Phenotypic Drug Discovery And Kinase Inhibitors

Question 1

For the following parameters: readout, detection, SAR and drawbacks, compare phenotypic vs structure based screening

Answer 1

Phenotypic:
Readout - cell viability/ phenotypic changes (IC/EC 50)
Detection - imaging/ colorimetry
SAR - complex, multiple targets, membrane permeability, metabolic enzymes
Drawbacks - MOA??

Structure-based screening
Readout - binding (Ki/Ka)/ functional changes (IC50)
Detection - X-ray/ NMR/ fluorescence/ colorimetry
SAR - very simple
Drawbacks - limited by application in biological model - often permeability/ lipophilicity

Question 2

What are Protein Kinases?

Answer 2

Enzymes that phosphorylate specific amino acids using ATP co-factor
Located within cytoplasm or cell membrane associated
Two types: tyrosine and serine-threonine residues

Question 3

How are membrane bound enzyme receptors activated?

Answer 3

Receptor has duel role of recognising chemical messenger (receptor) as well as catalysing phosphorylation (enzyme)

Question 4

What is the activation mechanism of the epidermal growth factor (EGF)?

Answer 4

EGF = tyrosine kinase receptor
Steps:
Binding with EFG
Dimerisation
Phosphorylation

Question 5

Describe the protein kinase signal transduction pathway

Answer 5

Phosphorylated tyrosine residues act as a binding site, highly complex process, interruption at any point disrupts entire pathway

Question 6

Describe the MOA and SARs for the ATP co-factor of protein kinases, in the epidermal growth factor receptor

Answer 6

Purine core - VDW with amino acid residue - allows correct orientation
Two hydrogen bonds with Hinge region
Ribose sugar in ribose binding pocket
Triphosphate in cleft - ionic and hydrophobic interactions

Question 7

Describe the MOA and SARs of protein kinase inhibitor (Gefitinib) in the ATP binding site

Answer 7

Question 8

What does the suffix of ‘tinib’ indicate about a drugs target?

Answer 8

Kinase inhibitor - anti-cancer

Question 9

Outline the optimisation of EGFR inhibitor (Gefitinib) from its hit molecule

Answer 9

Hit molecule (IC50 - 5nm) —P450–> inactive metabolites

Hit molecule (IC50 - 5 nm) —biostere or fluorination—> optimised lead (IC50 - 9 nm, stability increased but poor solubility) —incorporation of side chain, increase aq stability——> Gefitinib (IC50 - 33 nm)

Question 10

Name four EGFR inhibitors and indicate their SARs

Answer 10

Erlotinib - alkyne - metabolised by P450 to ketone with nucleophile
Icotinib - alkyne - metabolised by P450 to ketone with nucleophile
Lapatinib - dual action inhibitor
Vandetanib - extended spectrum inhibitor

Question 11

What is a covalent irreversible kinase inhibitor, as well as its MOA?

Answer 11

Afatinib - acylamide Michael acceptor - can accept nucleophile attack
Must be highly selective due to potency and consider target enzymes in host

Question 12

What is the synthetic procedure of Gefitinib and its analogues? (6-step process)

Answer 12

1) Addition of -OH group
2) conversion of -OH group into protecting group (OAc)
3) Conversion of carbonyl to Cl group
4) substitution of Cl to aniline substituent
5) Removal of OAc group to -OH group
6) Deprotection step