Mechanisms of Antivirals

Question 1

Why do we need anti-virals ?

Answer 1

because viruses are quick killers, eg. influenza, ebola, SARS

or viruses can be chronic and slow, leading to cancer, eg. HPV, Hep B, Hep C

To treat human immunodeficiency virus (HIV)

To treat acute inflammatory infections – herpes, shingles, chickenpox

Question 2

give some uses of anti virals?

Answer 2

1. Treating acute (influenza, chickenpox, herpes) and chronic infections (HCV, HIV)
2. Post exposure prophylaxis (PEP), taking antiviral medication after being potentially exposed (e.g. to HIV) to prevent infection
3. Pre-exposure prophylaxis (HIV)
4. Prophylaxis for reactivated infection: e.g. in transplantation CMV (ganciclovir)

Question 3

Principles of Anti-Virals as Therapeutic Agents

Answer 3

Selective Toxicity - exert their action only on infected cells

-taking into account differences in structure/metabolic pathways of host and pathogen

• drug should harm microorganism but not the host
• drug target should be in the microbe if possible and specific for the microbe

Question 4

Why is it hard to make anti-virals?

Answer 4

Because they are intracellular organisms that use cellular processes to replicate themselves.

Inhibiting the replicative processes inside the cell will stop the virus from replicating but will also damage the host cell

Viruses enter cells using cellular receptors which may have other functions

Viral enzymes may be very similar to host enzymes, and blocking cellular enzyme may kill the cell

Viruses have high mutation rate - quasispecies (large numbers of mutants of the same isolate of virus in the same human)

Some viruses are able to remain in a latent state e.g. herpes, HPV - difficult to target

Some viruses are able to integrate their genetic material into host cells e.g. HIV

Question 5

Modes of action of selected anti-virals

Answer 5

Preventing virus adsorption onto host cell

Preventing penetration

Preventing uncoating process, used to be blocked by Amantadine for influenza A, was an effective anti-flu drug BUT had toxic side effects

Preventing viral nucleic acid replication (nucleoside analogues), very effective
-Acyclovir, Ganciclovir, Ribavarin inhibit nucleic acid polymerisation by targeting reverse trancriptases/ DNA polymerases

Preventing virus maturation

Preventing virus release, Zanamivir inhibits Influenza release

Question 6

Selective toxicity viral targets - examples

Answer 6

Discovery of virally encoded enzymes sufficiently different from human counterparts
e.g.

Thymidine kinase and HSV/VZV/CMV
HIV Protease or Reverse transcriptase
DNA polymerases
Neuraminidase of influenza virus

Act as selective targets with minimal effect on host enzymes or processes

Question 7

Herpes viruses - examples and how to treat them

Answer 7

Herpes viruses include: 
Herpes simplex (HSV), 
Varicella Zoster Virus (VZV)
Cytomegalovirus (CMV)
Epstein-Barr virus (EBV)

ACICLOVIR

• IV/oral/topical
• good because of its low toxicity
• For HSV, VZV treatment/prophylaxis

GANCICLOVIR, CIDOFOVIR, FOSCARNET
-IV for CMV

Question 8

aciclovir - mechanism and uses

Answer 8

Acycloguanosine is a GTP analogue

1. Phosphorylated by a viral thymidine kinase - makes it stable within the cell
2. di and tri phosphorylated by cellular kinases. The tri-phosphate structure of aciclovir is the active drug
3. The active drug is a competitive inhibitor with normal GTP for viral DNA polymerase, which is synthesising the viral genome.
4. Incorporating aciclovir means termination of the viral DNA polymerase chain, because its a GTP analogue but without the ribose sugar, therefore acts as a chain terminator.

it has low toxicity

TREATS: 
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 9

acyclovir has a low toxicity - how?

Answer 9

• selectively activated in infected cells
• mainly activated by a viral thymidine kinase - unlikely for the drug to be activated by normal cell thymidine kinase
• selectively inhibits viral DNA polymerase, not human 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). This means you can accumulate the drug where you need it

Question 10

Ganciclovir

Answer 10

Active for CMV

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

Structurally similar to aciclovir
CMV does not encode TK but has UL97 kinase that activates ganciclovir, which is then di and tri phosphorylated by cellular kinases

Inhibits CMV DNA polymerase (competes with the natural substrate for DNA polymerase and inhibits DNA synthesis)

Question 11

Foscarnet

Answer 11

– not a pro-drug, doesn’t need to be activated

– May be used because of ganciclovir resistance (TK mutants) - can treat TK mutants in CMV because it targets a different part of the enzyme other than the target site, called the pyrophosphate binding site

selectively inhibits viral DNA/RNA polymerases and RTs

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

Question 12

Cidofovir

Answer 12

Chain terminator - targets DNA polymerase and competes with dCTP, acts as a monophosphate nucleotide analogue

Prodrug – phosphorylated by cellular kinases to di-phosphate

drug active against CMV, but MUCH MORE nephrotoxic

Treatment of retinitis in HIV disease

Question 13

Resistance to anti-virals in Herpes viruses

Answer 13

Two main mechanisms

Thymidine Kinase mutants
DNA polymerase mutants

If occurs in TK, no longer activate the pro drug, but drugs not needing TK phosphorylation are still effective (e.g. foscarnet, cidofovir)

If mutation occurs in DNA polymerase, all drugs rendered less effective as they cannot bind

VERY RARE in immune competent patients (low viral load) - Both of these mutants are only easily generated when you have a high viral load

Question 14

HIV stands for?

Answer 14

Human immunodeficiency virus

Question 15

Name the 7 steps in the life cycle of HIV

Answer 15

1. Attachment to host membrane, binding of viral gp120 via CD4 and CCRX
2. reverse transcription of RNA into dsDNA
3. Integration of proviral DNA into host chromosome
4. Viral gene transcription
5. Translation of viral mRNA into viral proteins
6. Virus assembly and release by budding
7. maturation

Question 16

Anti-HIV drugs

Answer 16

1. Anti-reverse transcriptase inhibitors - nukes block RT by mimicking nucleotides/nucleosides, non nukes block RT via allosteric sites
2. Protease inhibitors - Virus cant assemble itself. Virus proteases have a completely different structure to host proteases, so you block the viral ones without affecting the host ones – important part of selective toxicity
3. Integrase inhibitors – no cross linking of viral DNA into host DNA, POL gene codes for 3 separate components including integrase
4. Fusion inhibitors - prevent virus fusing with host cell membrane, preventing virus particle internalisation, complex long lipopeptides act as biomimicks and interrupt gp120-gp141 interaction.

HAART, highly active anti-viral therapy - combination of drugs to avoid resistance

Question 17

example of a nuke and non nuke?

Answer 17

Zidovudine
-analogue of nucleoside thymidine, acts as a chain terminator because they can’t be incorporated into synthesising chain

Nevirapine
Non-competitive inhibitor of HIV-1 RT
Synergistic with NRTI’s such as AZT because of different mechanism
Nothing to do with chain termination

Question 18

Resistance to anti-virals

Answer 18

use of single agents leads to rapid development of resistance, drug binding site is altered in structure by as few as one amino acid substitution

High mutation rate and viral load —–> resistance

Some mutations are lethal to the virus and it will die, but some mutations will confer resistance - selection pressure

Increased mutation rate seen in HIV due to lack of proofreading ability
Formation of quasispecies within an individual patient:-A viral swarm

Question 19

Influenza virus - drugs

Answer 19

Amantadine

• Inhibits virus uncoating by blocking the influenza encoded M2 protein when inside cells and assembly of haemagglutinin
• M2 protein involved in early endosome acidification when the virus gets into the cell - blocking M2 means virus capsid not released into cytoplasm
• rarely used because toxic + not very effective

Zanamivir/Relenza (IV/inhaled) and Oseltamivir/Tamiflu (oral)

• Inhibits virus release from infected cells
• targets and inhibits Neuraminidase at highly conserved site (reduces chances of resistance via mutation)
• prevents virus budding and release
• prevent release of sialic acid residues from the cell receptor

Question 20

Influenza resistance to anti-virals

Answer 20

Resistance sometimes only requires a single amino acid change - seen recently with swine flu (H1N1) and Tamiflu (oseltamivir)

Point mutation seen in immunocompromised patients; shed virus for weeks/months

Likely to be selected from among quasi species during treatment

antigenic shift and drift

Question 21

Occupational Infection - Hazards

Answer 21

Exposure prone incidents
-Sharps, Splashes and blood-born viruses

Hepatitis B
Hepatitis C
HIV

take universal precautions

Question 22

Post-exposure prophylaxis

Answer 22

Emergency, requiring rapid treatment

Hep B
specific Hep B immunoglobulin (passive immunity)
+ vaccination within 48 hours

Hep C
interferon- + ribavarin (anti-viral) for 6 months
within first 2 months of exposure
90% cure rate - now direct acting antivirals

HIV
	80% protection i.e. no sero-conversion
	must be FAST – hours
	antiviral drug treatment – 28 days
	2xNRTI + protease or integrase inhibitor

Question 23

Hepatitis C virus (HCV)

Answer 23

RNA virus, enveloped

transmitted via blood – infectious (mother to baby)

major cause of chronic liver disease

occupational risk groups – healthcare workers

needle-stick risk

vaccination NOT available

Question 24

Ribavirin

nucleoside analogue

Answer 24

HCV treatment

Ribavirin blocks precursor GTP pathway, blocks GTP synthesis and therefore blocks RNA synthesis

Inhibits IMP dehydrogenase, blocks conversion of IMP to XMP, stopping GTP synthesis and consequently RNA synthesis

Question 25

HCV - new treatments?

Answer 25

Direct-acting antivirals (DAAs) - new class of medication, targets specific steps in HCV life cycle

aim: shorten length of therapy, minimize side effects, target the virus itself

DAA with different viral targets, are synergistic in combinations

examples

NS3/4 protease inhibitors - block polyprotein (GAG) processing and protease synthesis

NS5A inhibitors block replication and assembly

Question 26

are all viral infections treatable?

Answer 26

no