Just like the drugs themselves, their metabolites have to be evaluated to succeed in a drug development and approval process. It is therefore essential to be able to predict drug metabolism and to ...synthesise sufficient metabolite quantities for further pharmacological testing. This study evaluates the possibility of using in vitro biotransformations to solve both these challenges in the case of testosterone as a representative component for steroids. The application of cells of Pichia pastoris with expressed membrane‐associated human liver cytochrome P450 enzyme (P450) 3A4 in two cycles of a preparative‐scale bioreactor experiment enabled the isolation of the common metabolites 6β‐hydroxytestosterone and 6β‐hydroxyandrostenedione on a 100 mg scale. Side‐product formation caused by enzymes intrinsic to P. pastoris was reduced. In addition more polar testosterone metabolites formed by a P450 3A4‐catalysed bioconversion, than the known mono‐hydroxylated ones, are reported and 6‐dehydro‐15β‐hydroxytestosterone as well as the di‐hydroxylated steroids 6β,16β‐dihydroxytestosterone, 6β,17β‐dihydroxy‐4‐androstene‐3,16‐dione and 6β,12β‐dihydroxyandrostenedione were isolated and verified by NMR analysis. Their respective biological significance remains to be investigated. Whole‐cell P450 catalysts expressed in P. pastoris qualify as a tool for the preparative‐scale synthesis of human metabolites. Biotransformation processes in combination with standard chemical procedures allow the isolation and characterisation even of minor drug metabolite products.
Efficient synthetic techniques for the diversification of natural products are incremental for drug discovery processes of the pharmaceutical industry because these complex bioactive compounds often ...require an adjustment of properties. Human liver P450 3A4, key player of the body's detoxification system and decisive factor of a drug's metabolic fate, is renowned for its broad substrate scope including many natural products. In this study, we investigated the synthetic potential of human P450 3A4 for the diversification of natural product classes and isolated the produced metabolites of six selected natural products at a preparative 100‐mg scale. Aided by efficient expression levels in P. pastoris, this whole‐cell biocatalyst was found to be highly effective at the intended job allowing the identification of a total of 31 authentic human metabolites, many of them for the first time. By revealing an unprecedented degree of diversification, this study extends the synthetic repertoire for efficient enzymatic natural product modification in a one‐step fashion and adds a completely new view to an old enzyme traditionally used for inhibition and toxicology studies.
Doing it all: In this study, the synthetic potential of human cytochrome P450 3A4 as a Pichia pastoris‐based whole‐cell biocatalyst for the diversification of natural products was successfully explored. A total of 31 authentic human metabolites of six selected natural products were isolated at a preparative 100‐mg scale, many of them for the first time, revealing an unprecedented degree of diversification.
Polycyclic aromatic hydrocarbons (PAHs) and their N- and O-containing derivatives (N-/O-PAHs) are environmental pollutants and synthetically attractive building blocks in pharmaceuticals. ...Functionalization of PAHs can be achieved via C-H activation by cytochrome P450 enzymes (e.g., P450 CYP3A4) in an environmentally friendly manner. Despite its broad substrate scope, the contribution of CYP3A4 to metabolize common PAHs in humans was found to be small. We recently showcased the potential of CYP3A4 in whole-cell biocatalysis with recombinant yeast
(
) catalysts for the preparative-scale synthesis of naturally occurring metabolites in humans. In this study, we aimed at exploring the substrate scope of CYP3A4 towards (N-/O)-PAHs and conducted a bioconversion experiment at 10 L scale to validate the synthetic potential of CYP3A4 for the preparative-scale production of functionalized PAH metabolites. Hydroxylated products were purified and characterized using HPLC and NMR analysis. In total, 237 mg of fluorenol and 48 mg of fluorenone were produced from 498 mg of fluorene, with peak productivities of 27.7 μmol/L/h for fluorenol and 5.9 μmol/L/h for fluorenone; the latter confirmed that CYP3A4 is an excellent whole-cell biocatalyst for producing authentic human metabolites.
Glioblastoma (GBM) is the most aggressive primary brain tumor with poor patient survival that is at least partly caused by malignant and therapy-resistant glioma stem-like cells (GSLCs) that are ...protected in GSLC niches. Previously, we have shown that the chemo-attractant stromal-derived factor-1α (SDF-1α), its C-X-C receptor type 4 (CXCR4) and the cysteine protease cathepsin K (CatK) are localized in GSLC niches in glioblastoma. Here, we investigated whether SDF-1α is a niche factor that through its interactions with CXCR4 and/or its second receptor CXCR7 on GSLCs facilitates their homing to niches. Furthermore, we aimed to prove that SDF-1α cleavage by CatK inactivates SDF-1α and inhibits the invasion of GSLCs. We performed mass spectrometric analysis of cleavage products of SDF-1α after proteolysis by CatK. We demonstrated that CatK cleaves SDF-1α at 3 sites in the N-terminus, which is the region of SDF-1α that binds to its receptors. Confocal imaging of human GBM tissue sections confirmed co-localization of SDF-1α and CatK in GSLC niches. In accordance, 2D and 3D invasion experiments using CXCR4/CXCR7-expressing GSLCs and GBM cells showed that SDF-1α had chemotactic activity whereas CatK cleavage products of SDF-1α did not. Besides, CXCR4 inhibitor plerixafor inhibited invasion of CXCR4/CXCR7-expressing GSLCs.
In conclusion, CatK can cleave and inactivate SDF-1α. This implies that CatK activity facilitates migration of GSLCs out of niches. We propose that activation of CatK may be a promising strategy to prevent homing of GSLCs in niches and thus render these cells sensitive to chemotherapy and radiation.
•Cysteine protease cathepsin K (CatK) cleaves and inactivates the chemokine SDF-1α.•CatK cleavage products of SDF-1α impair invasion of GSLCs in vitro.•CXCR4 inhibitor plerixafor inhibits invasion of GSLCs in vitro.•CatK and SDF-1α colocalize in glioma stem-like cell niches in glioblastoma tissue.•CatK activity can prevent homing of GSLCs to their therapy-protective niches.
The Cover Feature shows cytochrome P450 3A4 revealing its magic ability to diversify natural products such as nootkatone to many functionalised metabolites in one step, as illustrated by a grey ...butterfly transforming into coloured butterflies. In their Research Article, N. D. Fessner et al. explored the synthetic potential of promiscuous human liver P450 3A4 for the late‐stage diversification of natural products to produce oxyfunctionalised compound libraries of pharmaceutical or olfactory interest. The isolation of 31 authentic human metabolites at a 100 mg‐scale from the P450 3A4‐catalysed biotransformation of six selected terpenes, alkaloids and steroids represented an unprecedented degree of diversification. This study extends the synthetic repertoire for efficient enzymatic natural product modification. More information can be found in the Research Article by N. D. Fessner et al.
Abstract The aim of this work was to determine serum DPPIV activity as well as the percentage of CD26+ white blood cells and of CD26+ lymphocytes and the mean fluorescence intensity (MFI) of CD26 ...expression on lymphocytes in groups of patients with benign or malignant breast tumors and in healthy control people. Serum DPPIV activity was determined by colorimetric test, while CD26+ cells were counted using flow cytometer. Results of this study show that there is no statistically significant difference in serum DPPIV activity between examined groups of patients and healthy controls. However, two times higher frequency of patients with breast cancers had the enhanced DPPIV enzymatic activity in comparison to controls. Significant decrease in the percentage of CD26+ total white blood cells was found in the group of breast cancer patients and in patients with benign breast tumors compared to that found for healthy people. Although there was decrease in the percentage of lymphocytes in patients with breast tumors it was not statistically significant. The MFI of CD26 expression on these cells was significantly lower for cancer patients in comparison to healthy controls. In conclusion, this work showed the enhanced frequency of breast cancer patients with higher serum DPPIV activity. Decreased percentage of CD26+ white blood cells and decreased CD26 expression on lymphocytes are also characteristics of this group of patients. Determination of the clinical outcome of analyzed patients, 1 and 2 years after the surgical resection of the tumor, would clarify potential prognostic values of examined parameters for breast cancer.
Studies conducted on children and adults have pointed to the correlation of BMI (kg/m2) with risk factors for certain diseases. Other studies have stressed a more intense correlation between the risk ...factors and indicators of subcutaneous fat obtained in other ways. The aim Of the study was to compare the intensity of correlation between the risk factors and triceps and subscapular skinfold thickness in relation to BMI. The study included 53 postmenopausal and 107 premenopausal women, the risk factors were assessed upon systolic and diastolic blood pressure, glucose concentration, triglyceride and cholesterol levels. Statistically significant differences were established in almost all variables referring to premenopausal and postmenopausal women, except in body height, subscapular skinfold thickness and tryglyceride concentration. Significant correlation with the risk factors was detected in BMI and both skinfold thicknesses, while the subscapular thickness correlates more intensively with the risk factors than it is the case with the triceps thickness. The results indicate that BMI equally correlates with risk factors as well as skinfold thickness.
Despite the numerous benefits of milk constituents for human health a considerable number of the general population follow a milk-restricted diet due to clinically confirmed or self-assessed adverse ...reactions to cow's milk consumption. Recurrent aphthous ulcers (RAU) are currently one of the most common oral disorders, with a worldwide distribution and insufficiently defined etiology, which, among other factors, implies the immunological reaction to food proteins. The aim of this study was to determine the immune-reactivity to donkey's milk proteins in patients with RAU and compare it to the reactivity towards the proteins from cow's and goat's milks, in a set of simultaneous experiments. Levels of serum IgA, IgG and IgE antibodies to the same quantity of the examined antigens were determined by enzyme-linked immunosorbent assay. The results indicate that patients with RAU with increased immunity to cow's milk proteins could consider the use of donkey's milk as the best protein source.
Studies conducted on children and adults have pointed to the correlation of BMI (kg/m2) with risk factors for certain diseases. Other studies have stressed a more intense correlation between the risk ...factors and indicators of subcutaneous fat obtained in other ways. The aim of the study was to compare the intensity of correlation between the risk factors and triceps and subscapular skinfold thickness in relation to BMI. The study included 53 postmenopausal and 107 premenopausal women, the risk factors were assessed upon systolic and diastolic blood pressure, glucose concentration, triglyceride and cholesterol levels. Statistically significant differences were established in almost all variables referring to premenopausal and postmenopausal women, except in body height, subscapular skinfold thickness and triglyceride
concentration. Significant correlation with the risk factors was detected in BMI and both skinfold thicknesses, while the subscapular thickness correlates more intensively with the risk factors than it is the case with the triceps thickness. The results indicate that BMI equally correlates with risk factors as well as skinfold thickness.
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