lecture 12 - alkylation Flashcards
What is alkylation and how can damage caused by it be repaired?
Alkylation is a chemical reaction in which an alkyl group is transferred - this results in DNA damage.
Can be repaired by BER and NER, and by the MGMT repair protein
What is the process of nucleotide excision repair (NER)?
(A) DNA is damaged
(B) Damage is recognised by a protein called XPC, which is bound to another protein, R23
(C) The binding of XPC-R23 is followed by the binding of several other proteins
(D) The DNA duplex is unwound, creating a bubble. The second strand is untouched throughout this process and can be used to rebuild the DNA
(E) ERCC1-XPF binds and a completely assembled NER multi protein complex is formed
(F) XPG is an endonuclease that cuts the damaged strand at junctions 3’ to the site of base damage
(G) ERCC1-XPF is an endonuclease that sits the damaged strand at junctions 5’ to the site of base damage
(H) This fragment is excised from the genome, and a new section is synthesised. These events return the damaged DNA to its native state.
What are the two forms of NER?
Short patch repair - replaces a single nucleotide
Long batch repair - replaces a chain of nucleotides
What are the steps of base excision repair (BER)?
(1) The N-glycosidic bond of the damaged DNA is cleaved by a DNA glycosylase, leaving an abasic site in the DNA
(2) The sugar-phosphate backbone of the abasic site is then cleaved by a bi-functional glycosylase and/or an AP-endonuclease. If necessary, the 3’ strand break end is converted to hydroxyl, allowing DNA polymerases to reinsert new bases
(3) Synthesis of a single base is referred to as short patch BER, and synthesis of several bases is long patch BER
(4) The 5’ single strand end of the single strand break intermediate is then processed to allow for ligation by DNA ligases
What can BER be used for in addition to repairing base damage?
It is involved in the repair of single strand breaks (SSB) that are generated independently of the glycosylases and/or AP-endonucleases, such as DNA sugar-phosphate backbone cleavage by radiotherapy and chemotherapy
How do nitrogen mustards work to damage DNA?
They have several sites of DNA alkylation
A chlorine atom from the nitrogen mustard is lost, and the DNA becomes covalently bound to the drug - this occurs via nucleophilic attack onto the electrophiles of the drug
These drugs can be tagged with compounds (e.g. oestrogen) to target specific tumour cells (in this case, oestrogen-dependent tumour cells)
How do DNA methylating compounds (Temozolomide) work to damage DNA?
Temozolomide is broken down by water hydrolysis and releases a methyldiazonium ion
This is attacked by DNA, which acts as a nucleophile, and the DNA ends up getting methylated
How do cancer cells use O-6-methyl-guanine repair to become resistant?
O6MG is a protein that is crucial for genome stability
Cancer cells are able to repair O6MG to become resistant to temozolomide (specifically)
If the damage becomes too great for the repair mechanisms, a transversion or a DSB could occur
How are DNA minor groove binders used as therapeutics?
These compounds have a natural twist to fit the minor groove
Non-covalent compounds have been modified with DNA alkylation moieties (mustards, etc.) to give experimental covalent compounds
They can be used in very small doses because they are so potent
Includes, mitomycin C, PBDs, CPIs (covalent) and distamycin, netropsin (non-covalent)
What is the mechanism of action of mitomycin C?
Enzymatic/chemical reduction is the first step - then a molecule of methanol is released - the reduced mitosine interacts with DNA
N2 and N7 atoms of guanine in the minor groove of DNA are primarily alkylation sites
alpha attack occurs and two tags end up on the DNA
Intra-strand crosslinks are most commonly produced - the alkylation points are on the same strand, so BER and NER can be used to repair this damage
How do CPI-based anti-tumour antibiotics work?
They contain ethano-bridges that cause over-winding of DNA and lead to dose-limiting toxicity and death
They supercoil the DNA and lead to similar symptoms seen in delayed lethality
The drug was redesigned (ethanol-bridges removed) to form Adozelesin, but greater clinical outcomes have not been observed and synthesis is costly
How do PBD anti-tumour antibiotics work?
These compounds were traditionally recrystallised from methanol to give a stable methyl ether
all three forms can react with DNA, however it is widely considered that the imine or carbinolamine is the active form
Mechanism of action: the formation of a reversible aminal bond between the eco-cyclic NH2 of guanine and the C11 position of the PBD
What is the next step in this research?
- Targeted DNA alkylation
- Use of pro-drugs to restrict damage to specific tumour location
- Antibody based targeting
- Increase sequence recognition
- Further development of non-covalent DNA interactive compounds to DNA alkylating analogues
