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