Lecture 12 - Phenotypic drug discovery and kinase inhibitors

Question 1

What are protein kinases? What do they do? What do they use to aid their function?

Answer 1

• Class of protein
• Enzymes that phosphorylate specific amino acids in protein substrates using ATP as cofactor
• Protein kinase receptors have dual roles of receptor (recognising chemical messages) and enzyme (catalysing phosphorylation)

Question 2

Locations of protein kinases in the cell

Answer 2

• Within cytoplasm
• Associated with cell membrane

Question 3

Categories of protein kinases

Answer 3

• Tyrosine kinases
• Serine-threonine kinases

Question 4

How are membrane-bound kinase receptors activated? (3)(messenger, active site, substrate)

Answer 4

• Messenger binds to kinase receptor from outside the cell
• Active site on kinase receptor opens inside the cell
• Substrate binds / Reaction

Question 5

What is an EGF receptor? What is the activation mechanism for the EGF receptor? (Mention EGF ligands, activity, changes to receptors)

Answer 5

• EGF (epidermal growth factor) receptor = tyrosine kinase receptor
• EGF (bidentate ligand) binds to 2 EGF receptors forming dimer
• Tyrosine kinase activity increases
• EGF receptors phosphorylated using ATP

Question 6

Signal transduction pathway (complex, just know *)(tyrosine residues function)

Answer 6

• Phosphorylated tyrosine residues from EGF receptors act as binding sites*
• Highly complex process*
• Grb2 and SoS proteins bind to residues*
• GTP converted to GDP at SoS, activating Raf
• Raf activates Mek
• Mek activates Map kinase which activates the transcription factor for gene transcription
• Interruption at any point affects whole pathway*

Question 7

Factors of ATP making it a good cofactor

Answer 7

• Purine core; Van der Waals interactions
• 2 H bonds interactions in “Hinge region”
• Ribose sugar in the ribose binding pocket
• Triphosphate in the cleft

Question 8

How are EGFR inhibitors optimised? (What is changed chemically? Mention binding sites)

Answer 8

• fluorination and chlorination of methylbenzene group in the hydrophobic pocket (Gefitinib)
• occupy the binding site

Question 9

Differences between phenotypic and structure-based screening

Answer 9

Phenotypic:
- Screening object: Whole cells
- Readout: Cell viability/phenotypic changes (IC50/EC50)
- Detection methods: imaging/colorimetry
- SAR: Complex (multiple targets, membrane permeability, metabolic enzymes)
- Drawbacks: Mode of action

Structure-based:
- Proteins/enzymes
- Binding (Ki/Kd)/ functional changes (IC50)
- X-ray/NMR/fluorescence/colorimetry/TSA, etc.
- Simple
- Permability/availability

Question 10

What do covalent irreversible kinase inhibitors act against? What factors are key for this type of inhibitor?

Answer 10

• Act against mutated EGFR, bind irreversibly
• Selectivity/toxicity = key for covalent inhibitors