Novel Therapeutics Flashcards
(41 cards)
Compare traditional and more modern approaches to cancer treatment.
Traditional approaches include radiotherapy, chemotherapy and surgery. These can carry significant added risk, and surgery is not only invasive but also not always possible depending on location.
The genomic era is allowing for modern therapies to be more targeted and personalised, targeting specific oncogenic proteins depending on the deregulation specific to the tumour (eg targeting specific kinases/receptors, HER2 - trastuzumab).
What methods of immune therapy are available?
Antibodies/engineered fragments that activate the immune system or prevent T-cell inhibition by cancer antigens.
Adaptive transfer of engineered T-cells (CAR insertion/proliferation prior to autologous transplant).
Macrophage conversion to other immune types to prevent their inflammatory and metastatic roles.
How are new cancer drug targets systematically idenfied?
Cancer genome projects such as the Cancer Genome Atlas catalogue the mutations involved in cancer. Synthetic lethality screens add to the list of genes, a systematic and unbiased approach is used.
Potential targets are categorised depending on which class of oncogene they are, whether they have a catalytic site, whether there are structures available, and whether existing drugs for other diseases with the same target could be repurposed.
How many drug targets are kinases?
Kinases make up a third of all protein targets of cancer drugs, including ones targeting BCR-ABL, B-Raf and MEK, depite making up only 18% of cancer genes.
The large number of kinase drugs is however due in part to the need for second and third generation drugs to be used concurrently due to the cancer developing resistance through binding site mutation.
What makes kinases an attractive drug target?
Almost every signalling mechanism is wired through a phosphotransfer cascade, meaning that targeting this can have significant (and often broad due to pathway overlap) effects.
Despite enormous conservation in the ATP binding pocket, the active site can be targeted with highly specific small molecule competetive or covalent inhibitors.
Kinase signalling proteins are often the addiction oncogenes, so targeting them can be very effective (eg imatinib, 80% success in CML targeting BCR-Abl).
What are the disadvantages of targeting kinases with drugs?
Development of resistance is common. (second gen required)
Many inhibitors are insufficiently selective leading to serious side effects (editing side chains a priority)
Often inhibitors with great effect in vitro fail to perform in vivo (potentially due to metabolising of drugs)
What is the structure of a kinase active site?
The small N lobe and large C lobe are connected by a hinge region which contains the ATP binding pocket. This often contains Mg groups to stabilise the phosphates.
The activation loop is a short region thought to act as an autoinhibitor until phosphorylated, a regulatory event that allows for stimulation of kinase activity.
The N-terminus of the activation loop contains DFG motif which is flipped out when the kinase is active, exposing a hydrophobic binding pocket which can be utilised to make drugs more specific to a kinase.
How can activation loops be classified?
There are two broad, functional subcategories.
- Gated activation loops exhibit bifunctional properties restricting substrate access and controlling catalysis.
- Nongated activation loops allow free movement of the substrate in and out of the active site irrespective of phosphorylation state but potently modulate the phosphoryl transfer step.
What are type I kinase inhibitors?
Bind within ATP pocket, no interaction with hydrophobic DFG pocket so not dependent on kinase activation state but also less specific.
What are type II kinase inhibitors?
Contacts both the ATP cofactor binding site and an adjacent “allosteric” site available only when the DFG is flipped out and the kinase “active”. These are more specific as the DFG pocket is more varied between kinases.
What are type III kinase inhibitors?
These molecules bind outside but adjacent to the active site, in regions that are involved in the regulatory catalytic domain modulating the activity of the enzyme in an allosteric manner.
A high degree of kinase selectivity is exhibited because of the exploitation of binding sites and regulatory mechanisms that are unique to the target.
Additionally, allosteric modulators can provide subtle regulation of kinases controlled by multiple endogenous factors, something not easily performed with ATP-competitors
What are type IV kinase inhibitors?
These are totally allosteric inhibitors that bind in a totally different part of the enzyme - for example antagonists of RTKs.
What are type V kinase inhibitors?
AKA “bivalent inhibitors” these combine the binding actions of one or more of the other types in a single molecule.
What was the first kinase inhibitor approved, and how are they trending?
Imatinib in 2001. 1 or 0 a year after that until 2011, now ~30 on the market, including a lipid kinase inhibitor.
Basically they’re just so hot right now.
What is imatinib?
Inhibits the contisutively active fusion protein BCR-ABL produced by philadelphia chromosome translocation.
Imatinib is a Type II inhibitor, binding to the adenine pocket, hydrophobic pocket and allosteric pocket with its series of aromatic and often nitrogenous rings.
This is highly effective in CML (80% remission), but resistance often develops due to mutations, such as E225V, M351T and F485S within the adenine pocket.
What are second generation BCR-ABL inhibitors?
Nilotinib was designed to inhibit BCR-ABL proteins that developed resistance to imatinib.
This has the same struture other than the allosteric pocket binding region, where the addition of a bulky CF3 group allowd it to bind deeper and tighter into it, increasing potency of the drug 20x and overcoming the E225V, M351T and F485S mutations.
How do BCR-ABL proteins become resistant to nilotinib? How was this overcome?
Mutation of the gatekeeper residue, T315 to a bulkier residue such as isoleucine that precludes access of the drug to the active site.
Ponatinib was designed to overcome this. Instead of having a pyrimidineamino linker between the adenine and hydrophobic binding regions (where the gatkeeper resides), a slim alkyne linker that overcomes steric hindrance was used (still also using the CF3 group from nilotinib).
What are the disadvantages of ponatinib?
This is less specific to BCR-ABL, being designated a pan-kinase inhibitor. While some of these targets will be involved in oncogenic pathways many will not be, and will regardless lead to a great increase in side-effect severity.
Give an example of a type I kinase inhibitor.
Vemurafinib is a B-Raf inhibitor (used to treat BRAF mutant melanoma) that binds in the adenine pocket and hydrophobic pocket, but not the allosteric pocket. The side chain at that point instead points out into aqueous space to maintain solubility.
This too now has issues with developing resistance.
What approach is taken to vemurafinib resistance?
Targeting the downstream MEK with Trametinib - a Type III inhibitor. Being a downstream target this does reduce efficacy as it does not affect all the signalling pathways used by B-Raf.
How does trametinib work?
Being a type III inhibitor this binds in the active-site local allosteric pocket without an adenine pocket binding region.
This holds the activation loop of MEK in an inhibitory conformation, preventing it from being phosphorylated, preventing MEK activation in response to B-Raf signalling.
What kinase inhibitor targets EGFR?
Afatinib. This is notable in that it produces covalent cross links with the target, possessing a group which is targeted by the Cys797 residue for a nucleophilic attack.
This means that allosteric influences from mutations gained by the kinase will be unable to prevent Afatinib from inhibiting the receptor, and may be an important way forward in inhibitor design.
What drugs target Ras?
None, it is incredibly difficult to drug, often being called ‘undruggable’. This is unfortunate considering its prominent role in cancer, being mutated in ~30% of all cancers.
How is Ras regulated?
Ras can be bound to GTP and GDP, making it active and inactive respectively by inducing conformational changes allosing for activation of downstream signalling factors such as Raf.
While it does have some GTPase activity, its rate of hydrolysis is very low. Instead, the ratio of Ras-GTP to Ras-GDP is dependent upon the GAP (GTPase activating) and GEF (guanine exchange factor) proteins that act upon it. This is known as the canonical pathway.
Ras can also be non-canonically regulated by sequestration of the plasma-membrane embedded inracellular protein to cellular organelles, preventing colocalisation with both its upstream and downstream interactors.
