Andy Thompson Flashcards
Nitrogen mustards
Draw mechanism of action
Direct ring alkylation leads to a positively charged adduct that is liable to depurination and strand break
Nucleophilic sites on DNA
i.e. the sites of DNA alkylation
Guanine N7, N3, exocyclic NH2
Adenine N7, N3
Mustard gas
Too toxic for human use
Draw
Chlormethine
Aliphatic mustard, sufficient therapeutic index for human use
Draw
Chlorambucil
Aromatic mustard
Less electrophilic, reacts with DNA more slowly
Can be administered orally
Draw
Melphalan
Rationale for synthesis = enhance cellular uptake via Phe uptake mechanism
(draw)
Cyclophosphamide
Rationale for synthesis = release mustard agent through enzymatic degradation
(draw)
Estramustine
Rationale for synthesis = target oestrogen-dependent tumour cells
(draw)
Temozolomide
DNA-methylating compound
Draw mechanism of activation
Repair of DNA methylation by temozolomide
Methylation occurs on guanine O6
The DNA alkyltransferase enzyme AGT scans dsDNA for alkylation on guanine O6
Covalent transfer of the alkyl group to the conserve active site Cys in AGT restores guanine to normal (and inactivates AGT)
What can happen if a G-CH3 lesion is not repaired?
2 things can happen:
- Transversion = G to A transition mutation
- Strand break
DNA minor groove binders
Can bind covalently or non-covalently
Flat, planar, poly-aromatic structures - many also have a natural twist to fit into the groove
Alkylate a base within the mono groove
Secondary, non-covalent interactions are important in covalent binding
Some non-covalent compounds have been modified with DNA alkylation moieties (e.g. mustards, epoxides) to give experimental covalent compounds
Many covalent compounds have been linked to give dimer intra- and inter strand cross linkers
Covalent minor groove binders
Mitomycin C
Cyclopropapyrroloindoles (CPIs)
Pyrrolo-1,4-benzodiazepines (PBDs)
Non-covalent minor groove binders
Distamycin
Netropsin
Mitomycin C
Undergoes enzymatic or chemical reductive activation
Primary alkylation sites are guanine N2 and N7 in the DNA minor groove
Draw mechanism of alkylation
(+)-CC-1065
(draw)
CPI-based anti-tumour antibiotic
Ethanobridges cause DNA over-winding, leading to dose-limiting toxicity and delayed death
Adozelesin
Ethanobridges in CC-1065 removed
Adozelesin is effective but difficult to synthesise
(draw)
Bizelesin
Dimer of adozelesin
This allows 2 alkylation reactions to occur, therefore can form inter strand DNA cross-links
Very insoluble but very potent
High-field NMR studies on Adozelesin
Palindromic sequences used CGATTAATCG
Adozelesin binds at 2nd A (one adozelesin on each strand)
NMR showed the formation of 2 duplex adducts, which were both basically the same except one had Watson-Crick base pairing between the middle TA/AT and one had Hoogsteen base pairing
i.e. there were 2 base pair arrangements
High-field NMR studies on Bizelesin
Palindromic sequences used CGATTAATCG
One Bizelesin molecule forms inter stand cross-link at A
NMR showed 2 base pair arrangements: 1. Watson-Crick, 2. Open base pairing
Effect of open base pairing on DNA repair
Open base pairs (or Hoogsteen) affect the arrangement of bases
Open base pairing means the major groove is filled with bases and actually no longer a groove
The damage is no longer just within the minor groove - this contributes to the increased potency of Bizelesin
PBD-based anti-tumour antibiotics
Tomayamycin
Anthramycin
Sibiromycin
These 3 compounds are all naturally-occurring anti-tumour antibiotics, but none have been adopted clinically
Mechanism of action of tomayamycin with DNA
(draw)
Involves the formation of a reversible animal bond between the exocyclic NH2 of guanine and C11 on PBD
Reactive form of PBD compounds
PBD compounds are traditionally recrystallised with MeOH to give the stable methyl ether
Methyl ether imine carbinolamine
All 3 forms can react with DNA but it is widely considered that the immune/carbinolamine are the active forms
DC-81
Synthesised in the search for better PBD-based compounds
DSB-120
Dimer of DC-81
AT-486
More active PBD dimer but more difficult to synthesise
SJG-136
PBD dimer
Compromise in terms of reactivity and synthetic viability
Currently in the clinic
Potent anti-tumour and antibiotic - a “last line treatment” for MRSA
High-field NMR studies on SJG-136 DNA duplex adducts
SJG-136 can form inter- and intrastrand cross-links, but intrastrand are preferred
Next steps for DNA alkylators…
Use pro-drugs so damage is restricted to the tumour
Hypoxia-based targeting
Use of antibodies
Tumour cells have increased permittivity
DNA-specific structural targets e.g. telomeres/promoters
Bleomycins
A group of related basic glycopeptides that differ in the terminal amine substituent of the common structural unit (bleomycin acid)
Generally active against most tumours
Bind Fe(I) or Cu(I) and O2 - can catalyse the formation of ss and dsDNA lesions in the presence of a one-electron reductant
Similar damage to that generated by ionising radiation
In vitro studies indicate that a single molecule of bleomycin is sufficient to generate lesions on both DNA strands
Common outcomes of bleomycin treatment
Extended cell cycle arrest
Apoptosis
Mitotic cell death
Regions of the bleomycin molecule
The total bleomycin molecule is greater than the sum of its parts
The linker region between the metal-binding domain and the bithiazole DNA-binding domain, as well as the flexibility of the bithiazole moiety itself are essential for efficient dsDNA cleavage
Why are bleomycins unable to cross the cell membrane by free diffusion?
They are hydrophilic
Studies indicate the positively charged tail might play a key role in cellular uptake
Sites of dsDNA cleavage by bleomycins
Bleomycins firstly initiate ssDNA cleavage at pyrimidines 3’ to a guanine in a sequence-specific fashion
The secondary site of cleavage depends on the primary cleavage site:
5’-G-Py-Pu-3’ sequences = bleomycins generate 5’ staggered ends
5’-G-Py-Py-3’ sequences = bleomycins generate blunt ends
Mechanism for dsDNA cleavage by bleomycins
dsDNA cleavage by a single bleomycin molecule requires the reorganisation and reactivation of bleomycin during or after cleavage of the first strand of DNA in order to allow cleavage of the second strand
The key to this reorganisation is believed to be the linker and the flexibility of the bithiazole tail (bound by partial intercalation)
Rotation around the bond between the 2 thiazole rings in the bleomycin tail make the peroxide of the activated drug available for interaction with the second DNA strand
DNA DSB repair
There are several pathways to ensure the repair of DSBs in eukaryotic cells, but homologous recombination is the only inherently error-free pathway
Homologous recombination
- Homologous recombination is initiated by a DSB caused by an endonuclease/DNA-damaging agent
- DSB formation triggers cell cycle checkpoints through activation of ATM kinase
- The DNA ends are recognised by Rad52 protein
- The DNA is processed at the breakage site to yield regions of ssDNA
- Rad51 polymerises onto the ssDNA with the help of Rad52 and RPA (amongst other proteins) to form a nucleoprotein filament
- This “Rad51 nucleoprotein filament” searches for homologous sequences on the homologous chromosome/sister chromatid
- DNA strand exchange occurs, generating a joint molecule between the homologous damaged and undamaged DNAss
- The break is repaired by DNA polymerases, using the intact strand as a template
- Ligation and resolution of the recombination intermediates results in accurate DSB repair
Other methods for DSB repair
Non-homologous end joining
Break-induced replication
…but these are error-prone and can lead to deletion/insertion mutations - no regard for homology
However, NHEJ is the major DSB repair pathway because it can occur at any point during the cell cycle and doesn’t require a homologous chromosome
Homologous recombination can only occur later S phase/early G2 when the sister chromatid is in close proximity
Nucleotide Excision Repair
Operates on base damage cause by exogenous agents e.g. mutagenic/carcinogenic chemicals that alter the chemistry and structure of the DNA duplex
Involves the excision of damaged bases as part of an oligonucleotide fragment
Steps in nucleotide excision repair
- The recognition of base damage during NER requires disruption of the normal Watson-Crick base pairing and altered chemistry in the damaged strand, typically involving the bases
- DNA damage is recognised by the XPC-R23 complex
- The binding of XPC-R23 to the DNA is followed by the binding of several other proteins including XPA (3’ endonuclease), RPA, TFIIH and XPG
- Recruitment of ERCC1-XPF (another endonuclease) completes the NER complex and cuts the damaged strand at junctions 5’ to the site of base damage
- This bimodal incision generates an oligonucleotide fragment approx. 25-30 nucleotides in length that includes the damaged base
- The fragment is excised from the genome and the nucleotide gap is restored by repair synthesis involving DNA polymerase and its accessory replication proteins
- The covalent integrity of the damaged strand is restored by DNA ligase
Base Excision Repair
Removes and replaces the majority of damaged DNA bases
Steps in base excision repair
- The N-glycosidic bond of the damaged base is cleaved by a DNA glycosylase, leaving an abasic site in the DNA
- The sugar-phosphate backbone of the basic site is then cleaved by a bifunctional glycosylase or an AP-endonuclease
- If necessary, the 3’ strand break end is converted into an OH to allow DNA polymerase to reinsert new bases
- Synthesis of a single base = short patch BER, synthesis of several bases = long patch BER
- The 5’ single strand end of the ss break intermediate is then processed to allow for ligation by DNA ligases
Mechanism of action of cis-platin
Intracellular activation through substitution of the Cl groups by H2O
The resulting species can then covalently bind to DNA, forming DNA adducts
The major bis-adduct formed involves adjacent guanines on the same DNA strand (intrastrand cross-link)
The minor bis-adduct formed involves guanines on opposite DNA strands (interstrand cross-link)
Preferential binding occurs on guanine N7
Effect of cis-platin binding to DNA
The adducts cause unwinding/bending of the DNA
The final cellular outcome is generally apoptotic cell death/prolonged cell cycle arrest
What tumour types is cis-platin effective against?
Testicular/ovarian cancer
But is notoriously toxic to the kidneys :( so a less-toxic analogue that retained anti-cancer activity needed to be developed
Tumour resistance to cis-platin/carboplatin
Mediated by inadequate levels of Pt reaching the target DNA
Tumour cells acquire resistance resulting in reduced Pt accumulation
Mechanisms of tumour resistance to cis-platin
- Cis-platin causes a down-regulation of CTR1 expression in human ovarian cancer cell lines. CTR1 = transporter = substantial role in cis-platin influx. Loss of CTR1 means cis-platin must rely on passive diffusion to enter the cell (slow, because cis-platin is highly polar)
- ATPA7A/B = efflux proteins involved in Cu transport. Human ovarian cancer cells transfected with ATP7A showed resistance to cis-platin and carboplatin due to Pt sequestration in vesicles
- Increased cytoplasmic levels of glutathione/metallothioneins - these thiol-containing species bind to Pt (soft) before it can bind to DNA.
Conjugation of glutathione to cis-platin
Catalysed by GSTs (glutathione S-transferases)
Compound is made more anionic so is more readily exported from the cell by the GS-X pump
How do tumour cells mediate Pt resistance after DNA binding?
DNA repair
Removal of adducts
Tolerance mechanisms
What is believed to cause the increased sensitivity of testicular cancer to cis-platin?
A deficiency in DNA repair
Major pathway for removing cis-platin lesions from DNA
NER
Cis-platin resistant cancer cells show increased NER, associated with an increased expression/activity of ERCC1 and XPF
4 major DNA repair pathways
NER
BER
MMR
DSB repair
Enhanced replicative bypass
Another tolerance mechanism to cis-platin
Certain DNA polymerases can bypass cis-platin-DNA adducts by “translesion synthesis”
Ongoing strategies to circumvent cisplatin and carboplatin resistance
Increase delivery of Pt to tumour e.g. using liposomes
Combine existing Pt drugs with molecularly-targeted agents e.g. Avastin (bevacuzimab), Herception (trastuzumab)
Use novel platinum drugs that target resistance mechanisms e.g. oxaliplatin
Oxaliplatin
Can circumvent cisplatin-mediated resistance
draw
Function of diaminocyclohexane group in oxaliplatin
Provides increased resistance to cells producing glutathione
Topoisomerases
Participate in the over- or underwinding of DNA
Type I topoisomerases
Cut one strand of the DNA helix, leading to relaxation
Allows the molecule to rotate around uncut strand which reduces stress
The cut strand can then be re-ligated
Type 2 topoisomerases
Cuts both strands of the DNA helix, passes another unbroken DNA helix through the gap then re-ligates the cut strands
How do most clinically active drugs that target TOP2 kill cells?
By trapping an enzyme intermediate (“covalent complex”)
This generates enzyme-mediated DNA damage e.g. strand breaks, proteins covalently bound to DNA
Important side effect associated with targeting TOP2 with TOP2 poisons
Formation of secondary malignancies that arise from drug-induced translocations
i.e. patients treated with these drugs have a reasonably higher chance of developing secondary tumours
Doxorubucin
(draw) = adriamycin
Intercalator - inserts between planar DNA bases, distorting DNA structure
Targets TOP2
Structure of intercalators
Many flat, planar rings (min. 3) that can insert into major and minor grooves via non-covalent interactions
Have some sequence preference (i.e. preferred insertion sites)
Neighbour-exclusion principle
The 2 neighbouring sites of an occupied intercalation site in DNA must remain unoccupied
Every second intercalation site along the length of the DNA double helix remains unoccupied
Why can telomeres form G quadruplexes?
Because they are G-rich
Equilibrium between dsDNA of telomeric sequence and G-quadruplex
Dependent upon chaperone proteins required for G-quadruplex formation and helicases that unwind the quadruplex
Drugs that interact with G-quadruplexes
Inhibit helicases e.g. cationic porphyrins
Sequester newly-formed G-quadruplexes e.g. telomestatin
