Kinase Inhibitors Flashcards
How may Kinases are ther ein the human genome and how many families?
500 kinases
About 20 families
Why are kinases difficult to target?
Highly conserved catalytic domain amongst families (lack of specificity)
Inhibitors have to compete with high ATP concentration [2mM] so high doses would be needed
Kinases inhibition would produce many ADRs due to lack of specificity
Why is targetting kinases important?
Mutant kinases can become oncogenes and drive cancer = clinical need for specifically targeted kinase inhibitors
Name a disease which is has targeted drug therapy and what is this drug target?
Chronic Myeloid leukaemia targets the Bcr-Abl fusion protein which has tyrosine kinases activity
What is CML?
Myeloproliferative disorder of haemopoetic stem cells
CML blood film will have many granulocytes
What is some epidemiological data on CML?
1-2 per 100,000 person years
Median age of onset = 53
15-30% of all leukaemia
Incidence increases with age
Describe the clinical corse of CML
Chronic phase
Accelerated phase
Blast crisis
What are some features of the chronic phase?
85% of people diagnosed here
Lasts 4-5 years
Normally differentiated granulocytes but excess of them
What are some symptoms which occur is the accelerated phase?
Spenomegaly
Leukocytosis
Fever (infections)
Throbocytopenia or cytosis
How long does the accelerated phase occur for?
6-9 months
What are some blood characteristics on the accelerated phase?
15-29% blasts in blood
>20% basophils
What is the mean survival rate for the blast crisis?
3-6 months
What has happened to the granulocytes in blast phase?
They are non functioning poorly differentiated) with many molecular abnormalities within their DNA
What are the blood characteristics of the blast phase?
(Greater than or equal to) 30% blasts in blood
Symptoms of blast phase?
Fever, night sweats, bone pain, weight loss
What is the characteristic molecular abnormality in CML
Presence of Philadelphia chromosome (95% of cases)
What is the Philadelphia chromosome?
Reciprocal translocation between chromosome 9 and 22
T(9;22)(q34;11)
What genes are located on chromosome 9 and 22 which are important for CML?
9 = Abl
22=Bcr
What does translocation of part of 9 to 22 do?
Causes the juxtaposition of abl and Bcr on chromosome 22
Forms fusion protein Bcr-Abl when transcription and translation occurs
How is Bcr-Abl and oncogene?
It has constitutive tyrosine kinase activity which activate cell cycle proteins and enzymes which
Increase cell division,
Inhibits DNA repair
And promotes genetic instability which causes mote mutations in DNA (blast crisis)
What is the normal function of Abl?
A kinase with similar properties to Src kinase
What prevents physiological Abl activation?
There is autoinhibtion of the kinase activity by the SH2 +SH3 domain clamp
And the N terminal which is Myristoylated acts as a latch
Both prevent activation
What causes the normal Abl to unclamp?
Exposure to proline rich sequences or phosphorylated tyrosine
These bind to the SH3 and SH2 domains respectively
When able is unclamped what is allowed to occur?
Releases abl from autoinhibition which allows activation of the 2 kinase domains
These auto phosphorylate tyrosine 245 and tyrosine 412 (on activation loop)
This increases tyrosine kinase activity of the fusion protein which drives cancer
How does Brc disrupt normal Abl function?
Bcl will be fused onto he N terminus which means N terminal doe snot act as a latch to prevent activation.
This means that Abl will be active without the need for proline rich or phosphorylated tyrosine interaction and will be constitutively active
What is the implication of constitutively active Bcr-Abl?
You get defective cell adhesion and growth factor independence as RTKs do not need to be activated for protein to function
You get expansive growth and resistance of apoptosis
When will imatinib bind to Bcr-Abl?
When tyrosine 412 is not phosphorylated
What were the old therapeutic options for treating CML?
Allogenic stem cell transplant ion
Interferon alpha
Chemotherapy
(<20% cured)
What is the new treatment for CML?
Imatinib (Gleevec) which inhibits Bcr-Abl
What is the benefit of CML over chemo?
Chemo is no selective and targets all rapidly devising cells which included physiological haemopoetic stem cells. Thus normal stem cells not harbouring mutation will be destroyed. Imatinib only targets cells contains the bCr-Abl protein therefore benign cell survive and patient can have a fun ting immune system
What is the mechanism of imatinib?
Binds to the hydroxyl group on Threonine 315 via a hydrogen bind which blocks the ATP binding site
T315 regulates ATP site access this binding to imatinib inhibits ATP binding and kinase activity
What are the effectiveness stats of imatinib?
Complete cytogenetic response = 69% at 12 months
87% at 60 months
Only 7% progress to accelerated phase or blast crisis over 5 years
What are some side effects of imatinib?
Mainly mild to moderate ones Headache Dizziness Mouth ulcers Anaemia
Why is imatinib most effect in chronic phase?
Typically only Bcr-Able mutation in chronic phase
As disease progresses more mutations occur which imatinib does not selectively target.
These mutations are involve din other pathways which drive cancer
What are some disadvantages of imatinib?
Does not eliminate Bcr-Abl cell so life long therapy needed (expensive)
Less effective in blast crisis and Philadelphia + B cell acute lymphoblastic leukaemia
Resistant CML does exist
Name another cancer which has a Philadelphia chromosome?
Acute B cell lymphoblastic leukaemia
What causes CML resistance?
Additional mutations other than bcr-abl (>80% blast phase CML have mutations other than Philadelphia chromosome)
Point mutation in Bcr-abl fusion protein T315I (gate keeper mutation) which means imatinib does not bind
Role of other enzymes
Why does the isoleucine point causes resistance?
It a bigger amino acid and interacts with residues in the protein which would normally interact with imatinib. Thus imatinib can no longer bind but ATP still can get in
How can imatinib resistance T315I be targeted?
Less specific kinase blockers whic act on multiplekinases e.g. Pronatinib
Other kinases effected include Src
Advantage of pronatinib?
Non steric clash with I315 and blocks ATP binding site
Has relative efficacy
cytogenic response in all patients = 54%
Cytogenetic responce in those with T315I mutation = 70%
Disadvantages of pronatinib?
Less specificity = more ADRs
Abdo pain, N+V, infections, fever, myalgia
What is another common mutation in the later stages (blast phase) of CML?
P53 mutations
This is a tumour suppressor gene which is stabilised by DNA damage and promotes cell division arrest and apoptosis if defective DNA is detected
What does loss of p53 cause?
Blast crisis and cell is resistant to apoptosis
What are some other potential targets for imatinib?
Gastrointestinal stromal tumours caused by c-Kit mutation (RTK for stem cell factor) which arise from the intestinal cells of cajal
Glioblastomas which have PDGF-R mutation
Other leukaemias with constitutively active fusion proteins (Tel-PDGF-R)
Which is the responce rate for imatinib for GISTs?
60% in phase 1 clinical trials
What are future potential therapies?
RNAi
GPCR antibodies
Gene therapy
How does RNAi work?
Give small interfereing RNA (siRNA) or microRNA (double stranded). This binds to dicer which breaks them down into small 21 base long sections.
These then bind to argonaut which selects a single strand (guide strand). Complex formed = RNA induced silencing complex. The RISC binds specifically to mRNA of interest via base pair complementation and causes cleavage of mRNA which is degraded by ribonucleases
How can RNAi be used to treat CML?
SiRNA complementary for mRNA which forms Bcr=Abl
How might GPCR antibodies be useful?
Antibody to CCR5 GPCR
Prevent internalisation of HIV
