Screening Methods Flashcards
What’re bioassays used for?
Used for testing against an enzyme/protein
What’re the advantages of bioassays?
Requires little protein
If robust, in high concentrations of ligand, then its a rapid and quantitative method for detection
What’re the disadvantages of bioassays?
False positives due to compound aggregation
Solubility of compounds in assay buffers is a challenge
Often lack sensitivity
What is capillary electrophoresis?
It is a high-resolution technique that detects both high and low affinity molecular interactions.
(Pass through capillary)
What’re the advantages of capillary electrophoresis?
Very low protein consumption - no need for high purity
Solution based - no immobilisation needed
No false positives
Generates IC50 data
What’re the disadvantages of capillary electrophoresis?
Does not offer insight into the docked ligand that X-Ray crystallography might.
What is target-based NMR screening?
It monitors changes in H, C or O and N correlation signals for a labelled protein, when binding to a test compound
What’re the advantages of target-based NMR screening?
Is sensitive for weakly binding ligands - mM to nM interactions
Provides structural info on binding site
What’re the disadvantages of target-based NMR screening?
Requires a relatively large amount of isotopically labelled protein
Not all proteins are of correct size/solubility
What is ligand-based NMR screening?
It’s relies on changes in the ligand signals when binding to the target
What’re the advantages of ligand-based NMR screening?
Does not require isotopically labelled protein
Protein consumption considerably lower than target-based NMR screening
What’re the disadvantages of ligand-based NMR screening?
Has trouble detecting strong binding ligands
What’re the advantages of 19F NMR screening?
Faster and more robust the 1H based NMR screening - in ligand-based screening
What’re the disadvantages of 19F NMR screening?
Compounds must be 19F labelled
What’re the advantages of X-Ray crystallography?
False positives are reduced - can see the compound
Possible to assess how to enhance binding interactions through modelling techniques
Different ligands many occupy different volume of binding site, may allowing ‘linking’ to enhance binding affinity
What’re the disadvantages of X-Ray crystallography?
Very expensive and time consuming
Large proteins construct is needed, 10-50mg, with >95% purity.
No affinity information is obtained
Fragment ligand will need to be soluble in crystallisation medium - typically aqueous
What’re the advantages of mass spectrometry?
Process requires very small amounts of protein
Small protein-molecule complexes can be observed
No immobilisation or conjugation needed
What’re the disadvantages of mass spectrometry?
Can lead to false positives due to aggregation
What is Surface Plasmon Resonance and what are its (dis)advantages?
SPR is known to be a powerful tool for studying biomolecular interactions
It is sensitive and doesn’t require isotopic labelling.
Possible to evaluate larger libraries of compounds - however protein or ligand MUST be immobilised.
What are Thermal Shift Assays?
They monitor changes in protein stability and is regularly used to identify fragments.
As the temperature increases the protein unravels, exposing the hydrophobic centre - this can be measured using a fluorescent marker
Ligands that bind would be expected to increase protein stability
What’re the fastest screening methods?
Thermal shift
Capillary electrophoresis
Enzyme activity - bioassays
Surface Plasmon Resonance
What’re the slowest screening methods?
Protein NMR
Protein crystals
Mass spec
Ligand NMR
What is virtual fragment screening?
It is a computational method used to filter fragments with desired bioactivites from libraries
It docks fragments too targets and calculates binding affinities - saves time and money
Whats the significance of binding sub-pockets?
Smaller structural domains can bind to fragments, meaning 2 proteins may differ significantly but share similar subpockets.
Can therefore bind identical fragments
Whats the issue with the binding subpockets concept?
Other competitors may be building a similar drug using the same technique - as identical ligands will bind to similar subpockets in very different proteins
