the oxy complex of CYP17A1 with both proteins co-incorporated in lipid nanodiscs. For CYP17A1 with 17OH-PROG, a characteristic shift of the Fe-O mode is observed in the presence of Mn-b5, indicating reorientation of a hydrogen bond between the 17OH group of the substrate from the terminal to the proximal oxygen atom of the Fe-O-O moiety, a configuration favorable for the lyase catalysis. For 17OH-pregnenolone, no such shift is observed, the favorable H-bonding orientation being present even without Mn-Cytb5. These new data provide a precise allosteric interpretation for the more pronounced acceleration seen for the 17OH-PROG substrate.
Asunto(s)
Citocromos b5/química , Esteroide 17-alfa-Hidroxilasa/metabolismo , Regulación Alostérica , Biocatálisis , Dominio Catalítico , Citocromos b5/metabolismo , Humanos , Pregnenolona/química , Pregnenolona/metabolismo , Esteroide 17-alfa-Hidroxilasa/química , Especificidad por SustratoRESUMEN
Neuroactive steroids are potent modulators of microglial functions and are capable of counteracting their excessive reactivity. This action has mainly been ascribed to neuroactive steroids released from other sources, as microglia have been defined unable to produce neurosteroids de novo. Unexpectedly, immortalized murine microglia recently exhibited this de novo biosynthesis; herein, de novo neurosteroidogenesis was characterized in immortalized human microglia. The results demonstrated that C20 and HMC3 microglial cells constitutively express members of the neurosteroidogenesis multiprotein machinery-in particular, the transduceosome members StAR and TSPO, and the enzyme CYP11A1. Moreover, both cell lines produce pregnenolone and transcriptionally express the enzymes involved in neurosteroidogenesis. The high TSPO expression levels observed in microglia prompted us to assess its role in de novo neurosteroidogenesis. TSPO siRNA and TSPO synthetic ligand treatments were used to reduce and prompt TSPO function, respectively. The TSPO expression downregulation compromised the de novo neurosteroidogenesis and led to an increase in StAR expression, probably as a compensatory mechanism. The pharmacological TSPO stimulation the de novo neurosteroidogenesis improved in turn the neurosteroid-mediated release of Brain-Derived Neurotrophic Factor. In conclusion, these results demonstrated that de novo neurosteroidogenesis occurs in human microglia, unravelling a new mechanism potentially useful for future therapeutic purposes.
Asunto(s)
Microglía/metabolismo , Neuroesteroides/metabolismo , Receptores de GABA/metabolismo , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Línea Celular , Regulación de la Expresión Génica , Humanos , Neuroesteroides/química , Pregnenolona/química , Pregnenolona/metabolismoRESUMEN
The CB1 cannabinoid receptor is a G-protein coupled receptor highly expressed throughout the central nervous system that is a promising target for the treatment of various disorders, including anxiety, pain, and neurodegeneration. Despite the wide therapeutic potential of CB1, the development of drug candidates is hindered by adverse effects, rapid tolerance development, and abuse potential. Ligands that produce biased signaling-the preferential activation of a signaling transducer in detriment of another-have been proposed as a strategy to dissociate therapeutic and adverse effects for a variety of G-protein coupled receptors. However, biased signaling at the CB1 receptor is poorly understood due to a lack of strongly biased agonists. Here, we review studies that have investigated the biased signaling profile of classical cannabinoid agonists and allosteric ligands, searching for a potential therapeutic advantage of CB1 biased signaling in different pathological states. Agonist and antagonist bound structures of CB1 and proposed mechanisms of action of biased allosteric modulators are used to discuss a putative molecular mechanism for CB1 receptor activation and biased signaling. Current studies suggest that allosteric binding sites on CB1 can be explored to yield biased ligands that favor or hinder conformational changes important for biased signaling.
Asunto(s)
Agonistas de Receptores de Cannabinoides/química , Receptor Cannabinoide CB1/química , Sitio Alostérico , Sistema Nervioso Central/metabolismo , Humanos , Indoles/química , Ligandos , Modelos Moleculares , Piperidinas/química , Pregnenolona/química , Unión Proteica , Conformación Proteica , Transducción de SeñalRESUMEN
We explore here a long-standing mechanistic question by using quantum-mechanical/molecular-mechanical (QM/MM) methodology. The question concerns the mechanism of steroid hormone biosynthesis, whereby the P450 enzyme, CYP11A1, catalyzes the C20-C22 bond-cleavage in the 20,22-hydroxylated cholesterol, 20R,22R-DiOHCH, leading to pregnenolone, which is critical for the subsequent production of all steroid hormones. This is an unusual feat whereby the P450 enzyme breaks two O-H bonds and one C-C bond, while making two CâO bonds. How does the enzyme perform such a complex and highly energy-demanding reaction? Our computational results rule out the previously proposed Compound I (Cpdâ¯I) electrophilic attack mechanism via the formation of a peroxide intermediate as well as the H-abstraction-mediated C-C cleavage mechanism. Notably, oxygen-rebound cannot transpire, in spite of the fact that the classical active species, Cpdâ¯I, participates in the catalytic process. Our findings reveal a mechanism whereby C-C bond cleavage is mediated by an electron transfer from the C22-O--deprotonated substrate to Cpdâ¯I. As such, our QM/MM calculations demonstrate that Cpdâ¯I acts as an electron sink that facilitates the C-C bond cleavage.
Asunto(s)
Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Colesterol/metabolismo , Simulación de Dinámica Molecular , Pregnenolona/biosíntesis , Teoría Cuántica , Biocatálisis , Colesterol/química , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/química , Transporte de Electrón , Estructura Molecular , Pregnenolona/químicaRESUMEN
Cervical cancer is a leading cause of cancer-related deaths among women in developing countries. Therefore, development of new chemotherapeutic agents is required. Unlike normal cells, cancer cells contain elevated copper levels which play an integral role in angiogenesis. Thus, targeting copper via copper-specific chelators in cancer cells can serve as effective anticancer strategy. In this work, a copper chelator pregnenolone acetate nucleus-based tetrazole derivative (ligand-L) was synthesized and characterized by elemental analysis, ESI-MS, 1H NMR and 13C NMR. DNA binding ability of ligand-L was studied using UV-Vis and fluorescence spectroscopy. Fluorescence spectroscopy studies reveal that quenching constant of ligand-l-DNA and ligand-L-Cu(II) were found to be 7.4â¯×â¯103 M-1 and 8.8â¯×â¯103 M-1, respectively. In vitro toxicity of ligand-L was studied on human cervical cancer C33A cancer cells. Results showed that ligand-L exhibit significant cytotoxic activity against cervical cancer C33A cells with IC50 value 5.0⯱â¯1.8⯵M. Further, it was found that ligand-L cytotoxicity is due to redox cycling of copper to generate ROS which leads to DNA damage and apoptosis. In conclusion, this is the report where we synthesized pregnenolone acetate-based tetrazole derivative against C33A cells that targets cellular copper to induce pro-oxidant death in cancer cells. These findings will provide significant insights into the development of new chemical molecules with better copper chelating and pro-oxidant properties against cancer cells.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Quelantes/farmacología , Compuestos Organometálicos/farmacología , Especies Reactivas de Oxígeno/metabolismo , Neoplasias del Cuello Uterino/tratamiento farmacológico , Acetatos/química , Acetatos/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Quelantes/síntesis química , Quelantes/química , Cobre/química , Cobre/farmacología , División del ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Ligandos , Estructura Molecular , Compuestos Organometálicos/síntesis química , Compuestos Organometálicos/química , Pregnenolona/química , Pregnenolona/farmacología , Relación Estructura-Actividad , Células Tumorales Cultivadas , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/patologíaRESUMEN
A small and focused library of steroidal non-fused and fused pyrimidines was prepared from pregnenolone acetate and diosgenin, respectively. The key step was the cycloaddition reaction of nitrogen-containing 1,3-binucleophiles with the steroidal α,ß-unsaturated ketone. Urea, thiourea and guanidine reacted in a similar manner and afforded the steroidal pyrimidines in good yields. The antiproliferative tests against human tumor cell lines gave GI50 values in the micromolar range and had no effect on healthy fibroblasts. Additional experiments indicated that the compounds did not act as P-glycoprotein substrates, thus avoiding the rise of drug resistance. The fused steroidal pyrimidinethione was selected as drug lead for further testing due to its strong antiproliferative activities within the low micromolar range.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Pirimidinas/farmacología , Esteroides/farmacología , Acetatos/química , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Nitrógeno/química , Pregnenolona/química , Pirimidinas/síntesis química , Pirimidinas/química , Esteroides/síntesis química , Esteroides/química , Relación Estructura-ActividadRESUMEN
Cytochrome P450 (P450, CYP) 17A1 plays a critical role in steroid metabolism, catalyzing both the 17α-hydroxylation of pregnenolone and progesterone and the subsequent 17α,20-lyase reactions to form dehydroepiandrosterone (DHEA) and androstenedione (Andro), respectively, critical for generating glucocorticoids and androgens. Human P450 17A1 reaction rates examined are enhanced by the accessory protein cytochrome b5 (b5), but the exact role of b5 in P450 17A1-catalyzed reactions is unclear as are several details of these reactions. Here, we examined in detail the processivity of the 17α-hydroxylation and lyase steps. b5 did not enhance reaction rates by decreasing the koff rates of any of the steroids. Steroid binding to P450 17A1 was more complex than a simple two-state system. Pre-steady-state experiments indicated lag phases for Andro production from progesterone and for DHEA from pregnenolone, indicating a distributive character of the enzyme. However, we observed processivity in pregnenolone/DHEA pulse-chase experiments. (S)-Orteronel was three times more inhibitory toward the conversion of 17α-hydroxypregnenolone to DHEA than toward the 17α-hydroxylation of pregnenolone. IC50 values for (S)-orteronel were identical for blocking DHEA formation from pregnenolone and for 17α-hydroxylation, suggestive of processivity. Global kinetic modeling helped assign sets of rate constants for individual or groups of reactions, indicating that human P450 17A1 is an inherently distributive enzyme but that some processivity is present, i.e. some of the 17α-OH pregnenolone formed from pregnenolone did not dissociate from P450 17A1 before conversion to DHEA. Our results also suggest multiple conformations of P450 17A1, as previously proposed on the basis of NMR spectroscopy and X-ray crystallography.
Asunto(s)
17-alfa-Hidroxipregnenolona/metabolismo , Citocromos b5/metabolismo , Deshidroepiandrosterona/metabolismo , Modelos Moleculares , NADPH-Ferrihemoproteína Reductasa/metabolismo , Pregnenolona/metabolismo , Esteroide 17-alfa-Hidroxilasa/metabolismo , 17-alfa-Hidroxipregnenolona/química , Androstenodiona/química , Androstenodiona/metabolismo , Animales , Sitios de Unión , Biocatálisis/efectos de los fármacos , Inhibidores Enzimáticos del Citocromo P-450/química , Inhibidores Enzimáticos del Citocromo P-450/metabolismo , Inhibidores Enzimáticos del Citocromo P-450/farmacología , Citocromos b5/genética , Deshidroepiandrosterona/química , Humanos , Imidazoles/química , Imidazoles/metabolismo , Imidazoles/farmacología , Cinética , Ligandos , NADPH-Ferrihemoproteína Reductasa/genética , Naftalenos/química , Naftalenos/metabolismo , Naftalenos/farmacología , Oxidación-Reducción , Pregnenolona/química , Progesterona/química , Progesterona/metabolismo , Conformación Proteica , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Estereoisomerismo , Esteroide 17-alfa-Hidroxilasa/antagonistas & inhibidores , Esteroide 17-alfa-Hidroxilasa/química , Esteroide 17-alfa-Hidroxilasa/genéticaRESUMEN
The cytosolic sulfotransferase (SULT) SULT2A1 is known to mediate the sulfation of DHEA as well as some other hydroxysteroids such as pregnenolone. The present study was designed to investigate how genetic polymorphisms of the human SULT2A1 gene may affect the sulfation of DHEA and pregnenolone. Online databases were systematically searched to identify human SULT2A1 single nucleotide polymorphisms (SNPs). Of the 98 SULT2A1 non-synonymous coding SNPs identified, seven were selected for further investigation. Site-directed mutagenesis was used to generate cDNAs encoding these seven SULT2A1 allozymes, which were expressed in BL21 Escherichia coli cells and purified by glutathione-Sepharose affinity chromatography. Enzymatic assays revealed that purified SULT2A1 allozymes displayed differential sulfating activity toward both DHEA and pregnenolone. Kinetic analyses showed further differential catalytic efficiency and substrate affinity of the SULT2A1 allozymes, in comparison with wild-type SULT2A1. These findings provided useful information concerning the effects of genetic polymorphisms on the sulfating activity of SULT2A1 allozymes.
Asunto(s)
Deshidroepiandrosterona/química , Polimorfismo de Nucleótido Simple , Pregnenolona/química , Sulfotransferasas/química , Sulfotransferasas/genética , Humanos , Cinética , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes , Sulfotransferasas/metabolismoRESUMEN
Cytochromes P450 play a key role in the drug and steroid metabolism in the human body. This leads to a high interest in this class of proteins. Mammalian cytochromes P450 are rather delicate. Due to their localization in the mitochondrial or microsomal membrane, they tend to aggregate during expression and purification and to convert to an inactive form so that they have to be purified and stored in complex buffers. The complex buffers and low storage temperatures, however, limit the feasibility of fast, automated screening of the corresponding cytochrome P450-effector interactions, which are necessary to study substrate-protein and inhibitor-protein interactions. Here, we present the production and isolation of functionalized poly(3-hydroxybutyrate) granules (PHB bodies) from Bacillus megaterium MS941 strain. In contrast to the expression in Escherichia coli, where mammalian cytochromes P450 are associated to the cell membrane, when CYP11A1 is heterologously expressed in Bacillus megaterium, it is located on the PHB bodies. The surface of these particles provides a matrix for immobilization and stabilization of the CYP11A1 during the storage of the protein and substrate conversion. It was demonstrated that the PHB polymer basis is inert concerning the performed conversion. Immobilization of the CYP11A1 onto the PHB bodies allows freeze-drying of the complex without significant decrease of the CYP11A1 activity. This is the first lyophilization of a mammalian cytochrome P450, which allows storage over more than 18days at 4°C instead of storage at -80°C. In addition, we were able to immobilize the cytochrome P450 on the PHB bodies in vitro. In this case the expression of the protein is separated from the production of the immobilization matrix, which widens the application of this method. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.
Asunto(s)
Ácido 3-Hidroxibutírico/química , Bacillus megaterium/genética , Biotecnología/métodos , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/química , Proteínas Inmovilizadas/biosíntesis , Proteínas Mitocondriales/biosíntesis , Ácido 3-Hidroxibutírico/biosíntesis , Animales , Bacillus megaterium/enzimología , Biocatálisis , Bovinos , Colesterol/química , Colesterol/metabolismo , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Gránulos Citoplasmáticos/química , Liofilización , Expresión Génica , Proteínas Inmovilizadas/química , Proteínas Inmovilizadas/genética , Proteínas Mitocondriales/química , Proteínas Mitocondriales/genética , Pregnenolona/biosíntesis , Pregnenolona/química , Prohibitinas , Refrigeración , TransgenesRESUMEN
Selective binding of steroid molecules is of paramount importance for designing drugs that can target the biological pathways of only individual steroids. From this perspective, it is remarkable that progesterone (PRO) and pregnenolone (PRE), two structurally similar steroids, demonstrate a dramatically different propensity to interact with aromatic molecules. It has been recently reported that, in solid-state cocrystallization, PRO forms cocrystals with a wide variety of aromatic systems whereas PRE cocrystallizes only with a few. In this work, a simple yet effective computational procedure was developed to explain the fundamental origins of this surprising phenomenon. This procedure enables a direct comparison of the strength of intermolecular binding in the structurally similar cocrystals of PRO and PRE by generating experimentally inaccessible meta-stable cocrystals of PRE that closely resemble those observed for PRO. Direct comparative analysis shows that interactions between the α-face of the steroid and the π-electrons of aromatic molecules, the focus of previous studies, are not sufficiently different to explain the cocrystallization behavior of PRO and PRE. Instead, the observed difference is attributed to the different stabilities of the cocrystals relative to their pure components: organic and steroid crystals. It is calculated that the cocrystallization process is thermodynamically favorable in the case of PRO and unfavorable in the case of PRE. Furthermore, strong hydrogen bonds in the pure PRE crystal appear to be the major factor that makes the cocrystallization of PRE energetically unfavorable for a wide range of aromatic molecules. The fundamental analysis performed in this work has important practical implications for designing new steroid-containing crystals, selective biomolecular steroid receptors, and steroid-specific drugs. It suggests that a strategy for the selective binding of steroids should focus primarily on tuning the strength of hydrogen bonding.
Asunto(s)
Enlace de Hidrógeno , Pregnenolona/química , Progesterona/química , Cristalización , Cristalografía , Diseño de Fármacos , Electrones , TermodinámicaRESUMEN
Ablation of androgen production through surgery is one strategy against prostate cancer, with the current focus placed on pharmaceutical intervention to restrict androgen synthesis selectively, an endeavor that could benefit from the enhanced understanding of enzymatic mechanisms that derives from characterization of key reaction intermediates. The multifunctional cytochrome P450 17A1 (CYP17A1) first catalyzes the typical hydroxylation of its primary substrate, pregnenolone (PREG) and then also orchestrates a remarkable C17-C20 bond cleavage (lyase) reaction, converting the 17-hydroxypregnenolone initial product to dehydroepiandrosterone, a process representing the first committed step in the biosynthesis of androgens. Now, we report the capture and structural characterization of intermediates produced during this lyase step: an initial peroxo-anion intermediate, poised for nucleophilic attack on the C20 position by a substrate-associated H-bond, and the crucial ferric peroxo-hemiacetal intermediate that precedes carbon-carbon (C-C) bond cleavage. These studies provide a rare glimpse at the actual structural determinants of a chemical transformation that carries profound physiological consequences.
Asunto(s)
17-alfa-Hidroxipregnenolona/metabolismo , Andrógenos/metabolismo , Deshidroepiandrosterona/metabolismo , Pregnenolona/metabolismo , Esteroide 17-alfa-Hidroxilasa/metabolismo , 17-alfa-Hidroxipregnenolona/química , Andrógenos/química , Biocatálisis , Vías Biosintéticas , Deshidroepiandrosterona/química , Humanos , Enlace de Hidrógeno , Hidroxilación , Modelos Químicos , Modelos Moleculares , Estructura Molecular , Pregnenolona/química , Conformación Proteica , Espectrofotometría/métodos , Esteroide 17-alfa-Hidroxilasa/química , Esteroide 17-alfa-Hidroxilasa/genética , Especificidad por Sustrato , TemperaturaRESUMEN
A mycelial culture of the Kenyan basidiomycete Fomitiporia aethiopica was fermented on rice and the cultures were extracted with methanol. Subsequent HPLC profiling and preparative chromatography of its crude extract led to the isolation of five previously undescribed pregnenolone type triterpenes 1-5, for which we propose the trivial name aethiopinolones A-E. The chemical structures of the aethiopinolones were determined by extensive 1D- and 2D-NMR, and HRMS data analysis. The compounds exhibited moderate cytotoxic effects against various human cancer cell lines, but they were found devoid of significant nematicidal and antimicrobial activities.
Asunto(s)
Basidiomycota/química , Pregnenolona/análogos & derivados , Pregnenolona/química , Triterpenos/química , Animales , Antiinfecciosos/química , Antiinfecciosos/aislamiento & purificación , Antiinfecciosos/farmacología , Antineoplásicos/química , Antineoplásicos/aislamiento & purificación , Antineoplásicos/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Ratones , Pregnenolona/aislamiento & purificación , Pregnenolona/farmacología , Metabolismo Secundario , Triterpenos/aislamiento & purificación , Triterpenos/farmacologíaRESUMEN
Twenty-five novel pregnenolone/2-cyanoacryloyl conjugates (6-30) were designed and prepared, with the aim of developing novel anticancer drugs with dual NF-κB inhibitory and anti-proliferative activities. Compounds 22 and 27-30 showed inhibition against TNF-α-induced NF-κB activation in luciferase assay, which was confirmed by Western blotting. Among them, compound 30 showed potent NF-κB inhibitory activity (IC50=2.5µM) and anti-proliferative against MCF-7, A549, H157, and HL-60 cell lines (IC50=6.5-36.2µM). The present study indicated that pregnenolone/2-cyanoacryloyl conjugate I can server asa novel scaffold for developing NF-κB inhibitors and anti-proliferative agents in cancer chemotherapy.
Asunto(s)
Antineoplásicos/síntesis química , Cianoacrilatos/química , Diseño de Fármacos , FN-kappa B/metabolismo , Pregnenolona/química , Células A549 , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Células HL-60 , Humanos , Células MCF-7 , FN-kappa B/antagonistas & inhibidores , Relación Estructura-ActividadRESUMEN
16-Dehydropregnenolone (16-DHP) is an active compound with an unsatisfied in vivo behavior and poor water-solubility, which limits its clinical application. To improve its in vivo behavior and water-solubility, a Hydroxypropyl-beta-Cyclodextrin (HP-ß-CD) inclusion complex of 16-DHP was prepared in this paper. Pharmacokinetic studies after oral administration of 16-DHP-HP-ß-CD at doses of 37.5, 75, 150 mg/kg were carried out to investigate its dose proportionality in rats. The relative bioavailability was researched by comparing the area under the plasma concentration-time curve of 16-DHP-HP-ß-CD and free 16-DHP after oral administration in rats at the dose of 75 mg/kg. At the same time, tissue distribution of 16-DHP-HP-ß-CD after oral administration at the dose of 240 mg/kg in mice was also investigated. Consequently, 16-DHP-HP-ß-CD appeared to be a linear pharmacokinetic character after peroral administration to the rat at the doses tested. Compared to free 16-DHP, inclusion complex could significantly improve the relative bioavailability (467%). Tissue distribution studies indicated that 16-DHP-HP-ß-CD tended to distribute into stomach, intestine, lung, brain and liver.
Asunto(s)
Portadores de Fármacos/química , Pregnenolona/análogos & derivados , beta-Ciclodextrinas/química , 2-Hidroxipropil-beta-Ciclodextrina , Administración Oral , Animales , Disponibilidad Biológica , Liberación de Fármacos , Femenino , Ratones , Pregnenolona/administración & dosificación , Pregnenolona/química , Pregnenolona/farmacocinética , Ratas , Ratas Sprague-Dawley , Solubilidad , Distribución TisularRESUMEN
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
The human cytochrome P450 17A1 (CYP17A1) enzyme operates at a key juncture of human steroidogenesis, controlling the levels of mineralocorticoids influencing blood pressure, glucocorticoids involved in immune and stress responses, and androgens and estrogens involved in development and homeostasis of reproductive tissues. Understanding CYP17A1 multifunctional biochemistry is thus integral to treating prostate and breast cancer, subfertility, blood pressure, and other diseases. CYP17A1 structures with all four physiologically relevant steroid substrates suggest answers to four fundamental aspects of CYP17A1 function. First, all substrates bind in a similar overall orientation, rising â¼60° with respect to the heme. Second, both hydroxylase substrates pregnenolone and progesterone hydrogen bond to Asn(202) in orientations consistent with production of 17α-hydroxy major metabolites, but functional and structural evidence for an A105L mutation suggests that a minor conformation may yield the minor 16α-hydroxyprogesterone metabolite. Third, substrate specificity of the subsequent 17,20-lyase reaction may be explained by variation in substrate height above the heme. Although 17α-hydroxyprogesterone is only observed farther from the catalytic iron, 17α-hydroxypregnenolone is also observed closer to the heme. In conjunction with spectroscopic evidence, this suggests that only 17α-hydroxypregnenolone approaches and interacts with the proximal oxygen of the catalytic iron-peroxy intermediate, yielding efficient production of dehydroepiandrosterone as the key intermediate in human testosterone and estrogen synthesis. Fourth, differential positioning of 17α-hydroxypregnenolone offers a mechanism whereby allosteric binding of cytochrome b5 might selectively enhance the lyase reaction. In aggregate, these structures provide a structural basis for understanding multiple key reactions at the heart of human steroidogenesis.
Asunto(s)
Dominio Catalítico , Estructura Secundaria de Proteína , Esteroide 17-alfa-Hidroxilasa/química , Esteroide 17-alfa-Hidroxilasa/metabolismo , 17-alfa-Hidroxiprogesterona/química , 17-alfa-Hidroxiprogesterona/metabolismo , Androstenos , Androstenoles/química , Androstenoles/metabolismo , Sitios de Unión/genética , Cristalografía por Rayos X , Deshidroepiandrosterona/química , Deshidroepiandrosterona/metabolismo , Estrógenos/metabolismo , Hemo/metabolismo , Humanos , Enlace de Hidrógeno , Cinética , Modelos Moleculares , Estructura Molecular , Mutación , Oxidación-Reducción , Pregnenolona/química , Pregnenolona/metabolismo , Progesterona/química , Progesterona/metabolismo , Unión Proteica , Esteroide 17-alfa-Hidroxilasa/genética , Esteroides/química , Esteroides/metabolismo , Especificidad por Sustrato , Testosterona/metabolismoRESUMEN
Pregnenolone (P5) is a neurosteroid that improves memory and neurological recovery. It is also required for zebrafish embryonic development. However, its mode of action is unclear. Here we show that P5 promotes cell migration and microtubule polymerization by binding a microtubule plus end-tracking protein, cytoplasmic linker protein 1 (CLIP-170). We captured CLIP-170 from zebrafish embryonic extract using a P5 photoaffinity probe conjugated to diaminobenzophenone. P5 interacted with CLIP-170 at its coiled-coil domain and changed it into an extended conformation. This increased CLIP-170 interaction with microtubules, dynactin subunit p150(Glued) and LIS1; it also promoted CLIP-170-dependent microtubule polymerization. CLIP-170 was essential for P5 to promote microtubule abundance and zebrafish epiboly cell migration during embryogenesis, and overexpression of the P5-binding region of CLIP-170 delayed this migration. P5 also sustained migration directionality of cultured mammalian cells. Our results show that P5 activates CLIP-170 to promote microtubule polymerization and cell migration.