21 research outputs found
Bioactivity of Curcumin on the Cytochrome P450 Enzymes of the Steroidogenic Pathway.
No full textTurmeric, a popular ingredient in the cuisine of many Asian countries, comes from the roots of the Curcuma longa and is known for its use in Chinese and Ayurvedic medicine. Turmeric is rich in curcuminoids, including curcumin, demethoxycurcumin, and bisdemethoxycurcumin. Curcuminoids have potent wound healing, anti-inflammatory, and anti-carcinogenic activities. While curcuminoids have been studied for many years, not much is known about their effects on steroid metabolism. Since many anti-cancer drugs target enzymes from the steroidogenic pathway, we tested the effect of curcuminoids on cytochrome P450 CYP17A1, CYP21A2, and CYP19A1 enzyme activities. When using 10 µg/ml of curcuminoids, both the 17α-hydroxylase as well as 17,20 lyase activities of CYP17A1 were reduced significantly. On the other hand, only a mild reduction in CYP21A2 activity was observed. Furthermore, CYP19A1 activity was also reduced up to ~20% of control when using 1-100 µg/ml of curcuminoids in a dose-dependent manner. Molecular docking studies confirmed that curcumin could dock onto the active sites of CYP17A1, CYP19A1, as well as CYP21A2. In CYP17A1 and CYP19A1, curcumin docked within 2.5 Å of central heme while in CYP21A2 the distance from heme was 3.4 Å, which is still in the same range or lower than distances of bound steroid substrates. These studies suggest that curcuminoids may cause inhibition of steroid metabolism, especially at higher dosages. Also, the recent popularity of turmeric powder as a dilatory supplement needs further evaluation for the effect of curcuminoids on steroid metabolism. The molecular structure of curcuminoids could be modified to generate better lead compounds with inhibitory effects on CYP17A1 and CYP19A1 for potential drugs against prostate cancer and breast cancer
Metformin inhibits the activation of melanocortin receptors 2 and 3 in vitro: A possible mechanism for its anti-androgenic and weight balancing effects in vivo?
No full textMetformin is recommended as one of the first-line drugs for the treatment of type 2 diabetes and the metabolic syndrome. In addition to its insulin sensitizing effects, it has been shown to attenuate androgen excess in women with polycystic ovary syndrome (PCOS) or congenital adrenal hyperplasia (CAH), as well as to ameliorate obesity. The mechanisms of metformin action seem manifold. Preclinical studies suggest that it inhibits the cellular stress response at the level of the mitochondrial OXPHOS system and through AMPK dependent and independent mechanisms. Recent studies have shown that metformin decreases ACTH secretion from pituitary and reduces ACTH-stimulated adrenal secretion. In this study we investigated its specific effect through the melanocortin receptor 2 (MC2R) on signaling targeting adrenal steroidogenesis. To assess this effect, we used mouse adrenal OS3 cells, which do not express the MC2R. Cells were transfected with the MC2R and stimulated by ACTH. Downstream cyclic AMP production was then assessed by a co-transfected cAMP-responsive vector producing luciferase that was measured by a dual luciferase assay. The amount of luciferase produced in this assay corresponds to the amount of receptor activation with varying amount of ACTH. The effect of metformin was then tested in this system. We found a significant inhibition of ACTH induced MC2R activation and signaling with 10 mM metformin. The ACTH concentration response curve (CRC) was half-log shifted and a ∼30 % reduction in maximum receptor response (Rmax) to ACTH in presence of metformin was observed. This effect was dose dependent with an IC50 of 4.2 mM. qRT-PCR analyses showed that metformin decreased ACTH induced MC2R expression. Metformin did not affect cell viability and basal cAMP levels. We also tested the effect of metformin on homologous melanocortin receptors (MCRs). No significant effect was found on MC1R and MC4R activity. However, a log shift of EC50 of ACTH stimulation on MC3R was observed with metformin treatment. Metformin also inhibited melanocortin stimulating hormone (αMSH) induced MC3R activity. In conclusion, we show that metformin acts on MC2R and MC3R signaling directly. The role of MC2R for steroidogenesis is well established. MC3R is involved in energy balance and seems to act as a rheostat when the metabolism is challenged. Our study may explain how metformin helps in weight loss and attenuates the excess response to ACTH in androgen excess disorders such as PCOS and CAH
Altered CYP19A1 and CYP3A4 Activities Due to Mutations A115V, T142A, Q153R and P284L in the Human P450 Oxidoreductase
No full textAll cytochromes P450s in the endoplasmic reticulum rely on P450 oxidoreductase (POR) for their catalytic activities. Mutations in POR cause metabolic disorders of steroid hormone biosynthesis and affect certain drug metabolizing P450 activities. We studied mutations A115V, T142A, Q153R identified in the flavin mononucleotide (FMN) binding domain of POR that interacts with partner proteins and P284L located in the hinge region that is required for flexibility and domain movements in POR. Human wild-type (WT) and mutant POR as well as CYP3A4 and CYP19A1 proteins in recombinant form were expressed in bacteria, and purified proteins were reconstituted in liposomes for enzyme kinetic assays. Quality of POR protein was checked by cytochrome c reduction assay as well as flavin content measurements. We found that proteins carrying mutations A115V, T142A located close to the FMN binding site had reduced flavin content compared to WT POR and lost almost all activity to metabolize androstenedione via CYP19A1 and showed reduced CYP3A4 activity. The variant P284L identified from apparently normal subjects also had severe loss of both CYP19A1 and CYP3A4 activities, indicating this to be a potentially disease causing mutation. The mutation Q153R initially identified in a patient with disordered steroidogenesis showed remarkably increased activities of both CYP19A1 and CYP3A4 without any significant change in flavin content, indicating improved protein-protein interactions between POR Q153R and some P450 proteins. These results indicate that effects of mutations on activities of individual cytochromes P450 can be variable and a detailed analysis of each variant with different partner proteins is necessary to accurately determine the genotype-phenotype correlations of POR variants
Altered CYP19A1 and CYP3A4 Activities Due to Mutations in the Flavin Mononucleotide Binding Domain of Human P450 Oxidoreductase
No full textAbstract Cytochromes P450 proteins are responsible for the metabolism of many steroid
hormones as well as drugs and xenobiotics. All cytochromes P450s in the endoplasmic
reticulum rely on P450 oxidoreductase (POR) for their catalytic activities. Previously we and
others have shown that mutations in POR cause metabolic disorders of steroid hormone
biosynthesis and also affect certain drug metabolizing P450 activities. Human POR has
distinct subdomains, which bind flavin molecules and interact with redox partners. We
studied the mutations identified in flavin mononucleotide (FMN) binding domain of POR that
interacts with partner proteins. We found that mutations A115V, T142A located close to the
FMN binding site had reduced flavin content compared to wild type POR and lost almost all
activity to metabolize androstenedione via CYP19A1 and also showed reduced CYP3A4 acitivies
ALTERED STEROID AND DRUG METABOLISM BY A P450 OXIDOREDUCTASE VARIANT FOUND IN APPARENTLY NORMAL POPULATION
No full textP450 Oxidoreductase deficiency: Analysis of mutations and polymorphisms.
No full textCytochrome P450 oxidoreductase (POR) is required for metabolic reactions of steroid and drug metabolizing cytochrome P450 proteins located in endoplasmic reticulum. Mutations in POR cause a complex set of disorders resembling combined deficiencies of multiple steroid metabolizing enzymes. The P450 oxidoreductase deficiency (PORD) was first reported in patients with symptoms of defects in steroidogenic cytochrome P450 enzymes and ambiguous genitalia, and bone malformation features resembling Antley-Bixler syndrome. POR is now classified as a separate and rare form of congenital adrenal hyperplasia (CAH), which may cause disorder of sexual development (DSD). Since the initial description of PORD in 2004, a large number of POR mutations and polymorphisms have been described. In this report we have performed computational analysis of mutations and polymorphisms in POR linked to metabolism of steroids and xenobiotics and pathology of PORD from the reported cases. The mutations in POR that were identified in patients with disruption of steroidogenesis also have severe effects on cytochrome P450 proteins involved in metabolism of drugs. Different variations in POR show a range of diverse effects on different partner proteins that are often linked to the location of the particular variants. The variations in POR that cause defective binding of co-factors always have damaging effects on all partner proteins, while the mutations causing subtle structural changes may lead to altered interaction with partner proteins and the overall effect may be different for each individual partner. Computational analysis of available sequencing data and mutation analysis shows that Japanese (R457H), Caucasian (A287P) and Turkish (399-401) populations can be linked to unique founder mutations. Other mutations identified so far were identified as rare alleles or in single isolated reports. The common polymorphism of POR is the variant A503V which can be found in about 27% of alleles in general population but there are remarkable differences among different sub populations
Differential effects of variations in human P450 oxidoreductase on the aromatase activity of CYP19A1 polymorphisms R264C and R264H.
No full textAromatase (CYP19A1) converts androgens into estrogens and is required for female sexual development and growth and development in both sexes. CYP19A1 is a member of cytochrome P450 family of heme-thiolate monooxygenases located in the endoplasmic reticulum and depends on reducing equivalents from the reduced nicotinamide adenine dinucleotide phosphate via the cytochrome P450 oxidoreductase coded by POR. Both the CYP19A1 and POR genes are highly polymorphic, and mutations in both these genes are linked to disorders of steroid biosynthesis. We have previously shown that R264C and R264H mutations in CYP19A1, as well as mutations in POR, reduce CYP19A1 activity. The R264C is a common polymorphic variant of CYP19A1, with high frequency in Asian and African populations. Polymorphic alleles of POR are found in all populations studied so far and, therefore, may influence activities of CYP19A1 allelic variants. So far, the effects of variations in POR on enzymatic activities of allelic variants of CYP19A1 or any other steroid metabolizing cytochrome P450 proteins have not been studied. Here we are reporting the effects of three POR variants on the aromatase activities of two CYP19A1 variants, R264C, and R264H. We used bacterially expressed and purified preparations of WT and variant forms of CYP19A1 and POR and constructed liposomes with embedded CYP19A1 and POR proteins and assayed the CYP19A1 activities using radiolabeled androstenedione as a substrate. With the WT-POR as a redox partner, the R264C-CYP19A1 showed only 15% of aromatase activity, but the R264H had 87% of aromatase activity compared to WT-CYP19A1. With P284L-POR as a redox partner, R264C-CYP19A1 lost all activity but retained 6.7% of activity when P284T-POR was used as a redox partner. The R264H-CYP19A1 showed low activities with both the POR-P284 L as well as the POR-P284 T. When the POR-Y607C was used as a redox partner, the R264C-CYP19A1 retained approximately 5% of CYP19A1 activity. Remarkably, The R264H-CYP19A1 had more than three-fold higher activity compared to WT-CYP19A1 when the POR-Y607C was used as the redox partner, pointing toward a beneficial effect. The slight increase in activity of R264C-CYP19A1 with the P284T-POR and the three-fold increase in activity of the R264H-CYP19A1 with the Y607C-POR point toward a conformational effect and role of protein-protein interaction governed by the R264C and R264H substitutions in the CYP19A1 as well as P284 L, P284 T and Y607C variants of POR. These studies demonstrate that the allelic variants of P450 when present with a variant form of POR may show different activities, and combined effects of variations in the P450 enzymes as well as in the POR should be considered when genetic data are available. Recent trends in the whole-exome and whole-genome sequencing as diagnostic tools will permit combined evaluation of variations in multiple genes that are interdependent and may guide treatment options by adjusting therapeutic interventions based on laboratory analysis
Variability in Loss of Multiple Enzyme Activities Due to the Human Genetic Variation P284T Located in the Flexible Hinge Region of NADPH Cytochrome P450 Oxidoreductase.
No full textCytochromes P450 located in the endoplasmic reticulum require NADPH cytochrome P450 oxidoreductase (POR) for their catalytic activities. Mutations in POR cause multiple disorders in humans related to the biosynthesis of steroid hormones and also affect drug-metabolizing cytochrome P450 activities. Electron transfer in POR occurs from NADH to FAD to FMN, and the flexible hinge region in POR is essential for domain movements to bring the FAD and FMN close together for electron transfer. We tested the effect of variations in the hinge region of POR to check if the effects would be similar across all redox partners or there will be differences in activities. Here we are reporting the effects of a POR genetic variant P284T located in the hinge region of POR that is necessary for the domain movements and internal electron transfer between co-factors. Human wild-type and P284T mutant of POR and cytochrome P450 proteins were expressed in bacteria, purified, and reconstituted for enzyme assays. We found that for the P284T variant of POR, the cytochrome c reduction activity was reduced to 47% of the WT and MTT reduction was reduced to only 15% of the WT. No impact on ferricyanide reduction activity was observed, indicating intact direct electron transfer from FAD to ferricyanide, but a severe loss of CYP19A1 (aromatase) activity was observed (9% of WT). In the assays of drug-metabolizing cytochrome P450 enzymes, the P284T variant of POR showed 26% activity for CYP2C9, 44% activity for CYP2C19, 23% activity for CYP3A4, and 44% activity in CYP3A5 assays compared to the WT POR. These results indicate a severe effect on several cytochrome P450 activities due to the P284T variation in POR, which suggests a negative impact on both the steroid as well as drug metabolism in the individuals carrying this variation. The negative impact of P284T mutation in the hinge region of POR seems to be due to disruption of FAD to FMN electron transfer. These results further emphasize the importance of hinge region in POR for protein flexibility and electron transfer within POR as well as the interaction of POR with different redox partners
Variability in human drug metabolizing cytochrome P450 CYP2C9, CYP2C19 and CYP3A5 activities caused by genetic variations in cytochrome P450 oxidoreductase.
No full textA broad spectrum of human diseases are caused by mutations in the NADPH cytochrome P450 oxidoreductase (POR). Cytochrome P450 proteins perform several reactions, including the metabolism of steroids, drugs, and other xenobiotics. In 2004 the first human patients with defects in POR were reported, and over 250 variations in POR are known. Information about the effects of POR variants on drug metabolizing enzymes is limited and has not received much attention. By analyzing the POR sequences from genomics databases, we identified potentially disease-causing variations and characterized these by in vitro functional studies using recombinant proteins. Proteins were expressed in bacteria and purified for activity assays. Activities of cytochrome P450 enzymes were tested in vitro using liposomes prepared with lipids into which P450 and P450 reductase proteins were embedded. Here we are reporting the effect of POR variants on drug metabolizing enzymes CYP2C9, CYP2C19, and CYP3A5 which are responsible for the metabolism of many drugs. POR Variants A115V, T142A, A281T, P284L, A287P, and Y607C inhibited activities of all P450 proteins tested. Interestingly, the POR variant Q153R showed a reduction of 20-50% activities with CYP2C9 and CYP2C19 but had a 400% increased activity with CYP3A5. The A287P is most common POR mutation found in patients of European origin, and significantly inhibited drug metabolism activities which has important consequences for monitoring and treatment of patients. In vitro, functional assays using recombinant proteins provide a useful model for establishing the metabolic effect of genetic mutations. Our results indicate that detailed knowledge about POR variants is necessary for correct diagnosis and treatment options for persons with POR deficiency and the role of changes in drug metabolism and toxicology due to variations in POR needs to be addressed
