Mechanism of Antivirals

Question 1

List some different types of antimicrobials.

Answer 1

• Antibiotics
• Antivirals
• Anti-fungals
• Anti-protosoals
• Anti-helminths

Question 2

Why do we need antivirals?

Answer 2

There are many different types of viral disease with detrimental effects:

• quick killers e.g. influenza; EBOLA; MERS; SARS
• slow, progressive chronic disease leading to things like cancer (hepatitis B [350,000,000 carriers],
  hepatitis C [200,000,000 carriers],
  human papilloma viruses [cervical cancer, second commonest cancer in women])
• highly infectious (human immunodeficiency virus (HIV) [40 million infected])
• acute imflammatory diseases e.g. herpes

Question 3

List some uses of antivirals.

Answer 3

• treatment of acute infection (influenza; chickenpox; herpes infections - [with aciclovir])
• treatment/control of chronic infection (HCV, HBV, HIV [numerous different agents])
• post-exposure prophylaxis and preventing infection (HIV [PEP])
• pre-exposure prophylaxis (HIV [PrEP])
• prophylaxis for reactivated infection (e.g. in transplantation when you are immunocompromised; CMV [ganciclovir, foscarnet])

Question 4

List some principles of selective toxicity of antivirals as therapeutic agents.

Answer 4

It is based on the differences in structure and metabolic pathways between host and pathogen.

It harms the microorganisms, not the host.

We want the target to be in the microbe, not the host (if possible).

Selective toxicity is difficult for viruses (intracellular), fungi and parasites because they’re intracellular organisms.

We need to understand that there is variation between microbes, strains within the same species.

Question 5

Why is it so difficult to develop effective, non-toxic anti-viral drugs ?

Answer 5

• viruses enter cells using cellular receptors which may have other functions
• viruses must replicate inside cells – this obligates intracellular parasites
• viruses take over the host cell replicative machinery, so it is hard not to harm the cell as well
• viruses have high a mutation rate - this creates quasispecies (a large number of mutants of the same isolate of virus)
• antivirals must be selective in their toxicity i.e. exert their action only on infected cells
• some viruses are able to remain in a latent state so it’s harder to remove them as they are not presenting themselves e.g. herpes, HPV
• some viruses are able to integrate their genetic material into host cells e.g. HIV

Question 6

Briefly, recap the virus life cycle.

Answer 6

Essentially, the virus comes in and attaches to the membrane, and then get internalized, either by the mechanism of endocytosis or by membrane fusion. This means there are processes that associate the virus with the cell membrane, so this is a potential site of drug activity.

Once the virus is inside, it has to un-coat and release its genome. That genome has to replicate itself and also has to make mRNA, and goes to the ribosomes of the cell, where it starts to make viral proteins.

The virus will then reassemble, either by budding through the membrane, or they assemble completely inside the cell and get out via lysis of the cell.

Question 7

What are some considerations in developing safe antivirals?

Answer 7

Can certain stages of infection be targeted?

Cellular receptors may have other important functions.

Viral enzymes may be very similar to host enzymes.

Blocking the cellular enzyme may kill the cell.

Question 8

What are some modes of action of selected antivirals?

Answer 8

• preventing virus adsorption onto host
• preventing penetration
• preventing viral nucleic acid replication (nucleoside analogues)
• preventing maturation of virus
• preventing virus release

Question 9

List some examples of antivirals and how they work.

Answer 9

Amantadine blocks the process of uncoating. It’s an effective anti-flu drug, but it has a lot of toxicity associated with it.

Acyclovir, Ganciclovir and Ribavirin inhibit nucleic acid polymerisation, by targeting either reverse transcriptases or DNA polymerases.

Ribavirin blocks the access and is an analogue of GTP, and compromises genome replication.

Interferons inhibit the synthesis of RNA or the replication of the genome.

Protease inhibitors block the process of particle maturation and assembly of protein particles that are generated from the genome of the virus.

Zanamivir blocks the release of the virus from the cell.

Question 10

What are some examples of selective toxicity viral targets?

Answer 10

The discovery of virally encoded enzymes sufficiently different from their human counterparts means that they can act as selective targets with minimal effect on the host enzymes or processes.

Examples are:

• thymidine kinases (used for treating herpes-like infections) of HSV/VZV/CMV
• proteases of HIV
• reverse transcriptases of HIV
• DNA polymerases
• neuraminidases of influenza virus

Question 11

List the drugs that treat herpes viruses.

Answer 11

Aciclovir:

• for HSV, VZV treatment/ prophylaxis of CMV
• CMV/EBV prophylaxis

GANCICLOVIR:
- for CMV

FOSCARNET:
- for CMV

CIDOFIR:
- for CMV

Question 12

Describe the mechanism of action of Aciclovir.

Answer 12

Aciclovir is an analogue of GTP. It is missing it’s sugar phosphate ring, so it would be called a ‘chain terminator’. It will be inserted into the DNA and prevents it from polymerising.

First, it has to be activated.

It is first phosphorylated by a viral thymidine kinase (TK), then it gets di- and tri-phosphorylated by cellular kinases. It is only active in the tri-phosphate form.

It then becomes a competitive inhibitor of the viral DNA Polymerase. It will compete for standard GTP, and will stop the viral Polymerase from synthesising the viral genome.

Question 13

What two principles give Aciclovir selective toxicity?

Answer 13

• It is selectively activated inside cells that are infected; other cells do have their own thymidine kinase, but it has very poor activity against the acyclovir molecule.
• It is also selectively inhibits only viral DNA Polymerase, as it is 30 times more effective against them.

Question 14

Why is Aciclovir so effective and safe?

Answer 14

HSV thymidine kinase (TK) has 100x the affinity for Aciclovir compared with cellular phosphokinases.

Aciclovir triphosphate has 30x the affinity for HSV DNA polymerase compared with cellular DNA polymerase.

Aciclovir triphosphate is a highly polar compound - difficult to leave or enter cells (but Aciclovir is easily taken into cells prior to phosphorylation) thus, it will accumulate in cells that are infected.

It is a DNA chain terminator.

Question 15

What is Aciclovir used to treat?

Answer 15

HERPES SIMPLEX:

• treatment of encephalitis
• treatment of genital infection
• suppressive therapy for recurrent genital herpes

VARICELLA ZOSTER VIRUS:

• treatment of chickenpox
• treatment of shingles
• prophylaxis of chickenpox

CMV/EBV
- prophylaxis only

Question 16

Describe Ganciclovir.

Answer 16

It is effective against CMV:

• reactivated infection or prophylaxis in organ transplant recipients
• congenital infection in newborn
• retinitis in immunosuppressed

Ganciclovir is structurally similar to Aciclovir.
CMV does not encode TK (so it can’t activate Aciclovir) but it has UL97 kinase (or CMV phosphotransferase), which can activate Ganciclovir a bit better than it can Aciclovir.
It inhibits CMV DNA polymerase.

Question 17

Describe the mechanism of action by which Ganciclovir works.

Answer 17

Gancoclovir is taken up into infected cells. It is then, like Aciclovir, mono-, di-, and tri-phosphorylated to its active form. The first phosphorylation occurs via CMV phosphotransferase [UL97], and the subsequent phosphorylations occur via cellular kinases.

It then acts just like acyclovir, competing for the natural substrate for DNA Polymerase, and blocks the ability of the virus to make its own DNA.

Question 18

Briefly, describe Foscarnet.

Answer 18

It selectively inhibits viral DNA/RNA polymerases.

It does not require cellular activation.
It binds the pyrophosphate binding site – it is a structural mimic (thus it blocks the ability of the DNA Polymerase to polymerise and make DNA).

It is used for CMV infection in the immunocompromised e.g. pneumonia in solid organ and bone marrow transplants.

It may be used because of Ganciclovir resistance (due to TK mutants of CMV).

Question 19

Briefly, describe Cidofovir.

Answer 19

It is a chain terminator - it targets DNA Polymerase.
It competes with dCTP.

It is a monophosphate nucleotide analogue.
It’s a prodrug – it needs to be phosphorylated by cellular kinases to its di-phosphate form.

The drug is active against CMV, but MUCH MORE nephrotoxic that its alternatives.

It’s used for treatment of retinitis in HIV disease.

Question 20

How does resistance to antivirals in herpes viruses come about?

Answer 20

There are two main mechanisms:

• Thymidine Kinase mutants (so the prodrug cannot get activated)
• DNA polymerase mutants (so active drug can no longer bind)

If this occurs in TK, drugs not needing phosphorylation are still effective (e.g. foscarnet, cidofovir).
If it occurs in DNA Polymerase, all drugs will be rendered less effective.

This is VERY RARE in immune competent patients (due to the low viral load).

Question 21

List some structural features of HIV.

Answer 21

• reverse transcriptase
• ds RNA genome
• viral envelope
• envelope protein: gp120, with transmembrane protein: gp41
• membrane-associated matrix protein: Gag 17
• nucleocapsid protein: Gag p24

Question 22

List the 7 steps in the life cycle of HIV.

Answer 22

1) Attachment with binding of viral gp120 via CD4 and CCRX.

2) Reverse transcription of RNA
into dsDNA.

3) Integration into host chromosome of
proviral DNA.

4) Transcription of viral genes.
5) Translation of viral mRNA into viral proteins.
6) Virus assembly and release by budding.
7) Post-release maturation.

Question 23

List some anti-HIV drugs.

Answer 23

1) Anti-reverse transcriptase inhibitors:
- nukes (nucleoside/nucleotide RT inhibitors)
- non-nukes (non-nucleotide RT inhibitors [allosteric])

2) Protease inhibitors - multiple types

3) Integrase inhibitors:
the virus has a POL gene, which codes for three separate components: protease, reverse transcriptase and integrase (IN). The 3´end encodes for IN. (polynucleotidyl transferase allows for the cross-linking of the viral DNA with the host DNA)
The inhibitor will target the integrase in this gene, this means that the viral DNA will not be permanently established in the germline.

4. Fusion inhibitors:
   gp120/41 are transmembrane components that are involved in the membrane fusion, which allow the particle to be internalised - the drug is a biomimetic lipopeptide which blocks the process of fusion.

Question 24

What therapy do we use to treat HIV?

Answer 24

Treatment - HAART (highly active anti-retroviral therapy)

When treating HIV, we normally use a combination of drugs, because if we just use one that targets only one of the mechanisms, the virus can overcome that by rapid mutation and become resistant.

Question 25

Describe AZT (Zidovudine) as an example of a ‘nuke’.

Answer 25

Zidovudine is 3’-azido-3’deoxythymidime.

It is a synthetic analogue of nucleoside thymidine – when converted to tri-nucleotide by cell enzymes, it blocks RT by:

• competing for natural nucleotide substrate dTTP
• incorporation into DNA causing chain termination

Other examples of ‘nukes’ include ddI, ddC, d4T, and 3TC (2′,3′-dideoxy-3′-thiacytidine).

Question 26

Describe Nevirapine as an example of a ‘non-nuke’.

Answer 26

It is a non-competitive inhibitor of HIV-1 RT.

This molecule has nothing to do with chain termination – it has a completely different mechanism.

It is synergistic with (nukes) NRTI’s such as AZT because of different mechanism.

Question 27

Describe the applications of pre- and post-exposure prophylaxis for HIV.

Answer 27

PEP – within 72 hours post exposure
Take 2x NRTIs and an integrase inhibitor for 28 days.

PrEP – pre-exposure - blocks transmission. We use 2x NRTIs (Truvada).
Take two tablets 2 – 24 hours before sex, one 24 hours after sex and a further tablet 48 hours after sex - this is called ‘on-demand’ or ‘event based’ dosing.

The 2 x NRTIs are a combination of nuceloside RTIs:
emtricitabine (guanosine analogue) + tenofovir (adenosine analogue).

Question 28

What factors contribute to HIV resistance to antivirals?

Answer 28

• selection pressure and mutation frequency
• increased mutation rate seen in HIV.

They form a quasispecies (similar variants) within an individual patient, leading to a viral swarm.

The error rate in copying viral genome by reverse transcriptase enzyme is 1 base per 10^4-5 incorporations; it lacks proof reading capacity. So, for HIV with 10^9-10 viruses produced every day, ALL possible viral variants would be produced.

Question 29

Describe Amantadine as an antiviral combating influenza.

Answer 29

The M2 enzyme is involved in acidifying the early endosome when the virus goes inside the cell. If it cant be acidified, the membrane doesn’t fuse with the endosome, and it doesn’t release the viral nucleic capsid into the cytoplasm. Stopping that stops the virus at the early stages of its life cycle.

Amantadine inhibits the virus un-coating by blocking the influenza encoded M2 protein when inside cells and assembly of haemagglutinin.

It is now rarely used (as it is toxic and not too effective).

Question 30

Describe Zanamivir and Oseltamivir (Tamiflu) as an antiviral combating influenza.

Answer 30

Tamiflu inhibits virus release from infected cells via inhibition of neuraminidase.

Oseltamivir – oral
Zanamivir – inhaled or IV - less likely for resistance to develop

Question 31

Describe how neuraminidase aids in virus release.

Answer 31

Neuraminidase cleaves off sialic acid for the receptor associated with the surface of the cell.

Even through a virus is budded out of the cell, it is still attached to the sialic acid-containing receptor on the surface of the cell.

That is when neuraminidase cleaves the process, and allows the virus to be released and carry on. If this is inhibited, the virus can not longer be released, so there are no new virus particles occurring, and the cell will die as it is being clogged up.

Question 32

What do neuraminidase inhibitors do?

Answer 32

• target and inhibit NA at highly conserved site (reduce chances of resistance via mutation)
• prevent release of sialic acid residues from the cell receptor
• prevent virus budding and release and spread to adjacent cells

Question 33

Describe influenza resistance to antivirals.

Answer 33

Resistance sometimes only requires a single amino acid change - this was seen recently with swine flu (H1N1) and Tamiflu (oseltamivir).
A point mutation occured (H275Y; tyrosine replacing histidine) that made it reisstant to antivirals.

This was seen in immunocompromised patients; they shed the virus for weeks/months.

They were likely to be selected from among quasispecies during treatment.
It was transmissible and virulent.

But, it remained sensitive to Zanamivir;.

Question 34

Describe the post-exposure prophylaxis of Hep B, Hep C and HIV.

Answer 34

HEPATITIS B:
- specific Hep B immunoglobulin (passive immunity) + vaccination within 48 hours (HBV treatment includes antivirals 3TC/NRTIs)

HEPATITIS C:

• interferon-γ + ribavarin (anti-viral) for 6 months within first 2 months of exposure
• 90% cure rate - now we use direct acting antivirals which are very effective

HIV:

• 80% protection i.e. no sero-conversion
• it must be FAST – within hours
• antiviral drug treatment for 28 days (2xNRTI + protease or integrase inhibitor)

Question 35

Describe the Hepatitis C virus.

Answer 35

• it is transmitted via blood – infectious (mother to baby)
• increasingly common – high risk groups – drug users 20% +ve; – needles (sex?)
• major cause of chronic liver disease
• estimated 170 million people infected worldwide
• occupational risk groups – healthcare workers
• needle-stick risk – 3% to sero-conversion; chronic carriage almost certain (85%)
• long incubation – 1 - 6 months
• vaccination NOT available
• prevalence in UK - ~6000 per year ( 95% are i/v drug users)
• early treatment facilitates resolution

Question 36

Describe the mechanism of action of RIbavirin.

Answer 36

It block RNA synthesis by inhibiting inosine 5’-monophosphate (IMP) dehydrogenase – this blocks the conversion of IMP to XMP (xanthosine 5’-monophosphate)
and thereby stops GTP synthesis and, consequently, RNA synthesis.

It treats RSV and HepC (in combination with pegylated interferon).

Question 37

Describe DAAs (directly-acting antivirals).

Answer 37

• it is a relatively new class of medication
• it acts to target specific steps in the HCV viral life cycle
• it shortens the length of therapy, minimizes side effects, targets the virus itself, and improves the sustained virologic response (SVR) rate
• structural and non-structural proteins - replicate and assemble new virions
• HCV was the first chronic viral infection to be cured without IFN or Ribavirin

Question 38

List some DAAs and their mechanism of action.

Answer 38

NS3/4 protease inhibitors block polyprotein processing. If they can’t process the polyproteins, they cant make the enzymes necessary to assemble the virus. This is a very potent anti-viral effect.

Some of them inhibit Hep C polymerases. For this, we can use NS5B polymerase inhibitors, nucleoside/nucleotide and nonnucleoside inibitors.

We could also block the replication complex formation and assembly of the viruses by blocking the enzymes necessary for the process of budding, maturation and release. We would use NS5A inhibitors.

DAAs with different viral targets, are synergistic in combinations.