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Onko (Subject) / x16 Ausserlechner (Lesson)
- What means FRET? Explain the underlying mechanism and how this can be used for drug discovery screening (two examples). FRET (Fuorescence Resonance Energy Transfer): · If two chromophores are in close proximity, part of the energy of the excited state of the donor can be transferred via non-radiative transmission of energy through direct dipole-dipole interaction to the acceptor chromophore FRET requirements: · Overlap of the emission spectrum of the donor and the excitation spectrum of the acceptor (30 % or more are necessary to achieve sufficient FRET for reliable detection) · Close proximity of donor and acceptor ranging between 1-10 nm · An appropriate relative orientation of the dipole vectors of the two chromophores FRET applications: -Enzyme assays for the identifitcation of e.g. protease inhibtors -Protein-protein interaction in living cells -FRET kinases assay → change of protein conformation through Phophor./Dephosphorylation of a substrate sequence that links the two FRET chromophores → identification of enzyme inhibitors
- Which are the 3 main Fragen that have to be clarified / analyzed by a company before starting a drug discovery project? Give two examples for the tasks of each question that have to be addressed. Scientific and technical questionas (avalibity of targets, existing animal models) stragetic questions (market predictions, company strategy and franchise) and operational questions (resources need, timescale)
- What is “high throughput screening (HTS)” and which assays (two examples) are suitable for HT-screening? Explain the biological principles underlying these assays. 10000-100000 compounds per day can be tested on direct interaction with molecules. chromogenic assay: Chromogenic assays result in a colored reaction product that absorbs light in the visible range. The antigen-antibody complex formed on the solid carrier is separated from other substances by washing. The antibody was labeled by enzyme. After the substrate of the enzyme was added, the substrate becomes a colored product under catalysis of the enzyme-catalyzed, which is directly related to the amount of test substance fluorescence assay: example alamarblue
- What means “surface plasmon resonance (SPR)” and what can you measure with this method? Explain the technical background of this method. - Direct measurement of molecule – molecule interaction in realtime (association, equilibrium, dissociation) - No labeling of molecules - Biosensor (glas + thin goldfilm) - Gold is covered with hydrophilic matrix e.g. carboxymethylated dextran (100 nm) - Optical detector (LED, prism, diode-array detector) - Flow system SPR Principle - Light is reflected in a certain angle - Energy of light causes resonance oszillation of gold atoms - Induces a so called “evanescent field” and reduces the intensity of reflected light - Change of Mass (Receptor/Ligand) changes refraction and resonance
- What are the “properties of an ideal drug target” (3 examples) Target has a favorable assayability enabling high throughput screeningTarget/disease-specific biomarker exists to monitor therapeutic efficacyFavorable prediction of potential side effects according to phenotype dataTarget has a favorable IP situation ( no competitors on target/ freedoom TO operrate
- Which animals are suitable for “whole animal screening”? What are the two types of screen that may be used? C. elegans, D. melangogaster, D. rerio S. Voglio (!) Chemical genetics screen: Compounds to phenotype Therapeutic screen: Phenotype to compounds
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