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Pharm Decks > Lecture 11 - DNA Alkylation > Flashcards

Lecture 11 - DNA Alkylation Flashcards

1
Q

What are DNA alkylation based drugs?

A
  • Electrophiles which react with nucleophiles in the DNA
  • They make the electrophilic centre as the last step of synthesis, which will damage DNA
  • originate from natural products
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2
Q

What are the two single strand DNA repair mechanisms relevant to these drugs?

A

Nucleotide excision repair and base excision repair

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3
Q

What happens to DNA when you give these drugs?

A
  • The damage is too much for the repair mechanisms to cope with
  • Lead to apoptosis

(we hope)
(If they repair then drug has failed)

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4
Q

Why are these types of drugs usually given in combination?

A

To avoid resistance

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5
Q

Overview of NER

A
  • Damage to a single strand
  • Very efficient
  1. DNA is damaged at the base
  2. A recognition enzyme recognises the damage
  3. Other enzymes open up the DNA and form a complex around the damaged part
  4. 2 molecules cut the DNA, removing a chunk larger than the damaged part
  5. DNA ligases repair the strand using the undamaged strand as a template
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6
Q

Types of BER

A
  • Short or long patch repair
  • Similar method to NER, just different enzymes
  • Base has been damaged, usually causing a break in the back bone.
  1. Short - takes one base out and repairs
  2. Long - takes a few bases out around it and repairs
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7
Q

Overview of BER

A
  1. N-glycosidic bond of the damaged base is cleaved by a DNA glycosylase leaving an abasic site in the DNA.
  2. sugar-phosphate backbone of the abasic site is then cleaved by an enzyme
  3. Synthesis via short or long patch
  4. 5’ end strand break intermediate processed, ligation via DNA ligases
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8
Q

In the context of nitrogen mustards, what are the sites of possible DNA alkylation?

A
  • Guanine N7, N3
  • Adenine N7 and N3
  • Guanine exocyclic NH2
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9
Q

An overview of the development of nitrogen mustards for a cancer therapeutic…

A
  1. Sulphur mustard - too toxic
  2. Chlormethine developed - aliphatic mustard
  3. Chlorambucil developed - an aromatic mustard, less electrophilic so interacts with DNA more slowly
  4. Melphalan developed - enhanced uptake
  5. Cyclophosphamide - attempt to release through enzymatic degradation
  6. Estramustine - attempt to target oestrogen-dependent tumour cells
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10
Q

What is one of the most widely used mustards?

A

Chlorambucil

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11
Q

How do nitrogen mustards work?

A

Damage DNA by tagging the DNA twice

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12
Q

How do DNA methylating compounds work and what is an example of one?

A
  • temozolomide
  • Deliver methyl onto a DNA base
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13
Q

With regards to DNA methylation, what is AGT aka MGMT and what does it do?

A
  • O6-alkylguanine DNA alkyltransferase (AGT/MGMT) protein
  • Covalent transfer of the alkyl group to the active-site cysteine –> inactivates AGT and restores guanine to normal
  • scans double-stranded DNA for alkylation at the O6 position of guanine
  • Suicide enzyme
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14
Q

What do DNA minor groove binders do?

A
  • twist to fit the minor groove of DNA, reacting with it
  • hold a nucelophile and electrophile together
  • result in covalently modified DNA
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15
Q

Where is the alkylation centre of DNA Minor Groove Binders?

A
  • Bottom of the minor groove
  • NH2 of guanine
  • or N3 of adenine
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16
Q

Structure of DNA Minor groove binders

A
  • flat and slightly curved to fit in the groove of DNA
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17
Q

What interactions are also important in DNA Minor groove binders?

A
  • Covalent causes most damage of DNA
  • Secondary non-covalent interactions also important in causing cytotoxicity
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18
Q

How would you make a DNA Minor groove binder more potent?

A

Alter its fit to the minor groove of DNA so it binds more tightly

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19
Q

What is the main example of DNA minor groove binders?

A
  • Mitomycin C
  • Also PBDs, CPIs
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20
Q

What are the steps of mitomycin C reaction with DNA?

A
  1. enzymatical chemical reduction
  2. Releases methanol leading to reduced mitocene
  3. Get DNA alpha attack
  4. Then beta attack
  5. Reaction with a second molecule of DNA displacing the top arm ending up with 2 tags of DNA of mitomycin
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21
Q

What do you never get in mitomycin reaction?

A

Never get beta attack before the alpha attack

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22
Q

What is the final product in a mitomycin reaction?

A

Mitomycin with 2 DNA tagged off the rings

23
Q

What does mitomycin C target?

A
  • N2 and N7 of guanines in the minor grooves
24
Q

What cross links can Mitomycin C form?

A
  • Intrastrand cross links
  • 2 alkylation points are on the same strand
25
Q

What is an advantage of CPIs and PBDs?
(DNA Minor Groove Binder)

A

Act as anti tumour drugs and antibiotics

26
Q

What was the issue with safety of Cyclopropapyrroloindoles (CPIs)?

A
  • thought this could kill all tumours
  • killing every tumour line in vitro and in animal studies
  • found death by dissolving organs
27
Q

What was the mechanism behind delayed lethality in CPIs?

A
  • Ethano bridges cause DNA over-winding
  • lead to dose limiting toxicity and delayed death
28
Q

What was the 1st generation drug made of CPIs and its features?

A
  • Adozelesin
  • Ethano bridges were removed
  • Difficult to make and expensive
29
Q

What 2nd generation CPI was made by mistake?

A
  • Bizelesin
  • although had no more advantage over classical compounds
30
Q

What was the natural first CPI?

A

CC-1065

31
Q

How does CPI CC-1065 interact to alkylate DNA?

A
  1. CC10-65 alkylates DNA and is attacked by the N3 of adenine as a nucleophile
  2. The ring opens up, opening up the phenol, ending up with covalent complex
  • IMPORTANT – a positive charge remains in the adenine
32
Q

What is an issue with the mechanism of CPIs?

A

Alkylation onto a ring atom –> delocalised charge in the ring –> base unstable

  • they debase the DNA
33
Q

Features of the reaction of CPI Bizelesin?

A
  • two reactions can occur due to stereo-chemistry in the propyl ring –> 2 centres
  • can form inter-strand DNA duplex crosslinks
  • cannot be repaired by NER or BER
34
Q

What is an important stereochemistry feature in CPI reactions?

A
  • the stereochemistry of the cyclopropyl of the CPI
  • 2 stereochemistry’s they can adopt
  • One which can react with adenine and give reversible DNA complexes
  • One which doesn’t react due to positioning in the minor groove
35
Q

What centres/stereochemistry does Bizelisin (CPI) have?

A
  • cyclopropyl centres
  • both the right stereochemistry to react with adenine to give reversible adducts
  • enabling the drug to form interstrand cross links
36
Q

Intra strand vs Interstrand crosslinks and DNA repair

A
  • Interstrand cross links wont be able to repair by BER or NER
  • Intra can
37
Q

How many reactions take place in CC-1065 and adozelesin?

A
  • Only single reaction, unlike bizelesin
    due to the sterochemistry
  • do not form INTER-strand
38
Q

What is the base pair arrangements after treatment with Adozelesin? (CPI)

A

Normal watson crick –> Two base pair arrangements: Hoogsteen and Watson crick base pairs

39
Q

Overview of Hoogsteen base pairing

A
  • Base pairs open up their Watson crick formation into the major groove
  • Adenine rotates 180
  • Reforms in a Hoogsteen conformation
  • Can be short or longlived
40
Q

Examples of Pyrrolo(1,4)benzodiazepine (PBD) antitumour antibiotics

(DNA Minor groove binders)

A
  • Tomayamycin
  • Anthramycin
  • sibiromycin
  • all naturally occuring, none adopted clinically
41
Q

What compounds were synthesised in the search for better PBDs?

A
  • DC-81
  • dimersie to form DSB-120
  • use of right hand side alkene of tomayamycin to make AT-486
  • SJG-136 easier version to make of AT
42
Q

Issue with AT-486 (PBD)

A
  • Very faffy and difficult to make
43
Q

Why was SJG-136 easier to make than AT-486?

A
  • putting 2 protons at the end so the alkene is in the right configuration
44
Q

Are PBDs used in the clinic?

A
  • Not many as they are expensive and faffy to make
  • but SJG-136 has been used in the clinic
  • Antibacterial properties, last line of treatment for MSRA
45
Q

What form do PBDs come in?

A
  • carbinolamine, imine or a methyl ester
  • Interchangeable depending on what you dissolve the drug in
46
Q

How do the 3 forms of PBD react with DNA?

A
  • NH2 of guanine attacks PBD (all 3 forms)
  • PBD binds to the minor groove of DNA
  • Enters a dry environment and displaces water
  • Forming an imine
  • Imine thought to be active species
47
Q

What is the proposed mechanism of action of PBDs with DNA?

A
  • Formation of a reversible aminal bond between the exo-cyclic NH2 of guanine and the C11 position on the PBD.
48
Q

What can you form with PBDs and what crosslinks occur?

A
  • 2 PBDs can form a dimeric compound
  • can alkylate 2 guanines on opposite strands of DNA
  • INTER-STRAND
49
Q

Relavence of MGMT/AGT in Temozolomide resistance?

A
  • MGMT can repair the O6-MeG, removing methyl group restoring to guanine
  • DNA strand doesn’t break and apoptosis is not induced
  • Cells with absent/reduced MGMT more susceptible to TMZ
  • Upregulation of MGMT –> TMZ resistance
50
Q

What are the repair pathways of lesions (other than O6-MeG)?

A
  • N7 and N3 position repair by base excision repair
  • although not primary mediators of temozolomide toxicity
51
Q

Electrophile vs Nucleophile

A
  • Electrophile - Accepts A Pair Of Electrons To Form A New Covalent Bond
  • Nucleophile - Provides A Pair Of Electrons To Form A New Covalent Bond
52
Q

Examples of mustards in the clinic

A
  • Chlorambucil –> brand name Leukeran, chronic lymphocytic leukaemia
  • Cyclophosphamide –> multiple myeloma, sarcoma, and breast cancer
  • Estramustine –> metastatic or aggressive prostate cancer
53
Q

What happens if Temozolomide methylation is not repaired?

A
  • If repair of the CH3-G lesion does not occur, a G to A transition mutation or a strand break can occur, leading to apoptosis
  • Good thing - we don’t want repair

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