Antiviral agents Flashcards
What is meant by selective toxicity?
An important quality for an antimicrobial drug is selective toxicity, meaning that it selectively kills or inhibits the growth of microbial targets while causing minimal or no harm to the host.
Tell me about the prospects of selective toxicity with viruses.
What do viruses hijack?
What do viruses possesss?
How will the drugs have to vary and why?
- Since viruses hijack the host cell’s machinery, the prospects for selective toxicity do not look good.
- But viruses are different! They possess their own structural proteins and some of their own enzymes.
- Need to understand the molecular details of the virus life cycle to look for potential targets.
- The differences will be virus-specific, so we are unlikely to find a “broad spectrum” antiviral as they are specific to structural proteins or genetics of target
Why is immunisation not very useful with viruses?
- Only prophylactic- once infected vaccination is too late as something that is prophylactic is intended to prevent disease
- New viral serotypes i.e., mutation
- Non-immunogenic- some viruses can’t have vaccines against them e.g., HIV
What is meant by a positive stranded virus?
Positive-strand RNA viruses are a group of related viruses that have positive-sense, single-stranded genomes made of ribonucleic acid.
The positive-sense genome can act as messenger RNA and can be directly translated into viral proteins by the host cell’s ribosomes
Give some examples of RNA containing, positive stranded viruses
- Picornaviruses e.g., genera Enterovirus (including Poliovirus and Rhinovirus)
- Coronavirus
- Hepatitis C
What is meant by a negative stranded virus?
Negative-strand RNA viruses are a group of related viruses that have negative-sense, single-stranded genomes made of ribonucleic acid.
They have genomes that act as complementary strands from which messenger RNA is synthesized by the viral enzyme RNA-dependent RNA polymerase
Give some examples of RNA containing, negative stranded viruses?
- Myoxviruses e.g., Influenza
- Paramyxoviruses e.g., Measles, mumps, rubella, RSV (respiratory syncytial virus)
- Rhabdoviruses e.g., Rabies
- Filoviridae e.g., Ebola
- Retroviruses e.g., HIV (Human immunodeficiency virus)
Give some examples of DNA containing viruses?
- Papoviruses e.g., Warts
- Adenoviruses
- Herpes e.g., Zoster, Simplex, Epstein Barr, CMV (Cytomegalovirus)
- Poxviruses e.g., smallpox
How do eukaryotic viruses work (tell me about DNA and RNA)?
In eukaryotic cells, most DNA viruses can replicate inside the nucleus, with an exception observed in the large DNA viruses, such as the poxviruses, that can replicate in the cytoplasm.
RNA viruses that infect animal cells often replicate in the cytoplasm.
What is tissue tropism?
Tissue tropism is the cells and tissues of a host that support growth of a particular virus or bacterium.
Some bacteria and viruses have a broad tissue tropism and can infect many types of cells and tissues.
Other viruses may infect primarily a single tissue. For example, rabies virus affects primarily neuronal tissue.
What are the stages to the virus life cycle?
- Adsorption
- Penetration
- Uncoating
- Processing
- Packaging
- Release
In the first stage of the viral life cycle, attachment/adsorption, what are the three types of entry?
- Membrane fusion
- Endocytosis
- Viral penetration
Whats Membrane fusion?
Provide examples of viruses that do this?
Fusion of viral envelope with membrane and capsid uptake e.g., influenza, HIV and Herpes
Whats endocytosis?
Provide an example of a virus that does this
In the absense of envelope, capsid enter by hijacking endocytotic pathways
e.g., polio
Whats Viral penetration?
Provide an example of a virus that does this
direct injection of nucleic acids
e.g., bacteriophage
Tell me general information about the attachment/ adsorption stage of the viral life cycle
- To enter by membrane fusion the virus must first attach to the cell membrane
- Attachment is achieved between proteins on the capsid or viral envelope and complementary proteins (often receptors) on the cell membrane
- Presence of these proteins determines which cells/tissues will get infected.
Tell me about the uncoating stage of the viral life cycle?
What it is and what is requires
- It may be pH dependent via proton channels (e.g., influenza, rhinovirus) Or require new protein synthesis e.g., pox viruses
- host enzymes expose the core (virus specific RNA polymerase → mRNA for uncoating protein → degrades core releasing naked DNA)- used for making protein which comes back and then uncoats the virus
- This second stage of uncoating requires protein synthesis
Some viruses encode proteins that help synthesise nuclic acid precursors, give an example fo this and what it does
- Some viruses encode proteins that help synthesise nucleic acid precursors
- For example, thymidine kinase (TK) is responsible for the phosphorylation of any nucleosides to their monophosphates e.g., adenosine –> adenosine monophosphate
- TK is also naturally present in the host cell
- Despite its name, TK works on all nucleosides (A, G, C, T, U)
Tell me about the replication stage of the DNA and RNA virus life cycle
DNA viruses
- Often code for their own DNA polymerase e.g., herpes
RNA viruses
- -ve stranded (e.g., influenza) require RNA replicase (RNA→RNA) to make a complimentary strand and we don’t have this enzyme as it’s a viral enzyme
- Retroviruses (e.g., HIV) use reverse transcriptase (RNA –> DNA) and we don’t have that enzyme either
Tell me about the processing stage of the viral life cycle
Often the mRNA is translated into one long polyprotein (non-functional), which is the cleaved to produce the individual viral proteins (functional) – done by a viral protease (e.g., polio, HIV, HCV)
Tell me about the packaging of viruses in the viral life cycle
- Assembling the complete viral particle
- Many contain multiple nucleic acid strands (e.g., influenza contains 8 RNA strands)
Tell me about the release of viruses in the viral life cycle
The virus buds from the surface of the cell (influenza)
Give some examples of viral targets and the processes they are involved in
- Structural proteins (absorption, uncoating)
- Proteases (uncoating, processing)
- Thymidine kinase a metabolic precursor
- Polymerases (DNA polymerase, RNA replicase, reverse transcriptase)
Tell me the typical viral lifecycle targets at each stage of the virus life cycle
Tell me some general facts about interferon?
Interferons are a group of signaling proteins made and released by host cells in response to the presence of several viruses.
- Extremely potent
- Viral interference discovered in 1935, could be passed from one cell to another
- Cell specific glycoproteins mw 20-30k
- Very active 10-13-10-14 M
- Not itself an antiviral agent, but induces an antiviral state (shutting down protein synthesis and degrading mRNA)
What is the synthesis of interfron stimulated by?
dsRNA
- artificial inducers e.g., polyI.polyC
- Toxic and subsequent hyporeactivity
Tell me the mechanism of action of IFNs?
What does interferon induce the synthesis of?
2-5A synthase
Tell me about the activation of 2-5A?
What is 2-5A made from?
What type of activator is 2-5A?
Interferon Induces the synthesis of 2-5A synthase
This is inactive unless dsRNA is present
2-5A synthase is made from ATP
2’5A is an allosteric activator of RNaseL (L means the enzyme is inactive unless sees the polymer on right)
2-5A makes the polymer ‘ase’ shown in the diagram
Cell won’t degrade unless it sees 2-5A and dsDNA- just treating with interferon won’t turn off
- Induces the synthesis of a protein kinase
- This is inactive unless dsRNA is present
- The activated kinase phosphorylates eIF2 thereby inhibiting protein synthesis
- Cells that are exposed BOTH interferon AND dsRNA degrade mRNA and stop protein synthesis
Tell me about the structure of the influenza virus
Based on the structure, how many different subtypes are there?
How do these subtypes occur?
- Variety of flu vary dependent on Haemagglutinin and neuraminidase
- There are 16 different variants of haemagglutinin and 9 of neuraminidase. So, 144 different combinations of flu
- Antigenic drift causes point mutations in the amino acid sequence
Antigenic drift refers to the gradual accumulation of point mutations during annual circulation of influenza as a consequence of the high error rates associated with RNA-dependent RNA polymerase during virus replication.
How are new virus particles attached to influenza?
New virus particles are attached to the cell surface by interaction between haemagglutinin (on the virus) and sialic acid residues on the surface of the infected (host) cell.
What type of compound is Sialic acid?
A sugar
Sialic acids are a class of alpha-keto acid sugars with a nine-carbon backbone. … Sialic acids are commonly part of glycoproteins, glycolipids or gangliosides, where they decorate the end of sugar chains at the surface of cells or soluble proteins.
How are new virus particles released from the influenza?
The new virus particles are released from the surface by the action of neuraminidase (sialidase) – cleaving the terminal sialic acid residues from the remainder of the oligosaccharide chains (glycolipid or glycoprotein)
What does Neuraminidase cleave?
Neuraminidases are enzymes that cleave sialic acid from glycoproteins via the terminal sialic acid residue
What are the two forms of attachment that the sialic acid at the terminus of the cell surface glycoprotein can attach to the preceding sugar (galactose) via?
Tell me what type of neuraminidases are best for each attachment and where there are more common
SAα2,6Gal (human virus cleave best - found in nasal mucosa- upper respiratory tract)
SAα2,3Gal (avian virus cleave best – located in lower respiratory tract)
Tell me about the drug design for influenza…
- sialic acid at top. To cleave this bond, you start to flatten the structure
- The oxygen takes a +ve charge
- Make analogues that resemble the transition state. Put the double bond somewhere else on the ring rather than on the oxonium ion
- Via small chemical tweaks to structure you can change hydroxyl –> amino group
- Then make it even bigger by adding a guanidino group which means more nitrogens and more interaction
- Relenza is a good inhibitor of neuraminidase
- Problem with relenza is that its insoluble so can’t give orally, have to give via aerosol
Tell me some drugs developed to help treat influenza
As seen in the image the drugs relenza and tamiflu are sialic acid analogues (similar structure) and neuraminidase inhibitors (neuraminidase (NA) glycoprotein being responsible for cleaving the receptor to allow virus release)
Tell me some further drugs that have been developed against influenza
Peramivir still works against some viruses that have become resistant to Tamiflu and relenza
Tell me how the influenza virus generates resistance against tamiflu
Resistance may be specific to a drug and strain. Tell me about the H274Y mutation in the influenza virus and resistance
Name a drug that used to be used to treat influenza A
How did it work?
Amantadine
- Active against A not B
- Target: membrane protein M2 (ion, proton, channel involved in uncoating)
How did viruses become resistant to amantadine?
Through single amino acid changes at positions 26, 27, 30, 31 or 34
Where does the M2 channel sit?
In the capsid of the virus
Tell me about the M2 channel from influenza A and the amino acids roles in this channel
Name some other uncoating inhibitors for other viruses that are influenza
How do they work?
What is meant by the term “Cap Snatching”?
Viruses “steal” the cap structure from cellular mRNA
Why is the process of “cap snatching” useful for viruses?
Tell me the steps to this process
- Viruses “steal” the cap structure from cellular mRNA
- The cap-binding domain of the viral polymerase recognizes the cap of host mRNAs and cleaves 10–20 nucleotides downstream.
- The short-capped RNAs are then used as primers for viral mRNA synthesis by the viral polymerase.
- De-capping of cellular mRNA blocks the expression of cellular RNA while promoting the expression of viral RNAs.
- Capped with guanosine at 5’ end. often mesylated. Protects RNA and interaction with RNA initiation factors
Name two drugs developed which are inhibitors of the cap snatching mechanism. They are influenza antiviral drugs.
Tell me how the inhibitors of the cap snatching mechanism work? (what do they target?)
- These target the PB2 subunit of the viral polymerase and act by preventing the polymerase from binding the 7-methyl GTP cap structures on host pre-mRNAs.
- Inactive against influenza B virus due to structural differences in the PB2 cap-binding pocket. (J. Med. Chem. 57, 6668−6678)
- Mutations in PB2 confer resistance to pimodivir (Antimicrob. Agents Chemother. 59, 1569−1582.)
- These are for influenza A (not B)
What are Nucleoside analogues useful for when treating influenza infections?
- Look for analogues that are substrates for viral (not human) enzymes
- Note: nucleosides (not nucleotides, which are charged).
- Many used for treating herpes virus infections
- Monophosphate is a nucleotide, and you add a hydroxyl group to make a nucleoside
Name some nucleoside analogues
Idoxuridine
Vidarabine (Ara-A)
Tell me about Idoxuridine
Idoxuridine is similar to ‘T’ just has an ‘I’ present. Gets incorporated as if it’s a T in TTP
Tell me about Vidarabine (Ara-A)
Vidarabine is an analogue for ‘A’- competes with A. but not very selective or effective between virus and humans
Name some early nucleoside analogues
Acyclovir
Gancyclovir
Cidofovir
Ribavirin
An early nucleoside analogue is Acyclovir, tell me about this
- Guanine analogue, missing the 2’ and 3’- carbons
- First phosphorylated by the viral thymidine kinase
- Not a substrate for human TK (thymidine kinase)
- The monophosphate is converted to the di- and triphosphate by cellular enzyme
- AcyTP is a substrate for the viral (not human) DNA polymerases.
Acy –> AcyMP –> AcyDP –> AcyTP –> DNA
v c c v
How does Acyclovir work?
- Causes chain termination (missing the 2’ and 3’-OH)
- Competitive with G
- Active against Herpes simplex and zoster
IC50 ~ 0.1 - 1 µM
(cf IC50 for human DNA polymerase ~ 300 µM)
- Not active against CMV (cytomegalovirus) (does not possess its own thymine kinase)
- Resistance mutations in viral TK or viral DNA pol
- TK- mutants are less pathogenic
- Those undergoing immune suppression like cancer therapy, can have severe effects from CMV
How does Gancyclovir work?
- Active against CMV
- Another guanine analogue but missing just the 2’ -sugar carbon
- Active against CMV
- First phosphorylated but by a CMV virally encoded kinase (UL97 kinase)
- Still contains a 3’-sugar carbon so chain termination occurs by altering the conformation of replication complex
- Preferentially inhibits viral DNA polymerases more than cellular DNA polymerases
Tell me the following about Cidofovir:
What type of compound is it and therefore what does it not require
Where does chain termination occur
What does it act on
- Cytosine nucleoside phosphonate that does not require phosphorylation to its monophosphate
- Chain termination occurs after two successive additions of the nucleoside
- Acts on viral DNA polymerases 800 times better than human enzyme
- [Also, probably active against smallpox in the event of bioterrorism]
- Used against CMV
What is Ribavitin active against?
Both DNA and RNA containing viruses
Tell me the mechanism of action of Ribavirin against the DNA and RNA viruses
DNA: mechanism still unclear
RNA: It inhibits RNA-dependent RNA polymerase (mimicking RNA nucleotides- pairing with C or U)
Why is Ribavirin able to pait with C or U?
- The side chain on ribavirin can rotate. One pairs with C and then one pairs with U
- Mutagenic catastrophe (to work against RNA viruses by getting it to pair with anything)
What does Ribavirin do when used to treat against DNA viruses?
- Also blocks 5’-end capping of mRNA (as similar to G)
- But these do not explain its selectivity
- Ribavirin is a competitive inhibitor of IMP –> XMP
What drug is the closest thing we have to a broad-spectrum antiviral spectrum?
Ribavirin
Are Phosphonoformate (foscarnet) and Phosphonoacetate nucleoside analogues?
No
Foscarnet structure
What is phosphonoacetate active against?
Tell me the type of inhibition it has
What is the binding site?
What is it active against?
Name another drug that is used as an inhibitor against CMV
Letermovir
What is Letermovir an inhibitor of?
Its an inhibitor of the viral ‘Terminase’ complex
How is new viral DNA synthesised?
What happens after this stage?
- New viral DNA is synthesized as a long DNA chain of concatamers (a molecule made up of multiple copies of the same genome strung together in tandem)
- Maturation, packaging, and termination is performed by a group of proteins known as the “terminase complex”.
- The CMV terminase complex contains two proteins, pUL89 and pUL56.
- The main function of the terminase complex is to cleave CMV concatamers into single units of functional CMV monomers.
- The structure of the terminase complex is conserved across members of herpesviruses, but it is not shared with human cells
What is Hepatatis C?
A negative stranded RNA virus
consisting of around 9,600 bases
What does Hepatitis C produce?
A polyprotein of about 3,000 amino acids
Name 6 hepatitis C non-structural proteins?
- NS2
- NS3
- NS4A
- NS4B
- NS5A
- NS5B
What non-structural proteins of Hepatitis C are proteases?
NS2 and NS3
What non-structural proteins of Hepatitis C are cofactors for NS3?
NS4A
What non-structural proteins of Hepatitis C are important for changes in the cellular membranes?
NS4B
What non-structural proteins of Hepatitis C have no known enzyme function (regulator of replication)?
NS5A
What non-structural proteins of Hepatitis C is an RNA-dependent RNA polymerase (RNA replicase)?
NS5B
What is RNA replicase important for, however what are the downsides of this enzyme?
RNA dependent RNA polymerase (RNA replicase)- important for antiviral agents. The viral RNA replicase has no proof-reading activity so they make mistakes (this happens frequently)
Tell me about the mistakes that the NS5B has and why this occurs
- High rate of turnover 1012 viruses per day
- High mutation frequency
- ‘Every possible pre-existing mutation is present within any given patient prior to antiviral therapy’
Tell me the agents for targeting RNA replicase
Sofobuvir (GS-7977)
HCV-CS5A
What does Sofobuvir inhibit?
How does it do this function?
What is it often used in combination with?
- Hepatitis C inhibitor of RNA dependent RNA polymerase NS5B
- Thymine nucleoside analogue given as a prodrug (ProTide) metabolised to its monophosphate
- Substrate for RNA replicase and inhibits RNA replication (presence of fluorine)
- Used in combination with interferon and ribavirin or with daclatasvir for Hep. C infection
Tell me about the structure of the drug HCV-NS5A
- made up of 447 amino acids
- Has 3 domains
Tell me the role of each of the domains of HCV-NS5A
I – RNA binding motifs (tethers HCV to intracellular membranes?)- one to know about
II – binds various host factors (linked to RNA replication)
III – important for virus assembly, but not replication
Name some drugs that are used for the inhibition of domain 1 in HCV-NS5A
What type of virus is Hepatitis B?
A dsDNA virus
Name two drugs used for the treatment of Hep B?
How do they work?
Telbivudine and Adefovir
They both work via chain termination, acting on the viral DNA polymerase
What mutation in Hep B has been associated with Telbivudine resistance?
Resistance mutation M204I in the reverse transcriptase domain of the hepatitis B polymerase or L180M+M204V have been associated with Telbivudine resistance.
What method does Hep B copy its genome via?
- Hep B is a DNA containing virus which goes via a reverse transcriptase
- Hep B copied from DNA genome –> RNA (making new viral genome via reverse transcribing the RNA that has been transcribed from it)
Name two HCV (Hep C virus) protease inhibitors?
Telapravir
Bocepravir
Tell me about Telapravir
Tell me about Bocepravir
Tell me about the srtructure of Ebola
- Single-stranded, negative-sense RNA (has to be copied into complementary RNA which is used as the message to make viral proteins)
- 18,959 to 18,961 nucleotides in length
How many genes does the viral genome of Ebola encode?
Name them
The viral genome encodes 8 genes:
- nucleoprotein (NP)
- VP35
- VP40
- EBOV glycoprotein (GP)
- Soluble glycoprotein (sGP)
- VP30
- VP24
- RNA-dependent RNA polymerase (L)
Name some drugs that can be used to inhibit the replication of the Ebola virus
- Quercetin derivative
- FGI-106
- Favipiravir
- Brincidifovir
Name another drug which is an inhibitor of RNA-dependent RNA polymerase
Where does termination occur?
Name a drug that is the selective inhibiton of RdRNP, but does not cause inhibit of RNA polymerase
Tell me how Remdesivir is used against Ebola virus
- RNA containing sugar
- Has C attached to sugar (not N)
- Distorted A structure
- Pro-drug
- Phosphoramidate
- When cleaved the free nucleoside is released
- Metabolised to triphosphate which acts as a substrate for the RNA dependent RNA polymerase for diseases like the Ebola virus
Name some more drugs used against Ebola
Antisense oligonucleotides
- AVI-7537 (Against VP24)
- Piece of DNA that is designed to be complementary to the RNA of one of the viral proteins
siRNA (TKM-Ebola)
- combination of three siRNA
- molecules that targeted RNA-dependent RNA polymerase, virion protein 24 (VP24)
- and virion protein 35 (VP35)
Drugs with ‘unknown action’
- FGI-103, FGI-104
Tell me about the structure of coronavirus-SARS-Cov-2
Single stranded, positive sense (the genome of the virus as it infects the cell is an mRNA that can be translated to make viral proteins),
RNA genome (29-30kb) (the bigger the RNA genome the more likely mistakes are made as there are no proof reading activities, however corona has some proof reading ability)
In the Coronavirus structure what codes for the non-structural proteins 1a and 1b?
21kb at the 5’-end
What is this large polyprotein or coronavirus cleaved into and what is this done by?
They are cleaved by proteases into mature proteins
What does the next gene of coronavirus (at roughly 3.8kb) encode?
The spike (S) protein
name the other structural proteins and where are the encoded in coronavirus?
The other structural proteins (i.e., forming part of the virus particle) envelope (E), membrane (M) and nucleocapsid (N) proteins are encoded towards the 3’ end of the genome.
Tell me about the usefulness of the drug remdesivir is against coronavirus
Tell me what unusual about coronavirus
Coronaviruses (CoVs) have unusually large genomes (∼30 kb) and low mutation rates.
What does CoVs protein nsp14 have and what is their role?
CoVs protein nsp14, has RNA cap methylation activity (guanine N7-methyltransferase) and is also a 3′-5′ exoribonuclease (ExoN) that removes mismatched nucleotides at the RNA 3′-end.
What does mutation of the ExoN give?
Mutation of ExoN gives high rates of mutation and affects viral genetic stability.
What activity does nsp14 and nsp12 have?
nsp14 and nsp12 (the RNA polymerase) therefore have a proof-reading activity.
What protein is Ribavirin a substarte for?
Why is it not active against Covid?
Ribavirin is a substrate for nsp12, but it is excised by nsp14 – so is not active against CoVs
Why isn’t remdesivir excised by nsp14 ?
nsp14 and nsp12 (the RNA polymerase) therefore have a proof-reading activity.
Ribavirin is a substrate for nsp12, but it is excised by nsp14 – so is not active against CoVs
However, remdesivir doesn’t stop replication until i+3, so it isn’t excised
Unlike most nucleotide analogues remdesivir is more efficiently incorporated than ATP
Tell me about a drug that has recently been approved for use against COVID
Summary for lectures 1-3
For treating Influenza:
- Tamiflu and Relenza
- Neuraminidase inhibitors that prevent adsorption and release
- Amantadine
- M2 proton pump inhibitor that prevents uncoating
For treating DNA-containing viruses
- Aciclovir/Gancicovir/Cidofovir (Herpes)
- Nucleoside inhibitors of DNA polymerase (chain termination)
- Phosphonoformate/phosphonoacetate (Herpes)
- Non-nucleoside inhibitors of DNA polymerase
For treating RNA-containing viruses
- Ribavirin (‘broad spectrum’)
- Nucleoside inhibitor of IMG (DNA) and RNA replicase (RNA)
- Sofosbuvir/BX4430/Remdesivir (Hepatitis C/Ebola/Coronavirus)
- Nucleoside inhibitors of RNA replicase
Tell me the general features included in the structure of the HIV virus
The below diagram shows the rought life cycle of HIV
Also includes some inhibitos involved in the life cycle
Tell me the stages to how a HIV particle is adsorped to the host cell
The human immunodeficiency virus type 1 (HIV-1) gp120 exterior glycoprotein is conformationally flexible.
Upon binding the host cell receptor, CD4, gp120 assumes a conformation that is able to bind the chemokine receptors CCR5 or CXCR4, which act as coreceptors for the virus.
How do sulphated polysaccharides like heparin influence HIV adsorption?
Dextran sulfate, heparin, and certain other sulfated polysaccharides potently inhibit the adsorption of HIV to CD4+ cells.
The mechanism of this inhibition is unclear and, specifically, it is unknown if these agents act at the level of CD4-gp120 binding.
Tell me the effect that the compound Hyperricin and Pseudohyperricin have on HIV
The constituents hypericin and pseudohypericin effectively inhibit infection by a number of enveloped viruses of medical importance, including human immunodeficiency virus-1 (HIV), herpes simplex virus (HSV) and influenza A virus
Confer stability on the viral envelope
Name a drug that targets the CCR5 receptor antagonist?
What effect does it have?
MARAVIROC – CELSENTRI
Targets the CCR5 receptor antagonist (blocks entry of virus into cell by stopping interaction)
http://www.youtube.com/watch?v=95J4dLHXEzM
CCR5 is used as a co-receptor for HIV for getting the virus into the cell
[Gene modification to knockout CCR5 done in 2018 by a Chinese scientist]
Name two more inhibitors of HIV
Enfuvirtide (Fuzeon)
Lenacapavir (GS-6207)
Tell me about enfuvirtide (fuzeon)
What part of the HIV lifecycle does it inhibit
What does it bind to
Where is resistance seen
Tell me about Lenacapavir (GS-6207)
What part of the HIV lifecycle does it inhibit?
Tell me its EC50 value
What is HIV reverse transcriptase (RT) used to do?
Convert ssRNA to dsDNA which can integrate into the host genome
Tell me the three sequential HIV RT activities
(i) RNA-dependent DNA polymerase activity
(ii) RNase H activity (RNase H degrades RNA hybrid)
(ii) DNA-dependent DNA polymerase activity (dsDNA that matches ssRNA which was in viral genome)
What part of the HIV is the RT part of and what is it encoded by?
Part of the virion and encoded by the pol gene
What type of protein is RT and how big is it in kDa?
Mature protein is a dimer of 51 kDa and 66 kDa
What does HIV RT use as a primer?
tRNA-Lys
What does resistance emerge easily in HIV RT?
RT has no proof-reading activity so very error prone: 5-10 mutations per genome replication
Consequently, resistance emerges very easily
What are anti-HIV agents never used in?
Anti-HIV agents are never used in monotherapy they are only ever used in combination
Name some drugs that are clinically useful for treating HIV
Explain structure-activity relationships between AZT and ddT
AZT, Retrovir= An anti-HIV drug called a nucleoside analogue ‘nuke’
ddT, Dichlorodiphenyltrichloroethane= an insecticide used in agriculture
Where does reverse transcriptase work in the following reaction:
Z –> ZMP –> ZDP –> ZTP –> DNA
What are the above phosphorylation steps done by?
What is meant by a pro-drug?
Pro-drugs are molecules with little or no pharmacological activity that are converted to the active parent drug by enzymatic or chemical reactions or by a combination of the two
In the following reactions, what is the active form?
ZTP is the active form which is incorporated into the DNA by the RT
For most drugs, addition of the first phosphate is what?
However, how does this differ from AZT?
For most drugs addition of the first phosphate is rate-limiting step
BUT not for AZT - addition of the second phosphate is the rate limiting step.
What is the rate-limiting step of a reaction?
The RLS of a chemical reaction is defines as the slowest step out of all the steps that occur for a given chemical reaction
Some are metabolised to other nucleotides
ddI → ddIMP → ddAMP → →→
ABC → ABC-MP → guanosine derivative → →→
Z → ZMP → ZDP → ZTP → DNA
How do all of these reactions work by?
Chain termination
What is often the cause of a rapid emergence of resistance?
due to a rapid mutation rate
What can HIV do to become resistant to AZT?
What type of mutations are these?
- Reduced incorporation of the NTP
- Increased excision of the nucleotide analogue – removing the block
The HIV will acquire resistance to AZT by aquiring mutations in the RT
Some resistance to AZT requires multiple mutations – most are single point mutations
Tell me about increased excision and resistance to AZT
AZT and D4T M41L, D67N, K70R (conservative mutation), L210W, T215F (remove inhibiting terminator at the end), K219Q (cumulative high resistance); Thymine Analogue Mutants – TAMs
(Isolated enzymes show no change in sensitivity to AZTTP)
Tell me about descrimination and AZT resisitance
What can suppress this resistance?
ddI K65R, L74V; 3TC K65R, M184V
ABC K65R, L74V, Y115F, M184V
TFV K65R, K70E; FTC K65R, M184V
K65R or L74V suppress AZT resistance.
What do non-nucleotide reverse transcriptase inhibitors (NNRTIs) bind to?
Hydrophobic pocket on the surface of the enzyme (allosteric site)- not the active site
http://www.youtube.com/watch?v=h7V1eVwxV_c
What are the benefits of non-nucleotide reverse transcriptase inhibitors (NNRTIs)?
- No metabolic activation
- High affinity (IC50 values ~ low nM)
- Low toxicity (no equivalent cellular target)
- Non-competitive inhibition
Give some examples of NNRTIs
What type of HIV are they used against?
Why dont NNRTIs work against HIV-2, only HIV-1?
Hydrophobic pocket they bind to is not present in HIV-2, only HIV-1 hence why they don’t work against HIV-2
Is resistance rapid in NNRTIs, if so why?
Give examples where this is the case?
Very rapid emergence of resistance as the site is not necessary for catalysis
Especially K103N and Y181C (these are NNRTI mutations)
These are not active against HIV-2
What generation NNRTI is etravirine?
What is it active against?
Why is it able to bind multiple RT?
Etravirine (TMC125) is a second-generation NNRTI
active against HIV mutations that confer resistance to K103N for efavirenz (Y181C for nevirapine).
Flexible molecule that can bind reverse transcriptase in multiple conformations, allowing interaction even in the presence of mutations
Adapt to structure of region its binding to
Resistance…
Name a 3rd generation NNRTI, what is it active against?
Compare Doravirine (MK-1439 (Pifeltro)) to the following…
Efavirenz
Rilpivarine
Compared to Efavirenz
- 33-fold more active against K103N and 59-fold more potent against K103N/Y181C.
Compared to rilpivarine
- 5 and 7-fold more potent against
- Y181C and K103N/ Y181C.
What does the NH-N bond on Doravirina interact with?
K103
What is Delstrigo?
Delstrigo – a combination tablet with lamivudine, and tenofovir
What is the role of HIV integrase (IN)?
The role of HIV integrase is to integrate dsDNA into the host genome
When the process of integration starts to occur in a cell, what does this mean about what has to occur next?
What is the term used to describe what is made?
Integration is a point of no return for the cell, which becomes a permanent carrier of the viral genome (provirus)
Tell me the two sequential biochemical activites that HIV integrase undertakes
Has two sequential biochemical activities:
- 3’-processing to expose an invariant CA dinucleotide at both 3’-ends
- Cleavage + ligation into the host genome
What are both reactions of the HIV integrase catalysed by?
Both reactions are catalyzed by the same active site and occur via transesterification (water/magnesium)
What is HIV integrase a part of and what is it encoded by?
Part of the virion and encoded by the pol gene
Tell me the type of protein that HIV integrase is and what is its size?
Mature protein is a monomer of 32kDa (contains DNA binding domain)
Name an HIV inegrase inhibitor which has been licensed back in 2007
How does it work?
Mutations of the HIV integrase give resistance, but single mutations may reduce the enzyme’s activity. Give some examples of mutations that can occur in HIV integrase
What double mutants can occur?
Mutations
N155H
Q148H/R/K
Y143R/C/H
Double mutants
N155H with E92Q
Q148H with G140S
What is the oral dose of Raltegravir for HIV
How often does it need to be given
What is the outcome of taking this drug
Oral dosage (400 mg twice a day)
Reduces viral load below detection within few weeks
What type of enzyme is HIV protease?
a HIV encoded enzyme
What does HIV protease do?
It splices the poly protein made
Tell me some characteristics of HIV protease?
Really potent and anti-viral
What type of protease is HIV protease?
Why is this the case?
HIV protease is an Aspartyl protease (aspartic acid is the critical AA for the cleaving activity)
What encodes HIV protease?
The 5’ end of the pol gene
Tell me the structure and common sequence in aspartic proteases?
Dimer (two identical subunits which dimerise together) with one active site at the dimer interface and has the characteristic Asp-Thr-Gly (Asp25, Thr25 and Gly27) sequence common to aspartic proteases
Where does HIV protease cleave?
Cuts poly protein in 8 regions (creating 9 products)
Cuts at ‘*’ region (shown below)
What does HIV proteases recognise as opposed to individual AA?
it recognises particular structures
What is one of the substrates of the HIV integrase?
Phe-*-Pro
What do you want the inhibitors of HIV integrase to have a similar structure to?
The transitional state compound structure
Give some examples of protease inhibitors
Tell me the fold resistance of the following
L90M
G48V
BOTH L90M and G48V
What does this mean…
L90M – 3-fold resistance
G48V- 13.5-fold resistance
Both L90M and G48V – 419-fold resistance
Want to ensure double mutants don’t occur
What locations can resistance occur, provide an example
Resistance can be in…
- active site
- dimer interface
- in the substrate
e.g., Phe-Leu to Phe-Phe at p1-p6 gag
In general what do single point mutations give in sensitivity and secondary mutations to resistance?
In general, single point mutations give 5-fold difference in sensitivity; secondary mutations give high levels of resistance
What should be given to prevent the emergence of resistant strains?
High dose of inhibitors should be used (IC90- conc. Inhibiting 90% of growth) to give better response and to prevent the emergence of resistant strains.
What is bevirimat?
An anti-HIV drug
What does bevirimat bind to?
What does it inhibit?
Binds to polyprotein in site which would usually be cleaved also binds to the GAG protein and prevents maturation
So inhibits cleavage of where capsid protein would usually be made
Specifically, inhibits cleavage of the capsid protein (CA) from the SP1 spacer protein
Tell me about a type of combination therapy known as; highly active anti-retroviral therapy (HAART)
What is it and give an example of the most common type
Simultaneous treatment with three or four antiretroviral drugs to prevent the emergence of resistance.
The most common drug combination given to those beginning treatment consists of two NRTIs (Zidovudine and Lamivudine) combined with either an NNRTI (Efavirenz) or a protease inhibitor (Ritonavir)
Summary for treating HIV
Maraviroc
- Fusion inhibitor that prevents adsorption and entry
Zidovudine, Zalcitibane, Didanosine, etc.
- Nucleoside reverse transcriptase inhibitors (chain termination)
Nevaripine, Efavirenz, etc.
- Non-nucleoside reverse transcriptase inhibitors (allosteric)
Raltegravir
- Integrase inhibitor (active site)
Indinavir, Ritonavir, Berivamat, etc.
- Protease inhibitors (direct or indirect)
HAART
- Combination of NRTI/NNRTI/Integrase/Protease inhibitors
Antiviral summary
