Hits and Leads

Question 1

Hit

Answer 1

has desired effect on target protein

Question 2

Finding hits

Answer 2

• fragment screen
• literature
• random high throughput screen (HTS)
• structure-based
• natural products
• chemogenomics
• natural ligand

Question 3

High throughput screening assays

Answer 3

• GPCRs = reporter gene assays, enzyme complementation, radioligand binding
• enzymes = enzyme inhibition, label-free binding assay
• ion channels = electrophysiology, membrane potential-sensitive dyes

can confirm the effect is real if it is dose-dependent

Question 4

e.g. second messenger assays

Answer 4

• cells preloaded with calcium-sensitive fluorescent dye
• activation of GPCR = PLC = IP3 = Ca
• concentration-response curve to measure EC50

Question 5

e.g. enzyme activity assays

Answer 5

• bead-based assay
• FP = fluorescence polarisation
• free peptide = low FP
• bound peptide = high FP
• binding to bead based on charge increases the FP readout

Question 6

Requirements of an assay

Answer 6

• pharmacologically relevant
• simple
• cost-effective
• easy to automate
• preferably homogeneous
• good signal:noise
• robust
• lack of interference from coloured compounds

Question 7

Fragments

Answer 7

very small ligands which are components of a drug molecule
- small but no functional groups
- binds with low affinity
- must be soluble in aqueous buffer

Question 8

Ligand Efficiency

Answer 8

binding energy per heavy atom count

Question 9

Fragment screening

Answer 9

• hits selected based on affinity and so may not be quality leads
• whereas, fragments are low affinity but may be more chemically suitable
• highly selective screening required to detect fragment binding

Question 10

Structure-based design

Answer 10

• structure-activity relationship
• optimisation involves iterations
• empirical approach = change molecule and see what happens
• structure-based approach = use knowledge of protein structure to guide molecule design
• uses X-ray crystallography

Question 11

e.g. hepatitis C

Answer 11

• leading cause of chronic liver disease
• NS3 region of hepC is a protease
• crystallography showed protease active site is shallow + long + makes multiple weak interactions
• difficult to develop high affinity inhibitor for such a site
• move to structure-based approach
• boceprevir = potent HCV protease inhibitor with sufficient pharmacokinetics; withdrawn due to better drugs in the market