Lecture 14 - Intercalation and new targets Flashcards
What is TOP-2?
- Topoisomerase II
- Target of important classes of anticancer drugs
- a DNA processing enzyme
- make dsDNA break, to remove twists and knots in duplex DNA molecules
- Promising target in chemo
What drug targets TOP-2 and what is this type of drug?
- Doxorubicin
- Intercalator
What is a side effect of doxorubicin?
- formation of secondary malignancies that arise from drug-induced translocations
General overview of how intercalators fit into DNA
- Slot into the gaps in DNA through base pairs
- Via the neighbourhood exclusion rule where they fill every other gap
What is the structure of Doxorubicin?
- large aromatic core normally 3 rings fused together forming flat planar mass
- Sugars (multifunctional rings)
- stacks between the base pairs of DNA
- stacking of pi systems within the drug and the two base pairs either side
- very stable, cause a stabilisation of the duplex
What do DNA intercalators prefer to associate with on DNA?
- Protein DNA complexes rather than naked DNA
What do the sugars (multifunctional rings) of doxorubicin do?
- bind into the major groove of DNA
- form non-covalent interactions with the phosphate backbone of DNA stabilising the complex
- Important for increasing the potency
What increases potency of doxorubicin?
Stronger binding
Mechanism of action of doxorubicin
- TOP-2 complex is a dimer, with 2 binding pockets
- A kink in DNA forms for drug to be dropped into
- Drug slots into the two pockets, which are specific to the binding site
- Results in stabilisation
- Then get covalent modification by the protein of the DNA rather than the drug stuck to the DNA
- The TOP-2 protein stuck covalently, representing big challenge to DNA repair mechanism and thus DNA breaks
What intercalator targets TOP-1?
- SN-38 complex
- similar way to TOP-2
- interact with the complex TOP-1 and DNA
- single specific binding socket which opens up and drug fits in and binds tightly
- lock the complex of TOP-1 with DNA
What is pluramycin and what does it do?
- Intercalator/alkylator
- Binding of TBP to the TATA box facilitates intercalation of pluramycin
Overview of how doxorubicin works?
- stabilisation of a complex with eventual alkylation via a protein forming strand breaks at that site
How is SN-38 formed from natural products?
- Natural product camptothesin
- formed via hydrolysis of Irinotecan by carboxylesterases and metabolized via glucuronidation at the phenol
More than just alkylating DNA - principle of intercalators…
- Intercalators –> large molecule in the minor groove of DNA stabilises it, making it difficult for enzymes to pull apart and get into the DNA to repair it
- once complex formed itself may change the DNA
- Can form dimers with drugs, as they get bigger though they become less stable
- Secondary structures are also important within DNA
What do telomerases do?
- Take DNA and create a repeat end at the end of DNA to form telomeres
- adds DNA sequence repeats, “TTAGGG” to the 3’ end of DNA strands in the telomere regions
What forms at the end of telomeres by telomerases?
- G-quartet (complex between 4 guanines)
- Guanine tetraplex/quadruplex
Structure of a g-quartet?
- 4 guanines, 4 hydrogen bonds
- flat, planar structure
- metal in the centre to stabilise
What are telomeres?
- telomeres contains non-coding DNA material at end of chromosomes
- prevents constant loss of important DNA from chromosome ends
- regenerated by telomerase
Why could we target telomerases in cancer?
- They are active in cancer cells to maintain telomere length during uncontrolled cell divison
How could we target telomerases?
- Form telomerase inhibitor which fits across guanine quadruplex
What is name of best telomerase inhibitor and briefly how does it work?
- Telomerstatin
- stacks above guanine tetramers stabilising it and causing disruption
- sequester newly formed G-quadruplexes
What other 2 drugs can target the telomere/telomerase complex?
Perylene and cationic porphyrins
What do perylene and cationic porphyrins do?
- They are G-quadruplex-interactive drugs
- Which inhibit helicases or facilitate formation of new quadruplexes
General principle of Doxorubicin?
- Binds to sites up and downstream of TOP-2 interaction sites, in specific binding pockets
- damage done by formation of stable complex of drug, protein and DNA which sits with covalent modifications by the PROTEIN not the ligand
- Result in protein bound to DNA not the ligand
- Cell cannot process this damage and get SSB and DSB
How is the damage actually done by doxorubicin?
- Wasn’t the drug which alkylated DNA
- BUT the formation of a stable complex of drug, protein and DNA which sits in a way with covalent modifications by the protein not the ligand
- Protein bound to DNA not the ligand
- cell cannot process ssDNA and dsDNA breaks
What are the repair pathways for TOP-2 mediated DNA damage?
- double strand break repair pathways
- other pathways specific for removal of protein DNA adducts
Example of Intercalators which target TOP-2
- Doxorubicin
- Mitoxantrone
What is the intercalator SN-38 and cancer used in?
- irinotecan analog
- topoisomerase I inhibitor
- primarily used in the treatment of colorectal cancer
Mode of action of pluramycin
- binds peripherally to the TATA-binding protein (TBP)–TATA box complex
- Alkylation by pluramycin enhanced by TBP binding to the TATA box, and as a result TBP is immobilized on the DNA
Anticancer drugs which target TOP-1
- SN-38 (analogue of irinotcean)
- topotecan
Disadvantages of TOP-1 inhibitors
- side effects
- low solubility
- adverse drug interactions
What is the equilibrium between dsDNA and G quadruplexes dependent on?
- helicases that unwind the quadruplex and chaperone proteins that are required for its formation
What can G-quadruplex-interactive drugs do (telomerase inhibitors)?
- inhibit helicases or facilitate formation of new quadruplexes (perylene and cationic porphyrins)
- OR sequester newly formed G-quadruplexes (telomestatin)
Side effect of Doxorubicin
- cardiotoxicity by iron-related free radicals and mitochondrial disruption
Mode of action of SN-38
- SN-38 binds to and inhibits topoisomerase I by stabilizing the cleavable complex between topoisomerase I and DNA
- resulting in DNA breaks, inhibition of DNA replication, and apoptosis
