Kinases (bcr-abl and imatinib)

why do 2nd/3rd/4th generation kinase inhibitors exist?

why do 2nd/3rd/4th generation kinase inhibitors exist?

cancer develops resistance to treatment over time due to changes in protein structure

enzyme activation through kinases

phosphorylation by kinases → conformational rearrangement

receptor kinase domain autophosphorylation

occurs due to dimerisation as result of ligand binding eg EGF binding EGFR causes autophosphorylation. Active EGFR attracts proteins via phosphate group and phosphorylates other substrates for signalling

protein movement as a result of kinase action

phosphate attachment causes protein to be attracted to proteins with +ve motifs allowing them to be recruited to nucleus or membrane

kinase structure

bilobal global structure connected by flexible hinge-linker

how do kinases bind atp

as a cofactor at the hinge between lobes. Close to bind ATP and open to release ADP

kinase activation loop

binds protein substrate and positions it next to ATP so phosphate can be transferred by nucleophilic substitution

magnesium ions associated with kinase

coordinate with ATP terminal phosphate to make it more reactive towards transfer in the substrate

gatekeeper

first amino acid in hinge region at N lobe, gatekeeps potential kinase selectivity pocket

How does ATP associate with the hinge region?

binds amino acids by H bonds with GK+1 and GK+3

How can kinases be prevented from phosphorylating substrates?

design molecule which competes with ATP for the binding site

how to make sure druglike molecules only target the required kinase

need to know amino acid sequence in hinge region from gatekeeper onwards

staurosporine

kinase inhibitors which inhibit approx 90% of kinases - used as positive control in kinase assays

dual kinase inhibitors

can be beneficial as they may target multiple pathways in a certain cancer

how do most kinase inhibitors begin

as a heterocycle which targets H bonding groups of the hinge region

how is the heterocycle modified to exploit selectivity (GK)?

addition of groups to target the region behind the GK side chain

How is heterocycle selectivity and affinity improved (ATP binding site)?

differences in overall shape of ATP binding site exploited by addition of groups

gefitinib target

EGFR

gefitinib properties

potent inhibitor with poor solubility, low bioavailability and is rapidly metabolised

how does gefitinib compete with ATP

hydrogen bonds to GK+3 (met 793) and targets region behind GK side chain via benzene group with methyl chain (targets Thr 790)

gefitinib modification with hydrophobic site addition

blocks metabolism

gefitinib solvent exposed region modification

improves solubility

erlotinib

targets EGFR, similar core to gefitinib. Contains hydrophobic carbon-carbon triple bond and different solvent exposed region

Bcr-Abl

fusion gene formed be chromosomal translocation in chronic myeloid leukaemia

bcr-abl function

drives CEBP alpha mediated transcription → cell growth

how do imatinib/nilotinib bind bcr-abl

H bond to GK +3 of hinge (Met 318) and H bond to GK side chain (Thr 315)

properties of initial imatinib molecule

moderately potent (5 micromolar) with poor selectivity for Tyr kinases over Ser/Thr kinases, poor solubility and low bioavailability

imatinib modification with flag methyl group

forces bond rotation so amide group H bonds with Glu 286 in N lobe and Asp 381 in C lobe - increases affinity by 0.1 micromolar and selectivity

how is imatinib modified for increased solubility/bioavailability

addition of tertiary amine group so inhibitor can be formulated as water soluble hypochloride salt