Cancer Therapy - Topoisomerase Inhibitors

Mechanisms of action of Topoisomerase inhibitors

1. Inhibit Topoisomerase II

2. Bind to double stranded DNA

3. Contribute to the formation of reactive oxygen species » damage DNA, proteins and cell membranes » anti-tumour activity

How is the human genome packaged into the nucleus?

• Humans have 23 pairs of chromosomes (46 total), containing approximately 6.4 billion base pairs per diploid cell.

• DNA is approximately 2 nm wide and 2 m long when stretched out.

• DNA wraps around histone proteins to form nucleosomes.

• Nucleosomes undergo further coiling, folding and supercoiling to form higher-order structures (chromosomes).

• This allows approximately 2 m of DNA to fit inside a nucleus only around 10 µm in diameter.

  
  

What is DNA supercoiling important for?

• Allows DNA to be safely stored inside cells

• Allows DNA to be compacted during mitosis when chromosomes are separated into the 2 daughter cells

Why does DNA supercoiling need to be controlled?

• Parts of DNA need to be accessed without causing damage to the remaining genetic material during replication

• Topoisomerases control the supercoiling of DNA

What are topoisomerases, what are their functions, and how do Type I and Type II differ?

• Topoisomerases temporarily break DNA strands, allowing DNA to unwind

• They temporarily break the DNA backbone, allow DNA strands to move/unwind, then reseal the breaks.

• Functions:

  • Allow enzyme access to DNA during replication and transcription.

  • Help separate daughter chromosomes after DNA replication.

  • Maintain and regulate DNA supercoiling.

Two types:

• Type I: Breaks only ONE DNA strand.

• Type II: Breaks BOTH DNA strands.

Topoisomerase II

Functions

• Maintains DNA supercoiling

• Removes supercoiling formed during replication

• Separates interlocked double-stranded daughter DNA chains formed during replication

Topoisomerase II

Mechanism

• Cuts both strands of the DNA double helix

• Abother DNA double helix passes through the break

Mechanism:

Step 1: Topoisomerase attaches to the first double-stranded DNA, referred to as the G-segment.

Step 2: ATP binds to the N-terminal ATPase domain, priming the enzyme for action.

Step 3: A second DNA duplex, known as the T-segment, is captured and held above the G-segment.

Step 4: The enzyme cuts the G-segment and opens a pathway to let the T-segment pass through the break.

Step 5: Following ATP hydrolysis (ADP+PiADP+Pi​), the G-segment is resealed, and the T-segment is released out through the bottom C-terminal gate, resetting the enzyme for the next cycle.

Anthracyclines

Which bacterium are they derived from?

The Streptomyces bacterium

Anthracyclines

Structure

• Consist of a planar hydrophobic tetracyclic ring system

• Linked to an amino sugar

• Through a glycosidic bond

• All anthracyclines are positively charged at physiological pH, allowing intercalation with DNA (inserting itself between DNA base pairs)

  
  

Anthracyclines

Examples

• Doxorubicin

• Daunorubicin

• Idarubicin

Anthracyclines

How do doxorubicin and daunorubicin differ in their structures?

Differ only by a single hydroxyl group at the R1 position

Anthracyclines

What are Anthracyclines’ 3 mechanisms of antitumour activity?

1. Topoisomerase II inhibition

2. Binding to double stranded DNA (however, not the main mechanism that leads to cell death)

3. Formation of reactive oxygen species which damages DNA, proteins and cell membranes

Anthracyclines

Mechanism 1

How do Anthracyclines e.g. doxorubicin turn Topoisomerase II into a poison?

1. The anthracycline drug binds to DNA and Topoisomerase II, stabilising their interaction and forming a ternary complex. The sugar moiety of anthracyclines is essential for the formation of this complex

2. Anthracyclines stabilise the intermediate stage where DNA is cut and covalently linked to Topoisomerase II

3. This prevents the DNA strands from being resealed

4. This results in the DNA double-strand breaks

5. DNA repair enzymes attempt to repair the damaged DNA. However, if the damage is too extensive or cannot be repaired, double-strand breaks accumulate

6. Extensive, irreparable DNA damage triggers apoptosis (programmed cell death)

Anthracyclines

Mechanism 2

How do anthracyclines intercalate into DNA to have anti-tumour activity?

• The planar aromatic structure of the anthracycline intercalates into DNA

• The amino sugar moiety is positioned in the minor groove of DNA

• When anthracyclines become associated with DNA, there is separation of stacked bases

• This induces local structural changes to the DNA strand, such as partial uncoiling of the DNA double helix

• These structural changes lead to functional changes e.g. the inhibition of replication and transcription of cancer cells

• This leads to cell death

Anthracyclines

Mechanism 3

How do Anthracyclines form reactive oxygen species and how does this have anti-tumour activity?

• Anthracyclines contain the quinone moiety

• Reduction of the quinone moiety generates a semi-quinone (1 e- reduction) and dihydroquinone (2 e- reductions)

• When these reduced forms interact with O2, they transfer e- to it, producing superoxide (O2-) which is converted to hydrogen peroxide (H2O2)

• Reactive oxygen species can lead to:

  • DNA damage

  • Oxidation of proteins

  • Damage of cell membranes

= Triggering apoptosis

Anthracyclines

Hoes does the ability of Anthracyclines to produce reactive oxygen species explain their ability to cause cardiotoxicity?

• ROS accumulate within cardiomyocytes as they have low levels of detoxifying enzymes to neutralise ROS

• Oxidative stress damages DNA, proteins, mitochondrial function and cell membranes.

• Cardiomyocytes undergo apoptosis or other forms of cell death.

• Loss of heart muscle cells reduces the heart's ability to contract effectively.

• This can lead to reduced left ventricular ejection fraction (LVEF) and potentially heart failure.

Anthracyclines

Toxicities

• Nausea and vomiting (seen in 30%- 90% of patients)

• Alopecia

• Myelosuppression (leukopenia)

Anthracyclines

What is a dose-limiting toxicity of anthracyclines?

Cardiotoxicity

• Early stages: transient electrocardiographic changes

• Delayed stages: progressive cardiomyopathy leading to congestive heart failure

What are the short-term and long-term cardiac effects of anthracyclines?

• Short-term: Transient effects such as arrhythmias, ECG changes and temporary cardiac dysfunction.

• Long-term: Cardiomyocyte damage can cause reduced cardiac function and congestive heart failure (CHF)

Why do anthracyclines cause long-term cardiotoxicity?

• Anthracyclines generate ROS through their quinone moiety.

• ROS cause oxidative damage to DNA, proteins, mitochondria and cell membranes.

• This triggers cardiomyocyte damage and apoptosis.

• Loss of heart muscle cells reduces cardiac contractility and can lead to heart failure.

Why is cardiac tissue particularly vulnerable to ROS damage from anthracyclines?

• Cardiac tissue has relatively low catalase activity.

• Catalase breaks down hydrogen peroxide (H₂O₂).

• Reduced catalase activity means less effective ROS detoxification.

• This increases oxidative stress and makes cardiomyocytes vulnerable to lipid peroxidation and cellular damage.

How does cumulative anthracycline dose affect cardiotoxicity?

• Cardiotoxicity is dose-dependent.

• Increasing cumulative exposure increases the risk of congestive heart failure.

• Higher peak drug concentrations may also contribute to toxicity.

• Therefore, total lifetime anthracycline exposure must be monitored.

Why does combining doxorubicin and trastuzumab (Herceptin) increase cardiotoxicity?

• Doxorubicin causes cardiomyocyte damage through ROS generation and Topoisomerase IIβ inhibition.

• Trastuzumab blocks HER2 signalling, which normally helps protect cardiomyocytes.

• Combining them increases the risk of cardiac dysfunction and heart failure.

What cardiac monitoring is recommended for patients receiving anthracyclines?

• Echocardiograms to monitor LVEF

• ECGs to assess heart rhythm.

• Monitor for symptoms of heart failure (breathlessness, fatigue and peripheral oedema).

• Monitoring at 3, 6 and 9 months, although schedules vary according to patient risk and treatment regimen.

What are the main clinical uses of doxorubicin?

• Doxorubicin has broad-spectrum anticancer activity

• Used in both solid tumours and haematological malignancies.

• Examples: breast, bladder, endometrial, lung, ovarian and thyroid cancers.

• Also used for bone/soft tissue sarcomas, Hodgkin and non-Hodgkin lymphoma, and leukaemias.

What are the main clinical uses of daunorubicin and idarubicin?

• Primarily associated with the treatment of myeloid leukaemias

• Particularly used in acute myeloid leukaemia (AML)

What is etoposide and how does it work?

• Etoposide is a semisynthetic derivative of podophyllotoxin (plant compound).

• It is a Topoisomerase II poison.

• Stabilises the DNA–Topoisomerase II complex, preventing DNA resealing.

• Causes DNA double-strand breaks, leading to apoptosis.

What are the main clinical uses of etoposide?

• Small-cell bronchial carcinoma (small-cell lung cancer).

• Testicular cancer (often in combination with bleomycin and cisplatin).

• Can be used in combination with other chemotherapy drugs.

How can etoposide be administered?

• Orally (capsules).

• Intravenously (IV).

What are the main adverse effects of etoposide?

• Alopecia

• Hypotension particularly with rapid IV administration.

• Myelosuppression (reduced blood cell production).

• Nausea and vomiting

What is myelosuppression and what are its consequences?

Myelosuppression = reduced bone marrow activity and blood cell production.

• Reduced red blood cells → anaemia.

• Reduced white blood cells → increased infection risk.

• Reduced platelets → increased bleeding risk.