Lecture 4: Polymerase Inhibitors

HIV life cycle overview?

Virus enters the cell, copies its rna into dna via reverse transcriptase, this DNA is integrated and transcribed into mRNA,

Full life cycle?

1 - attachment: retrovirus attaches to host cell membrane

2 - Entry and uncoating: the virus fuses with eh membrane and endocytosis occurs and is uncoated, this releases viral rna plus its enzymes (reverse transcriptase, integrase)

3 - Reverse transcription: viral RNA is converted to double-stranded dna

4 - Integration: provial DNA is inserted into the host genome

5 - Transcription and translation: the host’s protein synthesis machinery makes viral proteins

6 - assembly and budding - new visions form and bud off

Define nucleobase, nucleoside and nucleotide? dNTP?

Nucleobase - just the base

Nucleoside - base and sugar

nucleotide - base sugar and at least one phosphate OR nucleoside monophosphate?

dNTP - deoxy nucleoside triphosphate

What is the central dogma?

DNA → mRNA → protein

The virus releases RNA, so the HIV rna must go backwards (reverse transcription) to make DNA before it can be integrated

This backwards step is what we inhibit today…

what is reverse transcriptase’s function?

Firstly it does the reverse transcription so rna → dna

destroys the original rna

then makes the dna double stranded

Jobs in order of reverse transcriptase?

1 - Rna-dependent dna polymerase reads the viral ssRNA template and makes DNA strand (RNA-DNA hybrid)

2 - Ribonuclease degreases the OG rna strand of the hybrid (leaves ssDNA)

3 - dna-dependent dna polymerase uses ssDNA as a template to make second strand dsDNA

Why is resistance so high?

the HIV reverse transcriptase is very sloppy so high error rate

no proofreading and also very fast so rapid mutation

(compared to dna polymerase)

do curly arrow mechanism

What are NRTI?

Nucleoside analogue RT inhibitor

It has no nucleoside

they must be activated via the addition of 3 phosphates in host cell

they act as chain terminator due to the absence of the 3’ oh-

Binds at active site

therefore work via chain termination

What are NtRTIs?

Nucleotide analogue RT inhibitor

They are a nucleotide as they already have one phosphate

Needs 2 phosphorylatios

Bind to active site

also work by chain termination

Advantage or NrRTI?

It skips the first phosphorylation step which is rate limiting (in NRTI)

What is the orthosteric site?

The classic enzyme active site where substrates bind

What are NNRTI?

Non-nucleoside RT inhibitor

these do not look like nucleosides or nucleotides and instead bind to allosteric pocket

they change the enzymes shape so the RT can no longer work as it can’t bind to the DNA/RNA

Why are mitochondria a weak point?

mtDNA is circular and semi-autonomously from nuclear DNA and replicated more frequency

it has no effective repair or histones to protect from reactive oxygen

so mtDNA is more vulnerable to mutations and to synthesis altering drugs

mtDNA encodes for oxidative phosphorylation (atp supply) so failure hits high energy organs

side effects linked to mitochondrial toxicity (hepatoxicity, cardiotoxicity)