Screening Methods

Question 1

What’re bioassays used for?

Answer 1

Used for testing against an enzyme/protein

Question 2

What’re the advantages of bioassays?

Answer 2

Requires little protein

If robust, in high concentrations of ligand, then its a rapid and quantitative method for detection

Question 3

What’re the disadvantages of bioassays?

Answer 3

False positives due to compound aggregation

Solubility of compounds in assay buffers is a challenge

Often lack sensitivity

Question 4

What is capillary electrophoresis?

Answer 4

It is a high-resolution technique that detects both high and low affinity molecular interactions.

(Pass through capillary)

Question 5

What’re the advantages of capillary electrophoresis?

Answer 5

Very low protein consumption - no need for high purity

Solution based - no immobilisation needed

No false positives

Generates IC50 data

Question 6

What’re the disadvantages of capillary electrophoresis?

Answer 6

Does not offer insight into the docked ligand that X-Ray crystallography might.

Question 7

What is target-based NMR screening?

Answer 7

It monitors changes in H, C or O and N correlation signals for a labelled protein, when binding to a test compound

Question 8

What’re the advantages of target-based NMR screening?

Answer 8

Is sensitive for weakly binding ligands - mM to nM interactions

Provides structural info on binding site

Question 9

What’re the disadvantages of target-based NMR screening?

Answer 9

Requires a relatively large amount of isotopically labelled protein

Not all proteins are of correct size/solubility

Question 10

What is ligand-based NMR screening?

Answer 10

It’s relies on changes in the ligand signals when binding to the target

Question 11

What’re the advantages of ligand-based NMR screening?

Answer 11

Does not require isotopically labelled protein

Protein consumption considerably lower than target-based NMR screening

Question 12

What’re the disadvantages of ligand-based NMR screening?

Answer 12

Has trouble detecting strong binding ligands

Question 13

What’re the advantages of 19F NMR screening?

Answer 13

Faster and more robust the 1H based NMR screening - in ligand-based screening

Question 14

What’re the disadvantages of 19F NMR screening?

Answer 14

Compounds must be 19F labelled

Question 15

What’re the advantages of X-Ray crystallography?

Answer 15

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

Question 16

What’re the disadvantages of X-Ray crystallography?

Answer 16

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

Question 17

What’re the advantages of mass spectrometry?

Answer 17

Process requires very small amounts of protein

Small protein-molecule complexes can be observed

No immobilisation or conjugation needed

Question 18

What’re the disadvantages of mass spectrometry?

Answer 18

Can lead to false positives due to aggregation

Question 19

What is Surface Plasmon Resonance and what are its (dis)advantages?

Answer 19

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.

Question 20

What are Thermal Shift Assays?

Answer 20

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

Question 21

What’re the fastest screening methods?

Answer 21

Thermal shift

Capillary electrophoresis

Enzyme activity - bioassays

Surface Plasmon Resonance

Question 22

What’re the slowest screening methods?

Answer 22

Protein NMR

Protein crystals

Mass spec

Ligand NMR

Question 23

What is virtual fragment screening?

Answer 23

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

Question 24

Whats the significance of binding sub-pockets?

Answer 24

Smaller structural domains can bind to fragments, meaning 2 proteins may differ significantly but share similar subpockets.

Can therefore bind identical fragments

Question 25

Whats the issue with the binding subpockets concept?

Answer 25

Other competitors may be building a similar drug using the same technique - as identical ligands will bind to similar subpockets in very different proteins