Drug Discovery: Case Study 6 - Gleevec and Kinase Inhibition

How many kinases are there and how many families

- 500 kinases in human genome which are split into approx 20 families

What do kinases do

- bind ATP and phosphorylate a protein substrate

- involved in huge amounts of signalling that alter gene expression and cell proliferation/survival

- large implications in cancer

Why target kinases?

- involved in signal transduction

- kinase mutations present in many cancers

- can intervene at multiple points in the phosphorylation cascades

Why were kinases originally thought to be poor drug targets

- making a molecule that can outcompete ATP for the binding site is very difficult

- requires very high affinity as ATP is always in cells

- off target effects - active sites well conserved and don't want to inhibit other kinases

oncogenes

genes that cause cancer by blocking the normal controls on cell reproduction

- activate kinase pathways that cause proliferation and survival in cancer cells

Kinase inhibitor used for CML

Gleevec (Imatinib mesylate)

What is CML

chronic myeloid leukaemia

- proliferative disorder of myeloid derived cells

- causes abundance of neutrophils

- leads to loss of functionality of neutrophils

- incidence increases with age

Phases of CML

Chronic Phase

- most people present in chronic phase

- myeloid cells still functioning but large abundance of them = causes symptoms

Accelerated phase

- increasing amounts of myeloid cells in the bone marrow

Blast Crisis

- end stage (3-6 month survival)

- full on leukaemia

- little to no function of neutrophils

What causes CML

Philadelphia chromosome

- truncated Chr 22 and elongated Chr 9

- present in over 95% of CML patients

- caused by translocation of chromosome fragments

- leads to the BCR-ABL gene fusion = produces a mutated protein with uncontrolled tyrosine kinase activity that activates cell proliferation and survival (STAT, MYC, RAS, PI3K pathways)

What is the normal state of kinase activity without the Philadelphia chromosome

- ABL is the normal kinase

- has SH2 and SH3 domains that wrap around the kinase domains

- the ligand binds the SH domains and this triggers unlatching of the kinase domains = activated and can phosphorylate

- BCR-ABL the SH domains don't latch the kinase domains so it is constitutively active

Consequences of BCR-ABL

- defective cell adhesion

- growth factor independence = expansion, growth and resistance to apoptosis

Therapeutic options for CML

- stem cell transplants

- interferon-alpha (reduces immune response)

- chemotherapy

- **inhibition of BCR-ABL = gleevec

How does Gleevec work

- BCR-ABL has a Thr315 near its active site

- Gleevec forms a hydrogen bond with the hydroxyl group of Thr315

- binds the inactive conformation of BCR-ABL and blocks ATP from binding = cant be activated

Gleevec trial outcomes

- 98% of patients in the trial (1998) saw massive reduction in white cells with minor side effects

- within one week patients on edge of death were leaving the hospital

- FDA approved in 2001

- miracle drug as other cancer treatments were not sophisticated

safety profile of Gleevec

- adverse events mostly mild to moderate

- long-term treatment required

- particularly effective in interferon-alpha-resistant chronic phase

What are the limitations of Gleevec

- doesn't eliminate the BCR-ABL expressing cells = patients have to take it everyday

- less effective in blast phase because at this stage other abnormalities can be acquired

reasons for resistance to gleevec

- BCR-ABL mutants - switch threonine for isoleucine = renders gleevec ineffective

- other mutations may block the inactive form of BCR-ABL so gleevec cannot bind and prevent activation

Drug developed for Gleevec-reistant mutants

- T315I mutant inhibitors

- Ponatinib = multi kinase inhibitor

p53 as a target in CML

- P53 is a tumour suppressor

- low levels of p53 - once damage is sensed p53 is stabilised and it orchestrates DNA repair and cell cycle arrest

- p53 mutations are present in cancers and they inactivate this process

- makes p53 a good target