Cancer 2 Flashcards
How do we use RNA as a target for anti-cancer drugs
The RNA in cancer cells is different to normal cells (as they are producing different proteins). We can subject this to antisense therapy which targets a section of the cancerous mRNA which blocks the mRNA from being transcribed into a cancerous protein
What are the advantages of antisense therapy
Advantages
- Same effect as an enzyme inhibitor or receptor antagonist without having to be physically designed to match the ligand. Just has to be an antisense to the mRNA
- Highly specific to where the oligonucleotide is 17 nucleotides or more (as only 16 oligonucleotides can ever be in the same exact sequence as another in the whole genome)
- Smaller dose levels required compared to inhibitors or antagonists for the binding sites
- Potentially less side effects as its so highly specific
What are the disadvantages of antisense therapy
Disadvantages
- ‘Exposed’ sections of mRNA must be targeted
- The instability and polarity of oligonucleotides (several nucleotides in a row) (pharmacokinetics)
- Short lifetime of mRNA oligonucleotides and there is poor absorption across cell membranes due to polarity issues
What is miRNA
Micro-RNA (miRNA)
Short segments of double stranded RNA
Recognised by enzyme complex RISC in the cell to produce single stranded RNA - a small interfering or small inhibitory RNA (siRNA)
This siRNA binds to complementary region of mRNA
mRNA is cleaved by enzyme complex – undergoes degradation. The antisense sequence is now bound to your risc complex which can go on to bind to the sense sequence on the mrna which then leads to degredation of thr mrna – no gene product synthesised
What are the advantages of miRNA as an anti cancer therapy
Advantages
siRNAs have potential to be used in gene therapy
Greater efficiency in silencing mRNA than conventional antisense therapy because of its stability
One siRNA could lead to cleavage of many mRNA molecules
What are the disadvantages of miRNA as an anti cancer therapy
Disadvantages
siRNAs need to be metabolically stable (need advantageous pharmacokinteics)
Difficult to reach target cells
Devise a mechanism to ensure entry to target cells
What are many of the hallmark processes driven by
Many of the processes behind the hallmarks of cancer are driven by signal transduction and the activity of tyrosine kinases. Thus, these are new therapeutic targets
What is the basic signal transduction process
Signal => reception => (amplicication) => transduction => response
Receptor proteins bind “signals” i.e. drugs & endogenous ligands with high affinity
Conformational changes in the structure of the receptor protein then convert the external signal into one or more intra-cellular signals.
This process is known as signal transduction
What can be a signal for signal transduction
Osmolarity change, temperature, growth factor etc.
What is the basic structure of a tyrosine kinase receptor
Receptors with an extra-cellular and intra-cellular domain
Intracellular domain capable of phosphorylating tyrosine residues in target proteins.
What does the activation of a tyrosine kinase receptor do
This can initiate a signalling cascade leading to changes in expression of genes that is important for:
Proliferation Evasion of apoptosis Induction of angiogenesis Generating metastasis (can also encourage protumor inflammation)
What are the 3 essential components of receptor tyrosine kinases
Ligand binding site (extracellular domain)
Transmembrane domain (α helix) (within the cell membrane)
Domain with tyrosine kinase activity (cytosolic) (inside the cell)
How are tyrosine kinase receptors activated
Ligand binds to the receptor site and induces association between adjacent RTKs (usually via dimerisation)
This causes activation of their kinase activity - the internal domains are joined together
They phosphorylate tyrosine residues in other molecules
Once the kinase receptor is phosphorylated, the phospho-tyrosine groups (are able to phosphorolate their own proteins) can act as binding sites for signalling proteins by attracting other molecules which need to be phosphorylated to induce activity
What happens once the tyrosine kinase activity on the receptor is activated
Each phosphorylated tyrosine region can bind a signalling protein
These molecules may also become phosphorylated and act go on to act as further binding sites
Phosphorylation cascade (kinases) inducing downstream signalling
Reversed by enzymes called phosphatases – negative feedback loop - this process is often inhibited in cancers
Describe the downstream signalling of the MAPK (ERK) signalling
Something binds to the receptor tyrosine kinase and activates it
This leads to RAS being phosphorylated
Leads to RAF, MEK1 and MEK2, ERK1, ERK2 and ERK5 being phoshphorylated all in turn
ERK1, ERK2, ERK5 are executer kinases which, when active, become transcription factors themselves or go on to activate other transcription factors which start gene expression changes leding to proliferation, prevention of apoptosis etc…
As a summary, what are the 2 ways in which kinases can be used as drug targets
The modulation of kinase activity can be
achieved through several strategies, including:
Disruption of ligand-receptor interactions
Inhibition of kinase (phosphorylation) activity (block ATP binding)
Give an example of a drug which disrupts kinase ligand-receptor interactions
Bevacizumab
This drug binds to a growth factor vascular endothelial growth factor (VEGF) thus preventing its binding to receptors (RTK)
This interferes with tumour blood vessel development.
First line treatment for colorectal cancers
Which receptor does tratuzumab (herceptin) target in the treatment of breast cancer
Trastuzumab (Herceptin) targets the HER2 receptor, which is over expressed in (5-20%) of all late stage breast cancers
Herceptin prevents binding of EGF (ligand) to the HER2 receptor by binding to the ligand and thus prevents signal transduction as the ligand can no longer activate the receptor
However its use is limited to cancers which test as HER2 +, since it only works on tumours in which this protein is over expressed.
Needs to be used in conjunction with a diagnostic test for HER2 overexpression to ensure its not used where its not needed
Give an example of a drug which inhibits kinase activity by blocking ATP binding
Imatinib mesylate (2001) Protein tyrosine kinase inhibitor through inhibiting ATP binding to the bcr/abl fusion protein (a super active kinase). This prevents any phosphate being available for the transduction of the cancerous cells signal
Chronic myeloid leukaemia, acute lymphoblastic leukaemia and intestinal stromal tumours
Give a second example of a drug which inhibits kinase activity by blocking ATP binding
Gefitinib (2002)
prevents ATP binding to EGFR (which stops the receptor geting hold of any phosphates which allow it to phsophorylate itself and other molecules) – useful in cancers with high EGFR expression
Give a brief outline of the the Epidermal growth factor receptor (EGFR) becomes active
Binding of EGF causes receptor dimerisation and activation of enzyme activity
Dimerisation means that the active site on each half of the receptor dimer catalyses the phosphorylation of the tyrosine residues in the other half
If dimerisation does not occur then phosphorylation cannot take place and signalling is inhibited
Important because EGFR is overexpressed in many solid tumours (ie lung cancers)
Summary:
How do we inhibit rtk signalling
We can inhibit RTK signalling in several ways using targeted therapies
Prevent ligand/receptor binding – blocks the initiation of the signal
Inhibit kinase activity – blocks intracellular transduciton of the signal and therefore associated response e.g uncontrolled proliferation, evasion of apoptosis etc (hallmarks of cancer)
What do phosphorylated tyrosine residues on receptors act as
The phosphorylated tyrosine residues on receptors act as binding sites for signalling proteins
Give an example of a targeted therapy which doesnt involve rtks
Disruption of oestrogen-receptor interaction
Breast cancer can be oestrogen-dependent
Oestrogen secretion can be reduced by surgery
An alternative to surgery is use of tamoxifen
- Competitive inhibition of oestrogen binding to its receptor with a higher affinity but ellicits no effect by the receptor
- FDA approved for advanced breast cancers and primary breast cancer
Inhibits expression of oestrogen regulated
genes
Give 2 examples of drugs used to inhibit MAPK signalling
. Dabrafenib and vemurafenib
When can Dabrafenib and vemurafenib be used
Used in the treatment of melanoma
Only suitable for patients with a mutation in the B-Raf gene
Approximately 50% of melanomas harbour B-Raf mutations
This mutation makes RAF-ERK signalling constitutively active
What is the most common B-Raf mutation
Diagnostic test is required to establish this
Most common mutation (90% of cases) is V600E (amino acid in position 600)
What part of the MAPK kinase cascade do dabrafenib and vemurafenib work on
The Raf section. In melanoma it can signal on its own without input from ras or the receptor. By turning it off by preventing it from binding phosphorous it cant signal downstream
What do all cancer cells posess
Associated inflammation
Inflammation can support all aspects of cancer:
Initiation - immune cells can release reactive oxygen species which goes on to damage dna which increases the number of mutations
Growth - immune cells can produce growth factors
Metastasis - immune cells can often be used to breqk down and remodel tissue in normal wound healing. These processes can be hijacked to allow for tumors to form and spread
Response to therapy - can alter all therapies work
What cells are asscoiated with tumors which arent actually tumor cells
Blood vessels, stromal cells, fibroblasts, an extracellular matrix, immune cells (trying to fight the tumor but often end up becoming a part of the tumor….)
What are the two ways in which we can target inflammation in cancer
Inhibit pro-tumour inflammation
Promote anti-tumour inflammation
Which inhibitors use the approach of inhibiting pro-tumour inflammation
MAPK inhibitors
Blocking certain pathways prevents pro-tumourigenic inflammatory signalling within the tumour
How does the checkpoint approach to promote anti-tumour inflammation
work (and give examples of therapy)
T cell immune responses (anti-tumour) are controlled through on and off switches, so called ‘immune checkpoints’
These protect the body from potentially damaging immune responses (hyper responses)
We have developed drugs to target these immune checkpoints
This illicits anti-tumour T-cell-mediated immune responses (which leads to a hyperactive immune response which cause side effects)
e.g. anti-CTLA4 and anti-PD1 or PDL1 therapy
What does CTLA4 do
Its the master switch (checkpoint) for T cell activation is CTLA4
CTLA4 is a receptor found on the surface of T cells and its activation inhibits T cell function. If we block it, it enables the activation of what it was inhibiting
When used as therapy - anti-CTLA4 monoclonal antibody (ipilimumab) – binds to CTLA4
This blocks CTLA4 signalling, enabling an anti-tumour T cell response
What is the PD-1/PD-L1 pathway and how does it work
PD-1 receptor and its ligand (PD-L1) are expressed on the surface of dendritic cells and macrophages
They are inhibitory factors, which act as a check-point for the dendritic-cell mediated T cell response (and are used to control it)
Therapy = monoclonal antibodies targeted against PD1 and PD-L1 (Nivolumab) – blocks the PD-1 signal hence inhibiting the inhibitor (we enable the t-cell response)
This releases the inhibitory checkpoint enabling a T-cell response, T-cell proliferation and therefore anti-tumour activity
What is the role of the pharmacists role in cancer treatment
Know about the disease and how thr medicines work to treat the cancer
Know how to communicate (and make complicated things simple). Always ask to see if the patient understands what were saying to them