sarah fawaz - antivirals

PART 1: FUNDAMENTALS OF VIROLOGY AND ANTIVIRAL STRATEGY

Section 1: Viruses as Obligate Intracellular Parasites (Page 1)

1.1. Key Characteristics of Viruses:

• Obligate intracellular parasites: Viruses cannot replicate or carry out metabolic processes independently. They are entirely dependent on the cellular machinery of a host organism to reproduce.

• Key Distinction from Bacteria: Unlike bacteria, which are free-living organisms capable of independent replication, viruses must hijack a host cell's ribosomes, enzymes, and energy supplies to produce new viral particles.

• The Therapeutic Challenge: This parasitic nature creates a fundamental challenge for antiviral drug design. The goal is to develop compounds that can interfere with the viral life cycle without disrupting normal cellular processes of the host. This requires targeting virus-specific enzymes or processes.

Section 2: The Viral Life Cycle – A Blueprint for Drug Targets (Pages 2-4)

2.1. General Viral Life Cycle Stages (Page 2):
The viral life cycle consists of several key stages, each representing a potential target for antiviral intervention:

1. Attachment: Virus binds to specific receptors on the host cell surface.

2. Entry/Penetration: Virus enters the host cell (e.g., by fusion, endocytosis).

3. Uncoating: Viral genetic material (DNA or RNA) is released from the capsid.

4. Replication/Transcription: Viral genes are expressed, and viral genome is replicated using host and/or viral enzymes.

5. Assembly: New viral components are assembled into complete viral particles.

6. Release: New viruses bud or are released from the cell to infect others.

2.2. Influenza Viral Life Cycle (Page 3):

• The slide likely shows a diagram of the influenza virus life cycle, highlighting specific stages and potential drug targets (e.g., neuraminidase inhibitors like oseltamivir block release; M2 ion channel blockers like amantadine block uncoating).

2.3. Strategy for Antiviral Drug Design (Page 4):

• Designing compounds to disrupt specific stages of the viral life cycle is a common and effective strategy.

• The choice of target depends on the specific virus and the stage where intervention is most effective, balancing efficacy with minimal host toxicity.

PART 2: HUMAN IMMUNODEFICIENCY VIRUS (HIV) AND ITS LIFE CYCLE

Section 3: HIV – The Virus and Its Impact (Pages 5-8)

3.1. What is HIV? (Page 5):

• HIV is a retrovirus that attacks the immune system.

• It specifically targets CD4 cells (T-helper cells) , which are crucial for coordinating the immune response. These cells have CD4 molecules on their surface that HIV uses for attachment (Page 7).

• Over time, the immune system begins to fail, leading to increased risk of opportunistic infections and tumours. This advanced stage of HIV infection is referred to as AIDS (Acquired Immunodeficiency Syndrome) .

3.2. Brief History of HIV (Page 6):

• 1981: Illness affecting the immune system first recognised.

• 1982: AIDS formally recognised.

• 1984: Virus responsible for AIDS discovered – named HIV-1.

• 1986: HIV-2 discovered in West Africa.

• Current Status: Over 40 million people infected with HIV worldwide.

• Transmission: Through blood, semen, vaginal fluids, breast milk.

3.3. Why is HIV Lethal if Left Untreated? (Page 8):

• HIV targets and progressively destroys CD4 T-cells, which are central to the adaptive immune response.

• This leads to immunodeficiency, leaving the body vulnerable to infections and cancers that a healthy immune system would normally control.

Section 4: The HIV Life Cycle in Detail (Pages 9-20)

4.1. Attachment and Entry (Page 9):

• HIV attaches to the CD4 molecule on the host cell surface via a viral envelope protein called gp120.

• HIV also requires binding to a co-receptor (either CCR5 or CXCR4) via another part of gp120 to successfully fuse with the cell membrane and enter the cell.

4.2. Reverse Transcription – A Unique Retroviral Process (Pages 10-20):

4.2.1. The Enzyme: Reverse Transcriptase (RT) (Page 10):

• Reverse transcription is a biological process unique to retroviruses (like HIV).

• Reverse transcriptase is the enzyme central to retroviral replication. Once HIV gains access to the cell, it releases its single-stranded RNA genome into the cytoplasm. RT then uses this RNA as a template to synthesise a complementary double-stranded pro-viral DNA.

4.2.2. The Building Blocks: Deoxyribonucleoside Triphosphates (dNTPs) (Pages 11-12):

• RT polymerises DNA by using deoxyribonucleoside triphosphates (dNTPs) as building blocks.

• Each dNTP consists of:

  • Three phosphate groups (α, β, γ).

  • A deoxyribose sugar (lacking a 2'-OH).

  • One of four nitrogenous bases.

• The Four dNTPs:

  • dATP (Deoxyadenosine Triphosphate) – base: Adenine.

  • dTTP (Deoxythymidine Triphosphate) – base: Thymine.

  • dCTP (Deoxycytidine Triphosphate) – base: Cytosine.

  • dGTP (Deoxyguanosine Triphosphate) – base: Guanine.

4.2.3. Mechanism of DNA Polymerisation by RT (Page 13):

1. Base Pairing: The choice of which dNTP to incorporate is determined by standard Watson-Crick base pairing with the template RNA.

   • Adenine (A) in the RNA template pairs with dTTP (incorporating T).

   • Uracil (U) in the RNA template pairs with dATP (incorporating A).

   • Guanine (G) pairs with dCTP (incorporating C).

   • Cytosine (C) pairs with dGTP (incorporating G).

2. Nucleophilic Attack: The 3'-hydroxyl (3'-OH) group of the last nucleotide on the growing DNA strand performs a nucleophilic attack on the alpha phosphate of the incoming dNTP.

3. Phosphodiester Bond Formation: This attack breaks the bond between the alpha and beta phosphates, releasing the beta and gamma phosphates as pyrophosphate (PPi) . The alpha phosphate remains, forming a new phosphodiester bond between the two nucleotides, extending the DNA chain by one base.

4.2.4. The Role of RNase H (Page 14):

• Reverse transcriptase has a second enzymatic domain: RNase H (Ribonuclease H).

• After the RNA/DNA hybrid intermediate is formed, RNase H becomes active and selectively degrades the RNA portion of the hybrid, preserving the newly synthesised DNA strand.

4.2.5. Second Strand Synthesis and Integration (Page 15):

• After the RNA is degraded, the remaining single DNA strand pairs with a complementary sequence on itself (or a second strand is synthesised), resulting in the formation of a double-stranded pro-viral DNA.

• This pro-viral DNA is transported to the host cell's nucleus.

• The viral enzyme integrase facilitates the integration of the pro-viral DNA into the host cell's genome, where it is now known as a provirus. This allows the virus to persist for the lifetime of the cell.

4.3. Summary of Reverse Transcriptase Activities (Page 16):
Reverse transcriptase possesses two key enzymatic activities:

1. DNA polymerase activity: Involved in synthesising both DNA strands (from RNA template and then from the new DNA template).

2. RNase H activity: Specifically removes the RNA template after it has been copied.

PART 3: NUCLEOSIDE REVERSE TRANSCRIPTASE INHIBITORS (NRTIs)

Section 5: Mechanism of Action of NRTIs (Pages 17-23)

5.1. What are NRTIs? (Page 17):

• Nucleoside Reverse Transcriptase Inhibitors (NRTIs) are a class of antiretroviral drugs used to treat retroviral infections like HIV.

• They are designed to mimic natural nucleosides (the building blocks of DNA/RNA) but are chemically modified.

• Common NRTIs: Zidovudine (AZT), Lamivudine (3TC), Tenofovir disoproxil fumarate (TDF), Emtricitabine (FTC), Abacavir (ABC).

5.2. The Key Modification – Lack of a 3'-Hydroxyl Group (Pages 17-23):

5.2.1. Zidovudine (AZT) as an Example (Page 17):

• AZT structurally resembles the natural nucleoside thymidine.

• However, it has a critical difference in its sugar moiety: AZT lacks a 3'-hydroxyl (3'-OH) group on its deoxyribose sugar. Instead, it has an azido (-N₃) group at the 3' position.

5.2.2. The Importance of the 3'-OH Group (Page 17):

• The 3'-OH group is essential for the formation of the phosphodiester bond with the next incoming nucleotide during DNA chain elongation. Without it, the chain cannot be extended.

5.2.3. AZT is a Prodrug – Activation by Phosphorylation (Pages 18-19):

• AZT itself is a prodrug. In its administered form, it cannot effectively inhibit reverse transcriptase.

• It must be sequentially phosphorylated within the host cell to become active.

• Phosphorylation Steps (Page 19):

  1. AZT → AZT-MP (monophosphate): Catalysed by cellular enzyme thymidine kinase (dThd kinase) .

  2. AZT-MP → AZT-DP (diphosphate): Catalysed by cellular enzyme thymidylate kinase (dTMP kinase) .

  3. AZT-DP → AZT-TP (triphosphate): Catalysed by cellular enzyme nucleoside diphosphate kinase (NDP kinase) .

• Active Form: AZT-TP (zidovudine triphosphate) is the active form that reverse transcriptase mistakenly uses as a building block.

5.2.4. Incorporation and Chain Termination (Page 18):

1. Mimicry: AZT-TP structurally mimics the natural nucleotide dTTP (deoxythymidine triphosphate).

2. Misincorporation: Reverse transcriptase mistakenly recognises AZT-TP as dTTP and incorporates it into the growing viral DNA chain.

3. Chain Termination: Because AZT-TP lacks a 3'-OH group, after it is incorporated, no further nucleotides can be added. The phosphodiester bond with the next incoming nucleotide cannot be formed, causing obligate chain termination and halting viral DNA synthesis.

Image Description (Pages 3-4 of Handout):

• These pages show a diagram of a growing DNA chain and ask whether a dNTP (with a 3'-OH) and AZT-TP (without a 3'-OH) will incorporate. The answer is that both will incorporate due to base pairing, but after AZT-TP is incorporated, the chain cannot be extended because the next dNTP has no 3'-OH to attack.

5.3. Other NRTIs (Pages 20-21):

5.3.1. Didanosine (ddI) (Page 20):

• Structure: Structurally resembles adenosine.

• Activation: Like AZT, it is a prodrug that undergoes sequential phosphorylation to its active triphosphate form, dideoxyadenosine triphosphate (ddA-TP) .

• Mechanism: ddA-TP is incorporated into viral DNA and, lacking a 3'-OH, causes chain termination.

5.3.2. Zalcitabine (ddC) (Page 21):

• Structure: A nucleoside analogue that mimics cytidine.

• Activation: Converted to zalcitabine triphosphate.

• Mechanism: Incorporated into growing DNA chain by RT, leading to chain termination due to the absence of a 3'-OH.

PART 4: HERPES SIMPLEX VIRUS (HSV) AND ACYCLOVIR

Section 6: Herpes Simplex Virus (HSV) Biology (Pages 23-24)

6.1. HSV Overview (Page 23):

• HSV is a group of viruses that cause a range of infections, including oral and genital herpes.

• Key Features:

  • Highly contagious.

  • Can establish latent infections in sensory neurons, persisting for life.

  • Periodically reactivates, causing recurrent symptoms.

6.2. Types of HSV:

• HSV-1: Typically spread through saliva, kissing, or sharing utensils – causes oral herpes (cold sores).

• HSV-2: Primarily spread through sexual contact – causes genital herpes. Can also be transmitted from mother to baby during vaginal delivery.

• Presentation: Clusters of small, painful, fluid-filled blisters that typically heal after a few weeks.

6.3. Latency and Reactivation:

• After initial infection, HSV infects nearby sensory neurons and travels to the nerve ganglia, where it establishes a latent (dormant) infection. This phase is typically asymptomatic.

• Periodically, the virus can reactivate, travel back down the nerve, and infect epithelial cells, causing recurrent blisters.

Section 7: Acyclovir – A Selective Anti-Herpes Agent (Pages 24-31)

7.1. Target: Viral DNA Polymerase (Page 24):

• Anti-herpes medications like acyclovir work by inhibiting viral DNA synthesis, thereby inhibiting viral proliferation.

• Acyclovir is a DNA polymerase inhibitor that specifically targets the replication of double-stranded DNA viruses like HSV.

7.2. Acyclovir is a Prodrug – Selective Activation (Pages 25-28):

7.2.1. Activation Pathway (Pages 25-27):
Acyclovir (ACV) is a prodrug and must be converted to its active triphosphate form within infected cells.

7.2.2. The Basis of Selectivity (Page 28):

• Acyclovir exhibits selective toxicity against HSV-infected cells because the first phosphorylation step is catalysed by viral thymidine kinase, an enzyme present only in infected cells.

• In uninfected cells, acyclovir is not phosphorylated (cellular TK does not recognise it) and therefore remains inactive. This explains why acyclovir has minimal toxicity to healthy, uninfected host cells.

7.3. Mechanism of Inhibition – Chain Termination (Pages 29-31):

7.3.1. Structural Mimicry (Page 29):

• ACV-TP is a guanosine analogue – it structurally resembles the natural nucleotide dGTP.

7.3.2. Incorporation into Viral DNA (Page 29):

1. Viral DNA polymerase mistakenly recognises ACV-TP as dGTP due to structural similarity.

2. The 3'-OH group of the previous nucleotide on the growing DNA strand performs a nucleophilic attack on the alpha phosphate of ACV-TP.

3. This forms a phosphodiester bond, incorporating ACV-MP (after release of pyrophosphate) into the viral DNA chain.

7.3.3. Chain Termination (Pages 30-31):

• Unlike dGTP, ACV-TP lacks a 3'-OH group (it has an acyclic sugar with no 3' carbon/hydroxyl).

• After incorporation, no further nucleotides can be added to the DNA chain because there is no 3'-OH for the next incoming nucleotide to attack.

• This causes obligate chain termination, halting viral DNA synthesis.

PART 5: REVIEW QUESTIONS AND ANSWERS

Section 8: Multiple Choice Questions (MCQs) from Lecture Slides (Pages 31-32)

MCQ 1: Why does acyclovir exhibit selective toxicity against HSV-infected cells?

• A. Acyclovir is preferentially activated in infected cells by viral thymidine kinase, an enzyme absent in uninfected cells. ✅

• B. Acyclovir directly inhibits human DNA polymerase...

• C. Acyclovir is phosphorylated by cellular enzymes only in the presence of HSV infection.

• D. Acyclovir binds specifically to viral surface receptors...

• E. Acyclovir is selectively delivered to infected tissues by immune system components.

MCQ 2: Which enzyme catalyses the conversion of acyclovir monophosphate to acyclovir diphosphate?

• A. Viral thymidine kinase

• B. Cellular guanylate kinase ✅

• C. Viral DNA polymerase

• D. Nucleoside diphosphate kinase

• E. RNA polymerase

MCQ 3: How does acyclovir triphosphate inhibit HSV replication?

• A. By blocking viral entry into the host cell

• B. By binding to viral surface proteins and preventing attachment

• C. By inhibiting viral DNA polymerase and causing chain termination ✅

• D. By interfering with host RNA polymerase activity

• E. By preventing viral protein synthesis at the ribosome

MCQ 4: What happens when acyclovir triphosphate is incorporated into the viral DNA?

• A. It causes strand displacement and misfolding of the DNA.

• B. It binds to the 5' end of the DNA and blocks replication.

• C. It prevents the addition of further nucleotides, halting elongation. ✅

• D. It causes the viral DNA polymerase to degrade existing DNA.

• E. It induces mutations in the viral genome, leading to loss of function.

MCQ 5: Why does acyclovir triphosphate cause chain termination during viral DNA replication?

• A. It binds irreversibly to the DNA strand, preventing further elongation.

• B. It lacks a 3'-hydroxyl group necessary for forming phosphodiester bonds. ✅

• C. It competes with ATP, depleting energy for DNA synthesis.

• D. It causes the viral DNA polymerase to misread the DNA template.

• E. It inhibits viral RNA synthesis, halting DNA elongation.

Section 9: Questions from Handout (Pages 1-2)

Page 1 Questions:

1. Which enzyme is responsible for catalysing the synthesis of the complementary DNA strand during the formation of the RNA/DNA hybrid?

   • Answer: b) Reverse transcriptase ✅

2. What is the primary function of RNase H during the viral life cycle?

   • Answer: b) Degrading the viral RNA in the RNA/DNA hybrid ✅

3. How do NRTIs interfere with the reverse transcription process in HIV-infected cells?

   • Answer: d) Incorporating into the growing viral DNA chain ✅

4. What is the consequence of NRTIs being incorporated into the growing viral DNA chain during reverse transcription?

   • Answer: b) Inhibition of DNA synthesis ✅

5. Which of the following is a characteristic feature of NRTIs in terms of their structure?

   • Answer: b) Nucleoside analogues ✅

6. How do NRTIs differ structurally from dNTPs?

   • Answer: a) They lack a 3'-hydroxyl group. ✅

Page 2 Questions (AZT-Specific):

1. AZT is a prodrug. Explain what that means and identify: the inactive form, the active form, the cellular enzymes responsible for activation, the viral enzyme that AZT ultimately targets.

   • Explanation: A prodrug is an inactive compound that must be metabolised in the body to become its pharmacologically active form.

   • Inactive form: AZT (zidovudine).

   • Active form: AZT-TP (zidovudine triphosphate).

   • Cellular enzymes responsible: Thymidine kinase, thymidylate kinase, nucleoside diphosphate kinase.

   • Viral enzyme targeted: Reverse transcriptase.

2. A patient's cells have reduced thymidine kinase activity. Predict how this would affect AZT's antiviral effectiveness and why. (Include which phosphorylation step is impacted.)

   • Prediction: AZT's antiviral effectiveness would be significantly reduced.

   • Why: Thymidine kinase catalyses the first phosphorylation step (AZT → AZT-MP) . If this enzyme has reduced activity, less AZT-MP is produced, leading to reduced production of the active AZT-TP. Therefore, less inhibition of reverse transcriptase occurs, and viral replication is less effectively suppressed.

3. AZT-TP competes with a natural nucleotide during reverse transcription. Which natural nucleotide is it mimicking? Why does incorporation of AZT-TP terminate DNA chain elongation?

   • Natural nucleotide mimicked: dTTP (deoxythymidine triphosphate).

   • Reason for chain termination: AZT-TP lacks a 3'-hydroxyl (3'-OH) group on its sugar moiety. This group is essential for forming the phosphodiester bond with the next incoming nucleotide. Without it, after AZT-TP is incorporated, no further nucleotides can be added, causing obligate chain termination.

SUMMARY TABLE: KEY ANTIVIRAL DRUGS AND MECHANISMS