Lecture 13 - Bleomycins and Platinums Flashcards
What is bleomycin and what did it originate from?
- mixture of cytotoxic glycopeptide antibiotics
- isolated from Streptomyces verticillus
- soluble in water
What is the structure/key features of bleomycin?
- Metal binding domain, with coordinated nitrogen atoms
- Linker region and bithiazole tail
- Disaccharide R
- +charged polyamine tail
- hydrophilic molecules, unable to cross cell membrane
How are bleomycins used in clinics?
- in combination chemotherapy
- against lymphomas, squamous-cell carcinomas and germ-cell tumours
What are the side effects of bleomycins?
- dose-dependent
- lung inflammation –> lung fibrosis
How do Bleomycins work as drugs?
- bind transition metals (Fe(II) or Cu(I)) and oxygen
- in the presence of a one-electron reductant, can catalyze formation of ssDNA and dsDNA breaks
- cause damage similar to ionizing radiation
What is an essential feature for dsDNA cleavage by bleomycins?
- The linker between the metal and the bithiazole DNA-binding domain
- flexibility of the bithiazole moeity
What are the most common outcomes of bleomycin treatment to cells?
- Extended cell-cycle arrest
- apoptosis
- mitotic cell death
Do we have a bleomycin synthesised?
- Not fully, we rely on natural products
What different cuts can bleomycins cause?
Staggered or blunt end cuts
Where do ssDNA cuts occur for bleomycins?
- cleavage at pyrimidines (Py) 3′ to a guanine
Where do secondary sites of cleavage occur for bleomycin and what cut do they cause?
- Depends on the residue 3′ to the primary cleavage site
- 5′-G-Py-Pu-3′, 5′-staggered ends,
- 5′-G-Py-Py-3′, blunt ends
Mechanism of interaction with DNA by bleomycin to cut DNA
- Bleomycin associates with DNA threading its bithiazole tail through the DNA bound by partial intercalation
- long polyamine tail interacts with the major groove on other side
- get cut event
- then regeneration
- rotation around the bond between two thiazole rings in the tail of bleomycin with other motions, make the peroxide available for interaction with second DNA strand - flips over the DNA and second cut occurs on the other side
What is the 3rd mechanism of DNA repair, for dsDNA?
Recombination
- for these reagents where we cut the DNA on both strands
- applies to agents which alkylate both strands of DNA, inter-strand crosslinkers, have to adopt this type of repair
Steps of Recombination to repair dsDNA break
- Recombination initiated by dsDNA break
- DNA processed at site of break to yield regions of ssDNA
- Rad51 and replication protein A (RPA), coats ssDNA to form a filament that searches for homologous sequences (on the homologous chromosome or the sister chromatid) and initiates the formation of a joint molecule
- Break is repaired by DNA synthesis using the intact strands as templates.
- Following branch migration and resolution, repaired recombination products are released.
- The final product contains cross over of both DNA material used
- Only way of repairing if have dsDNA breaks
What are platinum drugs?
- Antibiotics and antitumour drugs
- Interfere with DNA, react with individual bases
- forms duplex adducts crosslinking bits of DNA
Example of a platinum drugs
- Oxaliplatin
- Cisplatin
- Carboplatin
- Aroplatin
What is the mechanism of action of cisplatin?
- Reacts with water to form the hydrolysis product
- which reacts with N7 of guanine to form bridge links within the major groove
What crosslinks can cisplatin form?
- intrastrand crosslinks between 2 guanines on same strand
- or interstrand crosslinks between 2 on opposite strands
How does cisplatin cause DNA damage?
- kinks the DNA, interferes with ability of DNA to repair itself
How do tumours acquire resistance to cisplatin/carboplatin BEFORE DNA binding?
- mutations which block drug entering through copper transporters into cell
- DNA acts as a nucleophile against the electrophilic drug, upregulating , glutathione and metallothionein levels mopping up the drug
- Active export of platinum from the cells through copper exporters and through the glutathione S-conjugate export GS-X pump
How do tumours acquire resistance to cisplatin and carboplatin AFTER DNA binding?
- upregulate NER to remove major covalent bis-adduct that is formed with adjacent guanines on the same strand of DNA (the intra-strand crosslink)
- Bypassing of adducts by polymerase β and η
- Through down regulation of apoptotic pathways
What is the best way for tumours to acquire resistance to cisplatin or carboplatin?
- Upregulating NER
- Preventing the drug getting to the DNA in the first place
Once the platinum drug has entered the nucleus what happens?
- preferential covalent binding to the nitrogen on position 7 of guanine occurs
- major covalent bis-adduct that is formed involves adjacent guanines on the same strand of DNA (the intra-strand crosslink); a minor adduct involves binding to guanines on opposite DNA strands (the inter-strand crosslink)
What are some ongoing strategies to avoid resistance of cisplatin and carboplatin?
- increasing the levels of platinum reaching tumours e.g. liposomal platinum products
- combining existing platinum drugs with molecularly targeted drugs (for example, bevacizumab)
- using novel platinum drugs such as oxaliplatin
- using other drugs either alone (TLK286) or in combination (for example, decitabine), which exploit particular cisplatin-mediated resistance mechanisms.
What anti-angiogenic therapy can be used for renal-cell carcinoma?
- bevacizumab
- a recombinant human monoclonal anti-VEGF antibody that binds to and neutralizes all biologically active isoforms of VEGF
Important aspects of DNA recognition and targeting to consider
- secondary structures are important
- DNA not a rigid ladder
- Bases have properties depending on sequences around them
- Hoogsten or Watson crick
What do intercalators prefer to target?
- form complexes, prefer to react with DNA with a protein bound to it
How do bleomycins differ?
- differ in the terminal amine substituent of the common structural unit, bleomycin acid
What are the main components of bleomycin’s for injection
Bleomycins A2 and B2
How many molecules of bleomycin were determined sufficient to induce dsDNA lesions in an in vitro study?
A single molecule is sufficient to generate lesions on both strands of DNA
What structural feature of bleomycins are thought to play a role in cellular uptake?
Positively charged tail
What structural components of bleomycins interact with the grooves?
- +polyamine tail forms interaction in the major groove
- sugars sit in the minor groove
How does the bithiazole tail act?
- In an intercalator manner
What needs to happen to bleomycin in order for dsDNA cleavage to occur?
- Bleomycin reactivation and reorganization
- Must be a regeneration with this catalytic cutting centre to form the second cut
What other forms of dsDNA break repair are there?
- NHEJ
- BIR
What therapy are platinum drugs frequently combined with?
- Bevacizumab
- May stabilise the vasculature and reduce tissue interstitial pressure, enhancing penetration of chemotherapeutic drugs
- improve sensitivity to cytotoxic drugs like platinum’s
- reduce dose
Combination of cisplatin and decitabine…
- exploit cisplatin mediated resistance mechanisms
- Such as GSH, reduced glutathione and glutathione S-transferase
- In GASTRIC CANCER: induce Sox2 DNA demethylation to promote expression of the Sox2 gene –> anti-tumour
Overview of development of platins
- Cisplatin –>
- Carboplatin - improved safety
- and oxaliplatin - broader spectrum of antitumour activity
What is Aroplatin and the rationale for making it?
- an attempt to formulate liposomal preparations of cisplatin like agents
