Lecture 14 - Kinases in anticancer drug design Flashcards
what do kinases do?
kinase is an enzyme that uses ATP and transfers a negatively charged phosphate group onto a protein or lipid substrate.
They are usually involved in signalling cascade, where a ligand outside of a cell stimulates a membrane bound receptor, and a series of phosphate transfers are stimulated for message to be transmitted for response.
what are the consequences of phosphorylation by kinases?
enzyme activation - protein changes shape. the negatively charged phosphate attachment can cause conformational change from an inactive to an active form.
protein movement - negative charged phosphate can cause protein to be attracted to a positively charged protein. this can lead to recruitment of protein to the membrane or nuclear to transport signals around the cell.
how can signals be switched off?
phosphatases remove a phosphate from a protein, and reverts it back to its inactive state as a consequence of dephosphoryaltion. in the tumour environment, this balance is upset. the kinase is phosphoryalting and isn’t sufficient phosphatase to turn off signal
describe the structure of kinase molecule.
N-terminal domain at the top. C terminal domain at the bottom. hinge in between. at the hinge, this is where the kainse binds the ATP. hinge closes around the ATP once it is bound, and once ATP converted to ADP through the transfer of its phosphate, hinge opens and ADP can leave.
substrate binds next to where ATP is bound (next to the hinge)- the activation loop in the C terminus - so that the phosphate can be transferred.
ll kinases have same app binding sites, but different substrate binding region because substrate is unique to that kinase
what is the difference between tyrosine kinases and ser/thr kinase?
tyrosine kinases - a ‘tyrosine refers to the amino acid in the substrate that the kinase phosphorylates. eg VEGFR2 is a tyrosine kinase that phosphorylates a TYR in is substrate gastrin.
serine/threonine kinase is a kinase that phosphorylates ser or thr amino acids in their specific substrates
how do kinases transfer phosphates from ATP to their substrates?
kinases and substrate picks up ATP, phosphorylates the substrate, and phosphate taken up by substrate. phosphorylated substrate that is released is wheat is giving us signal in cancer
explain the molecule binding of kinase
hydrogen bonding to hinge is adenine head group of ATP
amino group at the top N terminal is hydrogen bonding to the GK+1
nitrogen of heterocyclic ring of ATP is hydrogen bonding to NH group of GK+3
GK - gatekeeper to potential selectivity pocket to the kinase that can used in drug development
two magnesium ions are involved in the binding of ATP within the active site of the kinase. magnesium play role in enhancing the reactivity of ATP, facilitating the transfer of the terminal phosphate group to the substrate
The substrate positions the hydroxyl group of a serine, threonine or tyrosine amino acid next to the activated terminal phosphate of ATP, and the phosphate is transferred by a nucleophilic substitution mechanism
When a protein is modified by adding a phosphate group (phosphorylated) by the kinase, it detaches from the kinase. Then, it goes on to interact with another protein to pass on the signal.
how do we stop the kinase from transferring at the phosphate to its substate?
design a molecule that competes with ATP from binding site.
Molecule needs to bind with higher affinity than ATP to outcompete it
Molecule therefore needs to make more interactions with the binding site to improve affinity:
Ionic
H-bonds
Van der Waals interactions
Pi-cation interactions
Aromatic ring stacking interactions
Aromatic ring – edge-to-face interactions
The side chain of GK - E, gK+1 - R1, GK+3, R3 can all be different. every kinase cant ahem different amino acids in that hinge sequence. so GK can be a serine in Kinase A, but could be a tyrosine in kinase B
Hinge potentially has different shapes as binding sites for different kinases will have different shapes
we can try build a molecule that has similar kinase N-lobe roof, C terminal floor and hinge wall binding to those sites, than an enzyme that has different amino acids in these sites and have different shapes.
Basic concept to design kinase inhibitors
- hinge binding region; molecular feature that can recognise hydrogen bonding groups in the peptide backbone of the hinge for your kinase, to compete with ATP which hydrogen bond sites to hinge.
e2. selectivity behind gatekeeper ; exploited the selectivity pocket behind gatekeeper of behind the roof and floor of the ATP binding site
- project towards the solvent exposed region to allow for addition of water soluble groups to aid with solubility and bioavailability
what is BCR-ABL
BCR-ABL is a kinase fusion protein binds atp. bcr-able transfers a phosphate from ATP onto a protein the is associated with ocogoened in MCL. looking to design a molecule that will compete with ATP.
