theme 4 screening Flashcards
What is the aim of High Throughput Screening (HTS)?
To take a wide range of molecules and assess their activity in an assay against a target.
What are the key characteristics needed for an HTS assay?
Reproducible, sensitive, simple, robust, reliable.
What types of biological activities can be assessed in HTS assays?
Agonism, antagonism, inhibition.
What are the two key types of assays in HTS?
- Target-based
- Phenotypic
What is a target-based assay?
An assay where the target is known, including biochemical, biophysical, and cellular assays.
What is a phenotypic assay?
An assay that assesses the cell as a whole, where the target does not need to be known.
What are the four key methods of readout in HT assays?
- Absorption
- Photoluminescence
- Chemoluminescence
- Radioluminescence
What is a homogeneous assay configuration?
An assay where no separation of components is needed to obtain results (‘mix and read’).
What is a non-homogeneous assay configuration?
An assay where separation of components via filtration/washing is required to obtain results.
What is Scintillation Proximity in HTS?
A radiometric analysis of activity that does not require washing steps or separation of components.
What biophysical techniques are increasingly applied in early drug discovery?
- Crystallography
- NMR
- Surface Plasmon Resonance (SPR)
- Isothermal Titration Calorimetry (ITC)
- Differential Scanning Fluorimetry
- Mass Spectrometry
What is fragment-based screening?
A method that utilizes smaller compounds with lower molecular mass that can be combined to create lead molecules.
What are the three main methods of using fragments to form lead molecules?
- Fragment evolution
- Fragment linking
- Fragment self-assembly
What is scaffold hopping?
A method that discovers structurally novel compounds by replacing parts of the scaffold to improve properties.
What are the two broad approaches in virtual libraries?
- Ligand guided
- Receptor guided
What does the signal to background measure assess in HTS?
Signal to background = mean of positive control signal / mean of background signal.
What is an acceptable value for the signal to noise ratio in HTS?
A value of 10.
What is the Z’ factor used for in HTS?
To evaluate the overall performance of the HTS assay.
What should the Z’ factor value be to indicate a good assay?
Close to 1 but above 0.4.
What is the purpose of developing a screening cascade?
To narrow molecules from hits to actives.
Fill in the blank: The nature of biological material can be either _______ or whole cells.
cell-free systems
True or False: Whole cell assays provide a simpler system than cell-free assays.
False
What is the purpose of identifying high quality hits during screening?
To eliminate false positives and prioritize true positives.
What is meant by false positives in screening?
Signals that indicate hits but do not correspond to actual active compounds.
