Kinases (bcr-abl and imatinib)

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

causes conformational rearrangement

cause and result of receptor kinase domain autophosphorylation

occurs due to dimerisation when ligand binds. Dimer attracts proteins via phosphate 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

make ATP terminal phosphate more reactive towards transfer to substrate

gatekeeper

first amino acid in hinge at N lobe. Gatekeeps kinase selectivity pocket

How does ATP associate with the hinge region?

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 region behind 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

H bonds GK+3 and targets region behind GK side chain via methylated benzene

gefitinib modification with hydrophobic site addition

blocks metabolism

purpose of gefitinib solvent exposed region modification

improves solubility

erlotinib target and structure

targets EGFR. Similar core to gefitinib but contains carbon-carbon triple bond and different solvent-exposed region

Bcr-Abl

fusion gene formed by 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 (Met 315) and 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

How does imatinib modification with flag methyl group increase affinity and selectivity?

forces bond rotation so amide group H bonds with Glu 286 in N lobe and Asp 381 in C lobe

how is imatinib modified for increased solubility/bioavailability

addition of tertiary amine group - makes it a water soluble hypochloride salt