Nucleic Acid Based Cancer Therapies 1

why is DNA a good drug target?

DNA possesses a number of nucleophilic sites :. target for electrophilic drugs

who has been targeting DNA for millions of years?

toxins that plants and bacteria release can target DNA

where is the major and minor groove in DNA?

what are the different types of DNA interacting therapies?

• DNA cleavage

• intercalation

• mono-adduct

• topoisomerase adduct

• interstrand cross link

• intrastrand cross link

what do intercalating agents do?

drug drives a wedge between 2 strands

what do inter/intrastrand cross linkers do?

creates a covalent bond between 2 sections of DNA that aren’t meant to be cross linked

how can DNA repair produce resistance to DNA-interactive anticancer therapies?

• if enzymes that repair DNA are in excess then the damage caused by anticancer drugs will be repaired :. counteracts action of drug

how does selectivity occur with DNA interacting agents?

• mainly due to difference in growth rate of cancer cells compared to normal cells

• could also come from reduced capability of cancer cells to repair DNA lesions compared to normal cells

• some agents target specific DNA regions e.g. GC-rich sequences that may be prevalent in some tumour cells

• may block action of key transcription factors that have been up-regulated in tumour cells

describe the DNA repair pathway and how this is relevant for DNA interactive agents

• p53 = transcription factor that surveys genome and identifies mutations → makes decision on whether to promote expression of DNA repair enzymes or induce apoptosis

• DNA repair pathways may become damaged in cancer cells as their genomes evolve to become increasingly mutated

what are the acute side effects associated with the DNA interactive class of anticancer agents?

• alopecia

• GI toxicity (mucositis and diarrhoea)

• nausea and vomiting

• reversible mone marrow suppression (most dangerous)

what are the side effects of long term use of DNA interactive anticancer agents?

• gametogenesis is severely affected → sperm and ovarian tissue storage is recommended

• increased incidence of acute non-lymphocytic leukemia and other cancers occurring later in life due to DNA damage caused by these treatments

what were the first DNA interactive agents? how were they discovered?

nitrogen mustards

• serendipity

• bombing of ship released mustard gas

• doctors treating troops found lymphoid and myeloid suppression

• realised it may be possible to use mustard gas to treat lymphoma and other leukemias since these are diseases of lymphoid cells

describe methylating agents as a class of anticancer drugs

adds methyl group to DNA

CH₃⁺ + DNA → CH₃-DNA

give 3 examples of methylating agents and where they act

• dacarbazine

• procarbazine

• temozolomide

guanine in the major groove at the N7 and O6 positions

describe how dacarbazine works

it is a prodrug for the CH₃ ion

• in the body, dacarbazine is converted by enzymes into a methyldiazonium ion

• methyldiazonium ion generates the CH₃⁺ ion which methylates the DNA

how is dacarbazine administered? why?

• IV → IV bag must be protected from light because dacarbazine is photolabile

• cannot be administered via injection into skin due to irritant properties of drug

what are the predominant side effects of dacarbazine?

• myelosuppression

• intense nausea and vomiting

how does procarbazine work?

it is a prodrug for the CH₃ ion

• in the body, procarbazine is converted by enzymes into a methyldiazonium ion

• methyldiazonium ion generates the CH₃⁺ ion which methylates the DNA

describe how temozolomide works

it is a prodrug for the CH₃ ion

• in the body, procarbazine is converted by CHEMICAL HYDROLYTIC CLEAVAGE (not enzymes) into a methyldiazonium ion

• methyldiazonium ion generates the CH₃⁺ ion which methylates the DNA

what is are the advantages of using temozolomide?

• very good oral bioavailability

• penetrates CNS :. used in brain tumours

how was temozolomide discovered?

in vitro screening

what does temozolomide’s action depend on at a biochemical level?

mismatch repair enzymes (MMRs) = DNA repair enzyme that detect the wobble base pair formed during replication of drug-modified DNA

• when guanine is methylated, it looks like adenine to DNA repair enzymes :. removes original C paired to G and replaces it with T

• this creates a kink in the DNA since G & T cannot pair → repetition of this causes MMR-induced strand breaks and causes arrest of cell cycle

how has resistance developed against temozolomide?

• repair proteins have evolved → alkyltransferases (ATases) cleave the O6 and N7 modifications :. restores native guanine base

what does the response of a tumour to temozolomide depend on? how does this help treatment selection?

relative levels of MMR and ATases

• can potentially screen patients to predict who would response: ideal = ATase -ve and MMR +ve

what are alkylating agents?

add >1 carbon to DNA

give examples of alkylating agents. how do they work?

• carbinolamines

• ET-743

alkylates N2 of guanine in minor groove using electrophilic attack :. adds bulky adduct that interferes with DNA processing

what makes ET-743 different from other DNA-binding agents available?

causes structural perturbation of the DNA molecule by bending it towards their site of interaction with DNA instead of away from it

• pulls it rather than pushes it

how do cross linking agents work?

• they contain 2 alkylating moieties separated by linkers

• moieties are some distance apart

• alkylates 2 nucleophilic functional groups either on same strand or opposite strand :. forming cross link

give examples of cross linking agents

• nitrogen mustards → interstrand cross linkers in the major groove

• aziridines

• mitomycin C

• nitrosoureas

what are the mechanisms of selectivity for nitrogen mustards?

• targets faster growing cells

• some cancer cells are not as good at repairing mustard adducts compared to healthy cells :. apoptosis induced

• GC-selectivity

what are some of the potential mechanisms of resistance for nitrogen mustards?

• multidrug resistance → MDR resistance (i.e. upregulation of p-glycoproteins)

• increased conc. of glutathione in tumour cells → glutathione reacts with nitrogen mustards :. no longer electrophilic :. unable to launch electrophilic attack

• up-regulation of repair processes where nitrogen mustards usually damage

what are the 2 types of nitrogen mustards?

aliphatic and aromatic

give an example of an aliphatic nitrogen mustard

chlormethine

• this is the only aliphatic nitrogen mustard in use because they are highly reactive and able to attack other nucleophiles :. high toxicity profile

how does chlormethine work?

• undergoes cyclisation in the body through chloride elimination

• forms cyclic aziridinium ion

• arizidinium ion attacks N7 of guanine in major groove of DNA

• compound contains 2 chloride atoms :. process is repeated with second chloroethyl group → attacks second guanine N7 on opposite strand

• causes interstrand cross link that locks 2 strands together

why are aromatic nitrogen mustards preferred over alipahtic?

• milder alkylating agents

• aromatic ring acts as an electron sink :. withdraws electrons from nitrogen atom :. discourages arizidinium ion formation

• less reactive :. makes travel to tumour site better → less toxicities observed

how do aromatic nitrogen mustards work?

• central nitrogen electron pair is delocalised with pi electrons of the aromatic ring :. not sufficiently basic to form a cyclic arizidinium ion

• :. alkylation most likely occurs via S_N1 mechanism → normal carbocation formation caused by chloride ion ejection = rate-limiting step

• occurs for first Cl then repeated for second Cl

• attacks N7 guanine on one strand followed by second :. interstrand cross link

how are aromatic nitrogen mustards administered?

orally since they can reach target site without interacting with other nucleophiles = big advantage

give an example of a conjugated aromatic nitrogen mustard

melphalan

• contains carboxylic acid giving it greater selectivity since it increases uptake in cells where protein synthesis is occurring faster

what are the dosing intervals like for melphalan?

every 6 weeks

• bone marrow toxicity is delayed :. have to wait longer to see effects

how do aziridines work? name an example

• instead of forming aziridinium ion, they have aziridine ring already incorporated into its structure

• e.g. thiotepa

compare aziridines with nitrogen mustards

ring opening of aziridines with nucleophiles is slower compared to aziridinium ions in nitrogen mustards

which 3 moiety components of mitomycin C are essential for its activity?

• quinone

• arizidine

• carbamate

describe the selectivity of mitomycin C

selective for hypoxia

describe how mitomycin C is administered

• IV

• 6 week intervals → delayed bone marrow suppression

what is the biggest benefit of using nitrosoureas over other cross linkers?

• high lipophilicity compared to other cross linkers :. can cross CNS :. can be used in brain cancers