Cell signalling +rational therapy Flashcards
What are the main problems associated with cytotoxic drugs?
- They are also toxic to normal, rapidly dividing cells causing patients nasty side effects.
- They do not necessarily target cancer cells.
- Cancer cells can easily acquire resistance to them e.g. resistance to carboplatin is a major problem due to resistance to apoptosis.
What are the two main pathways in which oncogenic mutations occur?
- MAP kinase
- - The MAP kinases phosphorylate serine/threonine kinases which are also called ERK which will act on transcription factors to elicit number of pathways. - PI3 kinase – its key function is to supress p53.
- - Cytokines can inactivate p53 by a PI3K – Akt – MDM-2 pathway.
- - PI3K activates Akt which then phosphorylates MDM-2 which allows it to translocate to the nucleus and to ubiquitinate p53.
- - Ubiq-p53 is then trafficked for degradation by the proteosome.
- - The phosphatase PTEN is a tumour suppressor gene and it dephosphorylates PI3K which inactivates it.
What is rational therapy and why are kinases a good target? Also explain mechanism of action of kinases.
Rational therapy consists of targeting signalling components.
- RTKs are commonly targeted –> including SMIs and monoclonal antibodies.
- Kinases are arguably the most commonly mutated genes in cancer.
- They are also drugable - have properties that facilitate small molecules to bind to them and inactivate their function.
- Kinases are a family of 512 genes (human) that encode serine/threonine or tyrosine kinases.
- They phosphorylate serine, threonine, and tyrosine residues. Each phosphate can then form up to 3 hydrogen bonds which will alter protein structure – inactivating or activating.
Explain the mechanism of action of Imatinib.
- Chronic myelogenous leukemia is a cancer primarily caused by chromosome translocation that creates the Philadelphia chromosome – defect in haematopoiesis.
- The chromosome results in mutant fusion protein BCR-ABL.
- Abl is a tyrosine kinase and the mutation causes it to be retained inside the cytoplasm and constitutively activated —> over-proliferation of white blood cells –> leukemia.
- Imatinib (Glivec) is small molecule tyrosine kinase inhibitor.
- It’s an ATP analogue, it competes with ATP to bind to the ATP binding pocket of the inactive form of BCR-ABL. Causes apoptosis of target cells.
- It is successful in treatment of CML – 96% of patients go into remission.
Explain resistance to Imatinib.
- In CML, majority are cured but 15% develop resistance – usually via re-activation of kinase activity due to mutations. The biggest problem is the T1351 mutation.
- CML has three phases –> chronic, accelerated and blast crisis.
- Effectiveness of Imatinib falls with the stage of CML.
- But small minority (5%) are not treated until they are at the ‘blast crisis’ stage, they will respond at first but then will most likely relapse due to developing resistance. The number of blast cells >10^10 meaning one cell can easily emerge with a mutation that will be selected for.
Explain function of nilotinib and dasatinib. What third generation kinase inhibitors are in development?
- Inhibit 32/33 of the known imatinib resistant mutations.
- However, neither inhibits T315I mutation – which is relatively common mutation.
- Imatinib resistant patients initially respond well but it is clear that a sizeable proportion become resistant due to the selection of T13151 mutations.
- The problem with CML and resistance is that there’s vast number of blast cells which means there’s a high chance of at least one of cells having the T13151 mutation for resistance.
- There are currently third generation inhibitors in development against this mutation. E.g. Ponatinib which has been shown to bind Bcr-Abl with the T1351 mutation.
Explain function of EGFR inhibitors.
- The EGFR is believed to be linked to at least 1/3 of carcinomas and hence is effective target.
- A number of tyrosine kinase inhibitors have been designed including Iressa and Tarceva
- They bind and block the ATP site of the associated kinase. Once signalling is blocked, no more mitogenic/anti-apoptotic signals are present in the cancer cells.
- Patients who initially responded to Iressa or Tarceva will eventually relapse due to further mutations that block binding of the drugs. e.g. Threonine to methionine mutation (T790M) that blocks Iressa binding.
How can we predict the response to EGFR inhibitors? How can we find new targets?
– A small subset of patients (10 to 20%) with NSCLC responded well to EGFR kinase inhibitors
– This subset had an oncogenic mutation in the kinase domain of EGFR – causing an altered form of the receptor.
The mutant EGF receptors preferentially activated the PI3K/Akt pathway.
– What about the 80% of patients with NSCLC who are not sensitive to EGFR inhibitors? We need to siRNA screens to identify new targets for cancers.
– siRNAs – can be used to screen for kinases that are essential for growth of cancer cell lines and later use them as therapy targets.
– Or we can develop inhibitors for more than one signalling pathway and use them in combination.
– Will the proposal to develop inibitors against more than one cell signalling pathway be successful? Merck and AstraZeneca - MET and AKT inhibitors currently in development.
Describe an example of the traditional method of developing kinase inhibitors.
a. Computer screening to find the smallest most effective candidate.
b. Tumour associated macrophages – shown to aid tumour progression by supressing T-cell killing of tumour cells.
- - Here the target is the receptor for the macrophage colony stimulating factor.
- - Screen several targets that will block the receptor and then carry out an assay of the cell lines to see the effects of these inhibitors on cell growth.
Describe high-throughput kinase profiling.
a. One of best assays used is bacteriophage plaque forming assay.
- - Each virus is coated with a different kinase and they are then incubated with the specific kinase inhibitor and the bacteriophage’s target - Ecoli.
b. If the inhibitor works, it will prevent your kinase coated virus from binding to Ecoli – which can be analysed by looking for plaques.
c. This will allow you to do kinase inhibition profiles for hundreds of kinase inhibitors.
d. 4 new kinase inhibitors have been developed by this method and are currently in trials.
