What is the typical error rate in DNA replication?
1 in 10 billion.
What’s the difference between purines and pyrimidines?
Purines (bicyclic heterocycles):
- Adenine (A)
- Guanine
Pyrimidines (monocyclic heterocycles):
- Thymine
- Cytosine
What are alkylating agents? Give examples.
- The alkylating mechanisms just comprises of nucleophilic substitution; but focussing on the attacking group, and not the species with the leaving group
- Attacking group has a hydrogen (H) replaced w/an alkyl group (e.g. M.E.P.B; methylation, ethylation, propylation, butylation)
E.g.:
• Bendamustine
• Cyclophosphamide
What determines which nucleophilic centres (-ve rich; attacking group) get ‘attacked’ WRT alkylating agents? Common sites of alkylation?
Depends on combination of natural chemical reactivity (electronegativity?) and their actual accessibility (major/vs. minor groove of DNA):
- N7 of guanine base (G) is most important target
- N3 of cytosine also common
»> Nitrogens; lone pairs, electronegative; NUCLEOPHILES susceptible to alkylation
What happens to N7 Guanine (and N3 Cytosine) once alkylated?
- Becomes positively charged (Nitrogen now has 4 bonds w/extra alkyl group)
- +ve charge is resonance stabilised (at N7 guanine AND N3 cytosine)
How do alkylating agents actually exert their anticancer effect?
- Alkylation allows them to bind to DNA (N7 guanine, N3 cytosine)
- The process of them binding involves nitrogen mustards, allowing chemical cross-linking of DNA strands; permanent covalent bonds, preventing uncoiling and thus DNA replication (transcription cannot occur)
What are nitrogen mustards? How do they result in chemical cross-linking?
- Alkylating agents bendamustine and cyclophosphamide both contain nitrogen mustards
- (Up to) two chloroethyl groups bound either side to a Nitrogen atom, which is bound to the rest of the alkylating agent (-R)
- Nitrogen ‘can’t get away’ from nucleophilic site (C - Cl) as it is bonded to it; nucleophilic attack occurs, forming Aziridinium ion (+ve, 3-membered ring with itself, Cl leaving group)
- Guanine N7 can easily attack the positive Aziridinium ion of the alkylating agents; 3-membered ring unfolds, Guanine N7 now covalently bonded (itself becomes positively charged, is resonance stabilised)
- Then nitrogen mustard (of bendamustine, cyclophosphamide etc.) is able to form another Aziridinium ion with its other chloroethyl arm, which in turn is attacked by another Guanine N7
- Resulting in cross-linking of DNA; chemical bridge (nitrogen mustard of alkylating agent) formed between 2 Guanine bases
What is the difference between a monofunctional alkylating agent and a bifunctional one?
Monofunctional alkylating agents:
- Contain just one reactive centre (e.g. one chloroethyl arm), can only attack ONE nucleophilic centre (G-N7, C-N3) of DNA
Bifunctional alkylating agents:
- Contain TWO reactive centres (two chloroethyl arms); can react twice with a region of DNA; cross-linking it.
What is the difference between intrastrand and interstrand cross-linking?
Intrastrand:
- Formed between two bases that are on the same strand of DNA
Interstrand:
- Formed between one strand of the DNA double helix and the other respective strand (parallel and antiparallel)
What are mutagenic agents?
Change the sequence of bases in DNA, but leave overall structure intact.
What are clastogenic agents?
Cause deletions, additions or rearrangements to DNA sequence.
What is a lesion?
Region of damaged DNA.
What are monoadducts/bisadducts?
- Chloroethyl groups (of nitrogen mustards) that may be hydrated by water instead of forming Aziridinium ion
- Mono; one chloroethyl; Bi; both chloroethyls (Cl is replaced by OH)
Why is DNA structure distorted in interstrand cross-links?
- Distance between Cls of chloroethyl group of un-Aziridinium’d nitrogen mustard = 5.1 Å
- Distance normally between guanine nucleobases on opposing strands = 6.8 Å
»> Thus DNA bends to accommodate cross-link.
How does the alkylating agent cyclophosphamide differ in its action from its fellow alkylating agent, bendamustine? Why?
- Cyclophosphamide itself is not an alkylating agent; it is a pro-drug
- As the electron-withdrawing phosphoramide group means nitrogen lone pair is not availible for formation of aziridinium ion of nitrogen mustard (drawn into N = P bond instead)
»> Phosphoramide groups pikeys electron lone pair
How the pro-drug cyclophosphamide bioactivated in the body?
- CYP450-mediated oxidation in the liver to 4-hydroxycyclophosphamide (like TAM is metabolised to 4-OH TAM)
- 4-hydroxycyclophosphamide is in equilibrium with its ring-opened form, Aldophosphamide (tautomerises)
- Aldophosphamide undergoes elimination reaction (w/an Acrolein elimination product)
- Yielding the active phosphoramide mustard, the active alkylating agent
»> Allows formation of aziridinium ion; phosphoramide group is less electron withdrawing, N still has lone pair for nucleophilic attack of C-Cl to form 3-membered ring
What are the common anthracycline antibiotics used in anticancer therapy? What do they share in common?
- Common anthracyclines: Doxorubicin, Daunomycin
- Both share planar tricyclic aromatic section (making them anthracyclines)
- Bulky 3D-structure of remaining molecule
How do they anthracyclines exert their anticancer effect?
- DO NOT form covalent bonds like alkylating agents
- Interact non-covalently w/DNA instead; planar anthracycline rings insert between the planes of successive base pairs; INTERCALATION
- Intercalation process itself is not cytotoxic; it inhibits the actions of Topoisomerase (II in Doxorubicin + Daunomycin, ) enzyme, inhibiting the religation of the cut section of DNA (final step of the enzyme mechanism)
»> Ultimately producing double strand breaks in the DNA
Why are Topoisomerase enzymes required in the DNA replication process in the first place? What’s the difference between TI and TII?
- As DNA unwinds for replication (transcription); twisting tension builds-up in the rest of the supercoil
- To keep unwinding DNA, tension needs to be released; topoisomerase enzymes cut DNA strands then splice them back together after
> Topoisomerase I; cuts a single strand of the ds double helix, passes the other strand through the cut, then reseals break (overall strand has one fewer twist, relaxing DNA)
> Topoisomerase II: cuts both strands of double helix, passes another double strand through, and then reseals the break (relieves tension from unwinding DNA - removes supercoil 2 twists at a time)
How does etoposide work? What therapeutic target does it share?
- Etoposide (for testicular cancer; salty sodium salicylate added to counter solubility issues) binds to Topoisomerase II complex, leading to formation of DNA ds-breaks (preventing re-ligation)
- Just like the Topoisomerase II inhibitor, Doxorubicin
»> But is NOT an anthracycline antibiotic; has no extended planar tricyclic aromatic ring section, does NOT intercalate into DNA (diff. mechanism)
How do platinum-based compounds work? Give examples.
- Bond covalently to DNA via their platinum ion, Pt.
- Both links to DNA are made through the SAME Platinum ion; nucleophilic centre attached must be close to each other
- Forms interstrand AND intrastrand cross-links
- Common if two nucleophilic centres are on adjacent bases of the same strand of DNA; forming a 1, 2 intrastrand cross-link
What is Topotecan derived from? What does it act on?
- Derived from camptothecins
- Topoisomerase I inhibitor
- Topotecan is the semi-synthetic analogue that is clinically useful
How is Topotecan clinically usefully compared to the naturally derived product, camptothecin?
- Dimethylamino group (side chain) of Topotecan is protonated at physiological pH
- This increases polarity of Topotecan and its water solubility, therefore bioavailability
- Topotecan -OH group also increases polarity
Give examples of some DNA damaging agents.
- Sunlight (UV)
- Ionising radiation (radon, potassium)
- Bioactive chemicals in the environment/diet
- By-products of metabolism (ROS)
- Errors in DNA replication
