Genetically engineering a new system for the expression of cytochrome P450 enzymes in insect cells using novel P450 reductases

Human cytochrome P450 enzymes (CYPs) are a superfamily of haem-binding monooxygenase enzymes involved in the metabolism of xenobiotics such as toxins, carcinogens and pharmaceutical drugs as well as in the biosynthesis of cholesterol, vitamins and steroids. In recent years, the area of drug metabolism in the drug discovery process has become crucial for the final clinical success of a drug candidate. Use of CYPs early in the drug discovery process can save significant amount of costs and time required in pre-clinical and clinical studies, thereby greatly facilitating the process. The human CYP superfamily of proteins comprises more than 50 enzymes, each enzyme being able to catalyse multiple reactions. With the exception of some plants, a single NADPH cytochrome P450 reductase (CPR) of a particular eukaryotic species interacts with all CYPs of the same species. For CYP catalytic activity, CPR is absolutely essential, however at the same time CPR is detrimental for the expression of CYPs. Therefore, understanding the process by which the interaction between CYP and CPR occurs is an important biological goal. I have cloned, expressed and studied the interactions of seven CYPs, CYP2D6, CYP3A4, CYP1A1, CYP1B1, CYP1A2, CYP2E1 and CYP2C8 in conjunction with different CPR species (the native human CPR, variants of human CPR and the yeast CPR) using the baculovirus expression system. In my studies I have found that different CPRs have different coupling efficiencies towards the individual CYP 3 isoforms. Use of a high-activity CPR from yeast in this study has allowed us to improve our understanding on CYP-CPR interactions. I have found that the ability of a CPR to reduce an artificial substrate like MTT is not directly proportional to its ability to reduce the physiological substrate, CYP. In other words, the strength of the reductase does not determine CYP activity but it is the ability of CPR to couple with CYP which is crucial. This study has resulted in the identification of ΔhRDM, a genetically engineered variant of human CPR, which couples with CYPs far better than the human native CPR and also offers advantage of better reaction rates for CYPs. ΔhRDM also offers an improvement in the ratio of spectrally active CYP2D6 to spectrally inactive CYP2D6. Identification of ΔhRDM has allowed us to devise an insect cell expression system that genetically provides an improvement in the levels and activities of the drug metabolising cytochrome P450 enzymes

Expressing the human proteome for affinity proteomics: optimising expression of soluble protein domains and in vivo biotinylation

The generation of affinity reagents to large numbers of human proteins depends on the ability to express the target proteins as high-quality antigens. The Structural Genomics Consortium (SGC) focuses on the production and structure determination of human proteins. In a 7-year period, the SGC has deposited crystal structures of >800 human protein domains, and has additionally expressed and purified a similar number of protein domains that have not yet been crystallised. The targets include a diversity of protein domains, with an attempt to provide high coverage of protein families. The family approach provides an excellent basis for characterising the selectivity of affinity reagents. We present a summary of the approaches used to generate purified human proteins or protein domains, a test case demonstrating the ability to rapidly generate new proteins, and an optimisation study on the modification of >70 proteins by biotinylation in vivo. These results provide a unique synergy between large-scale structural projects and the recent efforts to produce a wide coverage of affinity reagents to the human proteome

Sclerotium rolfsii Lectin Induces Stronger Inhibition of Proliferation in Human Breast Cancer Cells than Normal Human Mammary Epithelial Cells by Induction of Cell Apoptosis

Sclerotium rolfsii lectin (SRL) isolated from the phytopathogenic fungus Sclerotium rolfsii has exquisite binding specificity towards O-linked, Thomsen-Freidenreich (Galβ1-3GalNAcα1-Ser/Thr, TF) associated glycans. This study investigated the influence of SRL on proliferation of human breast cancer cells (MCF-7 and ZR-75), non-tumorigenic breast epithelial cells (MCF-10A) and normal mammary epithelial cells (HMECs). SRL caused marked, dose-dependent, inhibition of proliferation of MCF-7 and ZR-75 cells but only weak inhibition of proliferation of non-tumorigenic MCF-10A and HMEC cells. The inhibitory effect of SRL on cancer cell proliferation was shown to be a consequence of SRL cell surface binding and subsequent induction of cellular apoptosis, an effect that was largely prevented by the presence of inhibitors against caspases -3, -8, or -9. Lectin histochemistry using biotin-labelled SRL showed little binding of SRL to normal human breast tissue but intense binding to cancerous tissues. In conclusion, SRL inhibits the growth of human breast cancer cells via induction of cell apoptosis but has substantially less effect on normal epithelial cells. As a lectin that binds specifically to a cancer-associated glycan, has potential to be developed as an anti-cancer agent

Identification of A Novel Class of Benzofuran Oxoacetic Acid-Derived Ligands that Selectively Activate Cellular EPAC1

Cyclic AMP promotes EPAC1 and EPAC2 activation through direct binding to a specific cyclic nucleotide-binding domain (CNBD) within each protein, leading to activation of Rap GTPases, which control multiple cell responses, including cell proliferation, adhesion, morphology, exocytosis, and gene expression. As a result, it has become apparent that directed activation of EPAC1 and EPAC2 with synthetic agonists may also be useful for the future treatment of diabetes and cardiovascular diseases. To identify new EPAC agonists we have developed a fluorescent-based, ultra-high-throughput screening (uHTS) assay that measures the displacement of binding of the fluorescent cAMP analogue, 8-NBD-cAMP to the EPAC1 CNBD. Triage of the output of an approximately 350,000 compound screens using this assay identified a benzofuran oxaloacetic acid EPAC1 binder (SY000) that displayed moderate potency using orthogonal assays (competition binding and microscale thermophoresis). We next generated a limited library of 91 analogues of SY000 and identified SY009, with modifications to the benzofuran ring associated with a 10-fold increase in potency towards EPAC1 over SY000 in binding assays. In vitro EPAC1 activity assays confirmed the agonist potential of these molecules in comparison with the known EPAC1 non-cyclic nucleotide (NCN) partial agonist, I942. Rap1 GTPase activation assays further demonstrated that SY009 selectively activates EPAC1 over EPAC2 in cells. SY009 therefore represents a novel class of NCN EPAC1 activators that selectively activate EPAC1 in cellulae

Lymphadenopathy resulting from acute toxoplasmosis mimicking relapse of non-Hodgkin′s lymphoma on fluorodeoxyglucose positron emission tomography/computed tomography

We report a case documenting fluorodeoxyglucose (FDG) accumulation in cervical, supraclavicular and axillary lymph nodes resulting from acute toxoplasmosis. A 50-year-old Indian female with history of non-Hodgkin′s lymphoma (NHL) of left breast, postchemotherapy status, was found to have hypermetabolic right cervical, supraclavicular and axillary lymph nodes on a surveillance FDG positron emission tomography/computed tomography (PET/CT) scan. Her previous two PET/CT scans were unremarkable with no evidence of metabolically active disease. Therefore, a differential diagnosis of relapse of NHL versus infectious/inflammatory pathology was raised in the report. Biopsy of axillary lymph node demonstrated features characteristic of toxoplasmosis. The serological test results were also compatible with acute toxoplasmosis infection. Infective and inflammatory diseases are known to accumulate FDG, resulting in false positives for malignancy. This case demonstrates lymph nodal toxoplasmosis as a potential cause of false positive FDG PET/CT findings in patients with known malignancy and highlights the importance of histopathological and laboratory correlation for the accurate interpretation of FDG PET/CT scans

Composite adrenal medullary tumor: A rare cause of hypertension in a young male

Composite tumors of the adrenal medulla, containing pheochromocytoma and ganglioneuroma, are rare. A 27-year-old male presented to us with dyspnea and was found to have labile hypertension. A left suprarenal mass was detected on computed tomography. The patient was operated under the cover of alpha anti-adrenergic drugs. The histopathological examination demonstrated that the tumor consisted of pheochromocytoma and ganglioneuroma elements, and hence, a diagnosis of composite adrenal medullary tumor (CAMT) was made. To the best of our knowledge, this is the first case of CAMT reported from India

LC and LC–MS/MS studies for the identification and characterization of degradation products of acebutolol

The aim of the present investigation was to demonstrate an approach involving use of liquid chromatography (LC) and liquid chromatography-mass spectrometry (LC–MS) to separate, identify and characterize very small quantities of degradation products (DPs) of acebutolol without their isolation from the reaction mixtures. The drug was subjected to oxidative, hydrolytic, thermal and photolytic stress conditions as per International Conference on Harmonization (ICH) guideline Q1A(R2). Among all the stress conditions the drug was found to be labile in hydrolytic (acidic & basic) and photolytic stress conditions, while it was stable in water-induced hydrolysis, oxidative and thermal stress conditions. A total of four degradation products were formed. A C18 column was employed for the separation of all the DPs on a gradient mode by using high-performance liquid chromatography (HPLC). All the DPs were characterized with the help of their fragmentation pattern and the masses obtained upon LC–MS/MS and MSn analysis. All the hitherto unknown degradation products were identified as 1-(2-(2-hydroxy-3-(isopropylamino)propoxy)-5-(amino)phenyl)ethanone (DP-I), N-(4-(2-hydroxy-3-(isopropylamino)propoxy)-3-acetylphenyl)acrylamide (DP-II), 1-(2-(2-hydroxy-3-(isopropylamino)propoxy)-5-(hydroxymethylamino)phenyl)ethanone (DP-III) and 1-(6-(2-hydroxy-3-(isopropylamino)propoxy)-2,3-dihydro-2-propylbenzo[d]oxazol-5-yl)ethanone (DP-IV). Finally the in-silico carcinogenicity and hepatotoxicity predictions of the drug and all the DPs were performed by using toxicity prediction softwares viz., TOPKAT, LAZAR and Discovery Studio ADMET. The results of in-silico toxicity studies revealed that acebutolol (0.967) and DP-I (0.986) were found to be carcinogenic, while acebutolol (0.490) and DP-IV (0.437) were found to be hepatotoxic. Keywords: Acebutolol, Stress testing, LC, LC–MS/MS, Degradation pathway, In-silico toxicit

Endometrioid carcinoma of the upper urinary tract

Herein, we report a second case of endometrioid carcinoma of the upper urinary tract presenting 17 years after hysterectomy for high grade adenocarcinoma of ovary. A 51-year-old nullipara presented to us with a complaint of hematuria. After complete work up, she underwent right radical nephro-ureterectomy with bladder cuff excision. The histology showed endometrioid carcinoma of upper urinary tract without any evidence of endometriosis

Synthesis, antiinflammatory evaluation and docking analysis of some novel 1,3,4-oxadiazole derivatives

607-616In the present study, novel series of 5-{[4-(acetylamino) phenoxy] methyl}-1,3,4-oxadiazole-2-yl-sulfanyl-N-substituted-2-acetamide/2-propanamide/3-propanamide derivatives (8a to 8c, 9a to 9c and 10a to 10c) have been synthesized.The newly synthesized compounds have been tested for their anti-inflammatory and anti-ulcerogenic activities in vivo.Among the present series the compound 8c is found to be most active against inflammation with inhibition of 65.34% andhas been observed to be safe ulcerogenically. It is observed that introduction of an asymmetric centre near the sulfur atomdecreases the activity. Molecular docking simulations have been carried out for the compounds. Structures from all theseries fit into the active site of cyclooxygenase-2 enzyme with least binding energies and exhibit favorable bindinginteractions required for the selective inhibition of cyclooxygenase- 2