Polymerase inhibitors

What are the key stages of the HIV life cycle?

1) Attachment to host membrane 2) Fusion/endocytosis and uncoating 3) Reverse transcription of viral RNA to dsDNA 4) Integration of proviral DNA into host genome 5) Transcription and translation using host machinery 6) Assembly and budding of new virions

What enzyme converts HIV viral RNA into dsDNA?

Reverse transcriptase (reverse transcription)

What is proviral DNA?

The dsDNA made by reverse transcription of viral RNA, which is integrated into the host genome by integrase

What is the recommended ART for people not previously treated for HIV?

A combination of two NRTIs plus either a ritonavir-boosted protease inhibitor, an NNRTI, or an integrase inhibitor

What is the aim of lifelong antiretroviral therapy?

Reduce viral load below detectable levels, which stops transmission of the virus and progression to AIDS

What is the most important factor for effectiveness of ART?

Adherence (patient communication is key)

What is a nucleobase?

A nitrogenous base (purine or pyrimidine) attached to the pentose sugar via a beta-glycosidic bond

Which nucleobases are purines?

Adenine (A) and guanine (G)

Which nucleobases are pyrimidines?

Cytosine (C), thymine (T) and uracil (U)

What is a nucleoside?

Nucleobase + pentose sugar (no phosphate), e.g. adenosine, guanosine, cytidine, thymidine, uridine

What is a nucleotide?

A nucleoside with one or more phosphate groups (mono-, di- or triphosphate), e.g. dATP, AMP, UDP

What is a nucleoside triphosphate and why is it important for polymerases?

A nucleoside with three phosphates at the 5' position (e.g. dATP, dGTP, dCTP, dTTP); the incoming substrate that polymerases add to the growing strand

Which sugar is in RNA and which is in DNA?

RNA: ribose (2'-OH). DNA: 2'-deoxyribose (2'-H)

What is the base difference between DNA and RNA?

DNA contains thymine (5-methyluracil); RNA contains uracil (no methyl group)

What is the hydrogen bonding pattern in the DNA helix?

A pairs with T (2 H-bonds, A=T); C pairs with G (3 H-bonds, C≡G)

What is the central dogma of molecular biology?

DNA -> mRNA -> Protein

What is transcription?

Genomic DNA is used as a template to make pre-mRNA

What happens after transcription before translation?

Post-transcriptional modification converts pre-mRNA to mRNA, which is exported to ribosomes

What is translation?

Ribosomes use mRNA as a template for protein synthesis

Why must HIV undergo reverse transcription?

HIV has an RNA genome, so it must be converted to DNA before it can be integrated into the host genome and expressed

What is the structure of HIV reverse transcriptase?

A multifunctional enzyme made of p66 and p51 subunits, with polymerase and nuclease (RNase) activity

What are the three functions of reverse transcriptase?

1) RNA-dependent DNA polymerase 2) Ribonuclease (degrades RNA strand) 3) DNA-dependent DNA polymerase

What does RNA-dependent DNA polymerase activity do?

Builds a DNA strand from the viral RNA template, giving an RNA-DNA hybrid

What does the ribonuclease activity of reverse transcriptase do?

Degrades the RNA strand of the RNA-DNA hybrid, leaving ssDNA

What does DNA-dependent DNA polymerase activity of reverse transcriptase do?

Builds a second DNA strand using ssDNA as a template, forming dsDNA ready for integration

Why is HIV reverse transcriptase described as sloppy?

High error rate (~1 in 2000-5000 bases) and no proofreading, so HIV (~10 kbp genome) mutates rapidly, facilitating drug resistance

How does host replicative DNA polymerase compare with HIV reverse transcriptase for accuracy?

~1 error per 10^7-10^9 bases; proofreading corrects ~99% of errors (human genome ~6 Gbp)

What is the role of Mg2+ ions and Asp residues in reverse transcriptase?

Mg2+ ions and several Asp residues (Asp110, Asp185, Asp186) coordinate the incoming dNTP in the active site

Which residue catalyses phosphodiester bond formation in reverse transcriptase?

Asp185

Which groups react to form the new phosphodiester bond during polymerisation?

The 3'-OH of the primer attacks the alpha-phosphate of the incoming dNTP

Describe the curly arrow mechanism of nucleotide incorporation by reverse transcriptase.

1) Lone pair on primer 3'-OH attacks the alpha-phosphorus 2) P=O pi bond breaks 3) Electrons move back to reform P=O 4) P-O bond to the beta-phosphate breaks, releasing pyrophosphate (PPi)

What is the by-product of nucleotide incorporation by polymerase?

Pyrophosphate (PPi)

In which direction is DNA synthesised?

5' to 3' - new nucleotides are added to the 3'-OH end of the primer strand

What does NRTI stand for?

Nucleoside analogue Reverse Transcriptase Inhibitor

What does NtRTI stand for?

Nucleotide analogue Reverse Transcriptase Inhibitor

Why must NRTIs be activated?

They are nucleosides (no phosphate); cellular kinases must add three phosphate groups to the 5' end to form the active NRTI triphosphate

Which enzymes activate AZT (zidovudine)?

Thymidine kinase, thymidine monophosphate kinase and thymidine diphosphate kinase (using ATP). The first phosphorylation is rate-limiting

What is the rate-limiting step in NRTI activation?

The first phosphorylation (nucleoside to monophosphate)

How do NRTIs inhibit reverse transcriptase?

The triphosphate competes with the natural dNTP (e.g. dTTP for AZT), is incorporated into the growing DNA, and acts as a chain terminator

Why do NRTIs cause chain termination?

They lack a 3'-OH group (e.g. AZT has a 3'-azide), so no further phosphodiester bond can form

What is an NtRTI and how does it differ from an NRTI?

A nucleotide mimetic (or its prodrug) that already contains a phosphate, so it avoids the rate-limiting first phosphorylation. Mechanism is otherwise similar (incorporation and chain termination)

Name an example NtRTI and NRTI.

NtRTIs: tenofovir, adefovir. NRTIs: zidovudine (AZT), emtricitabine, lamivudine, abacavir

Why is tenofovir given as tenofovir disoproxil?

Tenofovir is polar (charged phosphate) and cannot cross membranes. Disoproxil is a masking prodrug group that improves membrane permeability and is removed by esterases

Why is tenofovir disoproxil formulated as the fumarate salt?

Fumaric acid salt improves stability and solubility and increases shelf life

What drugs are in PrEP (pre-exposure prophylaxis)?

An NtRTI (tenofovir disoproxil fumarate) plus an NRTI (emtricitabine)

Which NRTIs mimic dT?

Zidovudine and stavudine

Which NRTIs mimic dA?

Didanosine (and tenofovir as the nucleotide analogue)

Which NRTIs mimic dC?

Emtricitabine and lamivudine

Which NRTIs mimic dG?

Abacavir and amdoxovir

What do ribose and base modifications in NRTIs achieve?

Efficiency of 5' phosphorylation; efficiency of DNA incorporation; optimisation of lipophilicity; avoidance of metabolic deactivation

What is the orthosteric site?

The classical enzyme active site, where the endogenous ligand/substrate binds

What is the allosteric site?

A site distal to the active site where an inhibitor or activator binds, causing a conformational change in the protein

What does NNRTI stand for?

Non-Nucleoside Reverse Transcriptase Inhibitor

What is the mechanism of action of NNRTIs?

They bind an allosteric site distal to the active site, inducing a conformational change so the enzyme is no longer catalytically competent and cannot bind RNA/DNA

How do N(t)RTIs and NNRTIs differ in binding site?

N(t)RTIs are orthosteric (bind the active site like the substrate); NNRTIs are allosteric (bind a distal pocket and change protein conformation)

Name four NNRTIs.

Nevirapine, efavirenz, rilpivirine, etravirine

What is a pharmacophore?

The molecular features necessary for recognition and binding of a ligand by its target, allowing it to elicit a biological response

What is the NNRTI pharmacophore?

A 3-point pharmacophore: one common polar region and two separate lipophilic regions

Why do NNRTIs with different structures all bind the same pocket?

They induce subtly different shapes of the allosteric site but share the same 3-point pharmacophore

Which human DNA polymerase subtypes exist and what do they do?

Alpha, delta, epsilon: DNA replication. Beta and epsilon: repair. Gamma: mitochondrial DNA synthesis

How sensitive are human DNA polymerases to NRTIs?

Alpha, delta, epsilon: not inhibited. Beta: low sensitivity. Gamma: highly sensitive (main off-target of NRTIs)

Why does NRTI selectivity for viral over human polymerase occur?

Human replicative polymerases (alpha, delta, epsilon) do not incorporate NRTIs efficiently, whereas HIV reverse transcriptase does, and it lacks proofreading

Why is mtDNA especially vulnerable to NRTI toxicity?

It replicates more frequently, has no effective repair mechanism, no protective histones against reactive oxygen species, and is replicated semi-autonomously by polymerase gamma

What side effects may mitochondrial toxicity cause with antiretrovirals?

Anaemia, hepatotoxicity, cardiotoxicity

What does mtDNA encode and why does damage matter?

Genes for oxidative phosphorylation (ATP supply); failure affects almost all organs: liver, pancreas, heart, muscle, nervous system, haematopoietic system, kidney

What happened in the fialuridine trial (1993)?

Hepatitis B trial; 7 of 15 patients in Phase II developed severe liver toxicity, 5 of the 7 died and 2 survived after liver transplants. Fialuridine is a dUTP analogue causing mitochondrial toxicity

How does molnupiravir work (Covid-19)?

An RNA nucleoside prodrug that exists as two tautomers (hydroxylamine and oxime), so its base mimics both C (pairs with G) and U (pairs with A), causing catastrophic copying errors during RNA synthesis

How does remdesivir work (Ebola/Covid-19)?

An RNA nucleotide prodrug causing delayed chain termination: it is incorporated without stopping immediately, then polymerisation stops because the protruding CN group causes steric clashes