Identification of xenobiotics inhibiting AKR1C3

Androgen receptor (AR) activation is critical for the growth and function of the prostate and the testis but also for metabolic processes in skeletal muscle, bone and adipose. Inappropriate activation of the receptor by an overproduction of androgens is known to promote prostate cancer. In the US alone, 16% of all men will face prostate cancer during their life time, turning prostate cancer to the second most common malignancy.

Current prostate cancer treatments include the deprivation of androgens by chemical or surgical castration as well as treatment with anti-androgens. In castrate resistant prostate cancer, the AR is excessively activated by androgens produced within the tumor, by altered post-translational modification, the occurrence of mutations or due to splice variants. To block intra-tumor production of androgens, inhibition of enzymes responsible for the formation of potent AR ligands like testosterone and 11-dihydrotestosterone are under investigation. One of the key enzymes is the aldo-keto-reductase 1C3 (AKR1C3). Though inhibition of AKR1C3 in a cancer situation should be beneficial, this enzyme should not be inhibited during early development and puberty, where the male sex phenotype is determined. Thus, identification of xenobiotics unintentionally taken up and inhibiting this enzyme is important regarding anti-androgenic effects of such chemicals.  

In cooperation with the computational toxicology group of Dr. Martin Smiesko, in silico screening proposed chemicals inhibiting AKR1C3. In the proposed project, a cell-free enzyme assay using cytoplasmic fractions containing overexpressed recombinant protein will be established. After validating the new activity assay, a list of chemicals predicted in silico will be analyzed for inhibitory effects. Identified inhibitors will then be investigated in cells with endogenous enzyme expression and possibly for effects on cancer cells.

Methods: Culturing eukaryotic cells, transfection of cells, preparation of cytoplasmic fraction, western blotting to assess protein expression, enzyme activity measurements, RT-qPCR, transformation of E. coli, plasmid extraction and purification, construction of expression plasmids, PCR, agarose gels, scintillation measurements, cytotoxicity measurements

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Sensing NADPH in the endoplasmic reticulum

Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is an essential cofactor for many reactions in living organisms. It supports reducing capacity, macromolecular biosynthesis, superoxide and nitric oxide production and detoxification of drugs and xenobiotics. Perturbations in the NADPH metabolism lead to impaired cellular function causing increased risk of inflammation, cancer, diabetes mellitus and neurodegeneration.

The existing methods for NADPH measurements in the endoplasmic reticulum are often invasive and hence do not allow measurements in living cells; in addition they are prone to sample oxidation during the process. Other methods like Fluorescence lifetime imaging can distinguish between NADPH and NADH fluorescence in living cells; however, specialized equipment is needed and may not be readily accessible. In response to these challenges, Tao et al (2017) have recently developed a genetically encoded fluorescent sensor capable of sensing NADPH in the cytoplasm and mitochondria of the living cell.

The aim of this master project will be to modify the existing sensor to ensure appropriate expression and functionality in the endoplasmic reticulum. This will include a site directed mutagenesis based screening of different forms of the sensor to find the optimal form for the unique environment of the endoplasmic reticulum

Lab head: Professor Alex Odermatt, direct supervisor: Dr. Julia Birk

Molecular & Systems Toxicology

Pharmacenter, Klingelbergstrasse 50

4056 Basel, Switzerland

LC-MS/MS methods as a tool for biomarker discovery

Bile acids are involved in several important functions in the liver and the intestine. The formation of bile acids is important for the regulation of the maintenance of cholesterol homeostasis. They are regulatory molecules for a number of metabolic processes, excretion of endogenous and exogenous toxic substances. Bile acids and their derivatives are potential therapeutic agents for treating metabolic diseases of the liver. On the other hand, oxysterols are oxidised forms of cholesterol or of its precursors. They are involved in neurodegenerative disease (e.g. Huntington's, Parkinson's and Alzheimer's diseases) and in cancer. Disease, especially those involving oxidative stress, may alter the equilibrium, and oxysterols have to potential to serve as diagnostic markers.

Methods for the quantification of various bile acids and oxysterols are essential for understanding their mechanisms of action in vivo, and valuable for diagnosing rare diseases of cholesterol biosynthesis and metabolism as well as for detecting drug-induced toxicity. Liquid chromatography- mass spectrometry is considered a gold standard technique in Analytical Chemistry in both Academia and Industry.

The aim of the master project will be to optimize extraction protocols for the detection and quantification of bile acids and oxysterols in biological matrices. The newly established method will be applied to assess samples from in vivo studies. The master student will be able to learn the basics of chromatography and mass spectrometry techniques and data analysis.

Methods: LC-MS/MS, Solid-Phase Extraction, Liquid-Liquid Extraction, Derivatization

Lab head: Professor Alex Odermatt, direct supervisor: Dr. Cristina Gómez Castellà

Molecular & Systems Toxicology

Pharmacenter, Klingelbergstrasse 50

4056 Basel, Switzerland

Investigating xenobiotics-induced disturbances of hepatic sterol and bile acid metabolism

The liver is the central organ responsible for the selective uptake, metabolism and excretion of endogenous and exogenous compounds, including drugs. Drug-induced liver injury (DILI) can be caused by various chemicals and can present as an array of different pathologies, dependent on the specific function of the liver that is impaired. Numerous xenobiotics have been shown to cause liver injury but the manifestations of drug-induced hepatotoxicity are highly variable, ranging from asymptomatic alterations of liver enzymes to fulminant hepatic failure.

In the present project, we aim to investigate a selected range of xenobiotics (mainly drugs) screened by bioinformatics tools for potential inhibitory effects towards AKR1D1 and SRD5A1. AKR1D1 and SRD5A1 are two important enzymes involved in bile acid and steroid metabolism in the liver.  The disruption of their function has been associated with different liver pathologies including inflammation, NASH/NAFLD, fibrosis, cancer and infections. Upon establishing activity assays for these two enzymes the most promising hits will be investigated for their potential to inhibit their enzyme activities and the consequences for bile acid and steroid homeostasis. Besides, we will investigate the potential interference of selected azole antifungal drugs with cytochrome P450 enzymes involved in hepatic bile acid synthesis. The consequences will be studies in a hepatocyte cell line.

Methods: RNA/DNA/proteins extractions and quantification, bacterial transformation and eukaryotic cell culture, cloning, DNA amplification (mini/maxiprep), transfection, cell culture and maintenance, enzyme activity assays, western blot, PCR/qPCR, protein purification.

Lab head: Professor Alex Odermatt, direct supervisor: Dr. Julien Allard

Molecular & Systems Toxicology

Pharmacenter, Klingelbergstrasse 50

4056 Basel, Switzerland

Selectivity modeling of fungicides inhibiting cytochrome P450 enzymes   

Several Cytochrome P450 (CYP) enzymes play a role in steroid hormone synthesis, e.g. aromatase or aldosterone synthase. They can be potently inhibited by fungicides containing azole moieties, thereby potentially interfering with hormone homeostasis. However, the selectivity of CYP inhibition and the respective binding modes of the inhibitors are still poorly understood. This master thesis aims to rationalize the selectivity of CYP inhibition using computational approaches such as pharmacophore modeling and docking studies. The results could help to understand and possibly avoid unintentional CYP inhibition by future fungicides.

Contact: daniela.schuster@clutterpmu.ac.at  Tel: 0699/14420025

Univ.-Prof. Daniela Schuster

Paracelsus Medizinische Privatuniversität

Leiterin Abteilung Pharmazeutische und Medizinische Chemie

Strubergasse 21, 5020 Salzburg, Österreich 

Dihydrochalcones as multi-target anti-inflammatory and anti-cancer targets: SAR modeling on selected targets    

Dihydrochalcones are natural products with multiple biological activities. For example, they have been shown to act on anti-inflammatory and anti-prostate-cancer targets. This multi-target activity could be an attractive strategy to combat multi-factorial diseases such as cancer in the future. In this project, computational approaches such as docking and pharmacophore-based screening will be used to rationalize the activity data of a series of dihydrochalcones against several targets and to propose improved compounds for future synthesis.

Contact: daniela.schuster@clutterpmu.ac.at Tel: 0699/14420025

Univ.-Prof. Daniela Schuster

Paracelsus Medizinische Privatuniversität

Leiterin Abteilung Pharmazeutische und Medizinische Chemie Strubergasse 21, 5020  Salzburg, Österreich 

Investigation of effects of combination therapy in cancer treatment  

The research focus of the Department of Pharmaceutical Biology and Clinical Pharmacy at the PMU Salzburg lies on the study of mechanisms involved in cancer pathogenesis, progression and therapy resistance. The aim of our group is to optimize cancer treatment strategies through combination therapy of biogenic or synthetic drugs. Currently, the effects of combination therapy on a wide range of tumor cells derived from liver and thyroid carcinomas are evaluated with state of the art methods including proliferation, migration and clonogenic survival assays as well as diverse biochemical techniques and confocal microscopy.

Contact: Johanna.pachmayr@clutterpmu.ac.at

Paracelsus Medizinische Privatuniversität

Strubergasse 21, 5020  Salzburg, Österreich