SL32113-MD2 - Medicines Design - Alkylators & DNA Repair

Why is DNA an effective target for cancer chemotherapy despite being present in normal cells?

1. Rapid Division: Cancer cells divide quickly, creating a high demand for DNA replication and leaving less time for repair before division.

2. Susceptibility: DNA is more vulnerable to damage during mitosis.

3. Defective Repair: Many cancers have pre-existing repair weaknesses (e.g., BRCA1/2 mutations) that can be exploited by drugs like PARP inhibitors.

Compare the two primary DNA repair pathways: NER vs. BER.

• Nucleotide Excision Repair (NER): Repairs "bulky" lesions (e.g., those caused by radiation or specific alkylators) by excising a fragment of \sim 27\text{--}30 nucleotides.

• Base Excision Repair (BER): Repairs damage to a single base. It involves DNA glycosylases cleaving the base to create an "abasic site," followed by short-patch (1 base) or long-patch (several bases) synthesis.

What are the most common sites of DNA alkylation on nucleotide bases?

• Guanine: N7 (most common), N3, and exo-cyclic.

• Adenine: N7 and N3.

• Note: Alkylation on a ring nitrogen can destabilize the base, leading to "de-purination" and subsequent DNA strand breaks.

How has the structure of Nitrogen Mustards evolved for clinical use?

• Mustard Gas (Sulphur Mustard): Too toxic for humans; used as a chemical weapon.

• Aliphatic Mustards (e.g., Mustine): Sufficient therapeutic index for human use.

• Aromatic Mustards (e.g., Chlorambucil): Less reactive and slower acting; can be administered orally.

• Carrier-linked (e.g., Melphalan, Estramustine): Use transport mechanisms (Phenylalanine) or hormones (Oestrogen) to enhance cellular uptake or target specific tumors.

Key clinical details: Cyclophosphamide.

• Cancers: Breast, Lung, Leukaemia, Lymphoma, Ovarian, Ewing sarcoma.

• Side Effects: Increased infection risk, bruising/bleeding, anemia, hair loss, and notably bladder irritation.

Key clinical details: Estramustine and Chlorambucil.

• Estramustine: Used for relapsed Prostate cancer. Side effects include GI disturbance and Gynecomastia (male breast development) due to its oestrogen component.

• Chlorambucil: Used for Leukaemias (CLL) and Lymphomas. Side effects include infection risk and bruising.

What is the mechanism and specific repair protein associated with Temozolomide?

• Mechanism: Acts as a pro-drug that releases methyldiazonium ions to methylate DNA (primarily at Guanine N7 and O^6).

• Repair: The AGT protein (O^6-alkylguanine DNA alkyltransferase) restores Guanine by scanning DNA and "sacrificing" itself to remove the methyl group.

• Clinical: Used for high-grade gliomas (e.g., Glioblastoma multiforme).

Describe the unique activation and use of Mitomycin C.

• Activation: Requires reductive activation; it is unreactive at neutral pH but forms DNA cross-links rapidly in the presence of reductants.

• Site: Targets Guanine N2 and N7 in the minor groove.

• Cancers: Gastric, breast, lung, pancreatic, and bladder.

What are PBDs (Pyrrolobenzodiazepines), and how do they interact with DNA?

• Mechanism: Form a reversible aminal bond with the exo-cyclic NH_2 of Guanine in the minor groove.

• Development: Newer PBD dimers (e.g., SJG-136) are designed to form inter-strand and intra-strand cross-links.

• Side Note: Phenol groups in older PBDs caused dose-limiting cardiotoxicity.

What are the "Next Stage" strategies for DNA alkylating agents?

• Hypoxia-based targeting: Exploiting low oxygen levels in tumors.

• Antibody-Drug Conjugates (ADCs): Using antibodies to deliver CPI or PBD payloads specifically to tumor cells.

• Size-based targeting: Using PEG chains to exploit increased tumor permeability.

• Sequence Recognition: Increasing recognition to 14\text{--}18 base pairs to target a single specific site in the human genome.

What are the three steps of Base Excision Repair (BER)?

1. Step 1: DNA glycosylase cleaves the N-glycosidic bond of the damaged base, creating an abasic site.

2. Step 2: AP-endonuclease cleaves the sugar-phosphate backbone.

3. Step 3: DNA polymerase reinserts bases (Short Patch = 1 base; Long Patch = several bases) and DNA ligase seals the strand.

Contrast the binding sites of Mitomycin C vs. CPI anti-tumour antibiotics.

• Mitomycin C: Targets the N2 and N7 atoms of guanines in the minor groove.

• CPIs (e.g., CC-1065): React specifically with the N3 position of adenine.

Why is the stereochemistry of the cyclopropyl ring in CPI drugs (like Adozelesin) critical?

Only one specific isomer will react covalently with DNA; the stereochemistry dictates the drug's ability to fit into the minor groove and undergo alkylation.

Explain the clinical significance of Hoogsteen base pairs in DNA alkylation.

• Sequence Dependency: For Hoogsteen pairs to form, bases must "open" from their standard Watson-Crick conformation and rotate 180^{\circ}.

• Drug Interaction: Some sequences (A-tracts) rarely open, while others (TAATTA) are prone to it, affecting how drugs like Bizelesin interact with the DNA duplex.

What is the specific mechanism of action for PBD (Pyrrolobenzodiazepine) antibiotics?

They form a reversible aminal bond between the exo-cyclic NH_2 of guanine and the C11 position of the PBD. The imine or carbinolamine forms are considered the active species that react with DNA.

What are the main side effects and indications for Temozolomide?

• Indications: Glioblastoma multiforme, malignant glioma, and astrocytoma.

• Side Effects: Nausea/vomiting, headache, fatigue, hair loss, and seizures (fits).

How do Minor Groove Binders interact with DNA?

These ligands are typically flat (planar) poly-aromatic structures with a natural twist to fit the curve of the minor groove. They provide access to base pairs for covalent alkylation at the base of the groove (e.g., Guanine-NH_2 or Adenine N3).

What are the advantages of Aromatic Mustards (e.g., Chlorambucil) over Aliphatic Mustards?

Aromatic mustards are less electrophilic, meaning they react with DNA more slowly. This allows for a better therapeutic index and the ability to be administered orally.

How does the AGT protein restore DNA after Temozolomide treatment?

AGT scans double-stranded DNA for O^6 alkylation; it restores the guanine by covalently transferring the alkyl group to its own active-site cysteine, which inactivates the protein permanently.