ANTIVIRAL AGENTS

DR A CHAUDHURY

Antiviral Drugs-Problems

• Need knowledge of replication at molecular level to define targets Viruses as intracellular parasites make targeting more

difficult to avoid host toxicity.• Lack of culture systems for some agents hinders

development.• Resistance due to rapid mutation of many viruses• Intracellular – can’t use non-permeable drugs• Symptoms usually occur at height of viral replication• Latency or non-replicating phases hard to target• Limited capability for rational drug development; Labour

intensive and expensive.

History

• Early works focused on the use of the

current bacterial antibiotics, including sulphonamides. The futility of these early attempts led to the dogma that viruses are not susceptible to ‘antibiotics’.

• Virologists were taught that selective toxicity for these obligate intracellular parasites was unattainable

History

• In 1957 came the famous first description by Isaacs and Lindenmann of interferon. However, the discovery of the interferons in the late 1950s was something of a false

dawn.

• Idoxuridine: the first useful antiviral .The description of 5-iodo-2′-deoxyuridine by Dr Bill Prusoff in 1959 and the realization of its antiviral properties.

History

• The first publications on this and similar nucleoside analogues appeared in cancer journals.

• The development of IDU from laboratory inhibitor to useful antiviral drug was driven by several notable pioneers, especially the ophthalmologist Dr Herbert Kaufman who proved its clinical value in 1962 for treating viral keratitis.

History

• Antiviral activity of adenine arabinoside (vidarabine, ara-A) by M. Privat de Garilhe

and J. De Rudder also dates from the year 1964.• By providing treatment early in the disease, it

was possible to curtail herpes zoster in the immunosuppressed and reverse the potentially lethal progression of herpes encephalitis and the overwhelming herpes infections that occasionally occur in the newborn.

History

• β-thiosemicarbazone was first reported to be aninhibitor of a related poxvirus, vaccinia virus,in 1951 by D. Hamre, K.A. Brownlee and R. Donovick.

• Dr John Bauer at the then Wellcome Foundation Laboratories in Beckenham, UK, led the team which developed the drug marboran, a β-thiosemicarbazone derivative.

• Marboran was shown in several trials to have some clinical efficacy both for the treatment of smallpox and the complications of vaccinia following vaccination

Many well-known antiviral compounds are nucleoside and nucleotide analogs:

• Acyclovir is a nucleoside analog similar to guanosine, but contains an acyclic sugar group

• AZT is a nucleoside analog similar to thymidine, but contains an azide group

Antivirals other than anti-HIV agents

A. Herpesviruses

B. Respiratory viruses

C. Hepatitis viruses

A. Agents for Herpesvirus: HSV, CMV, VZV.

• Acyclovir : HSV, VZV• Valacyclovir: HSV, VZV• Famciclovir: HSV ,VZV• Ganciclovir: HSV, CMV, VZV• Valganciclovir; CMV• Foscarnet : , HSV,CMV• Cidofovir : HSV, CMV• Trifluridine: Topical eye HSV Keratitis• Idoxuridine: HSV Keratitis

ACYCLOVIR

• Close to a perfect antiviral drug (specific, nontoxic).• Highly effective against herpes simplex virus (HSV),

less so against varicella-zoster virus (VZV).• Highly selective and extremely safe.• Acyclic guanine derivative (nucleoside analogue )

that inhibits viral DNA synthesis.• It is a pro-drug, a precursor of the antiviral

compound.• Activation of the drug requires three kinase activities

to convert acyclovir to a triphosphate derivative, the actual antiviral drug.

Acyclovir: MOA

– Preferentially taken up by virally infected cells– Monophosphorylated by virally encoded thymidine

kinases– Di- and triphosphorylation completed by cellular

kinases– ACV-TP is the active moiety • Competitive inhibitor of viral DNA polymerase– Cellular DNA polymerases much less susceptible to

inhibition• Leads to viral DNA chain termination

Acyclovir-MOA• Inhibition of HSV DNA Polymerase is a

three step process:

1. ACV-TP competitively inhibits dGTP incorporation.

2. Next ACV-TP acts as a substrate and is incorporated in the growing DNA chain

3. Polymerase translocates to the next position on the template, but cannot add a new dNTP because there is no 3’-hydroxyl on ACV-TP→ Dead end complex→Enzyme inactivation.

Acyclovir :Resistance

– Mediated by mutations in viral thymidine kinase and/or viral DNA polymerase genes

• TK-deficient and TK partial virus can be produced. These viruses fail to phosphorylate acyclovir.

• Acyclovir resistance mutations can also alter pol to be less inhibited by the drug.

– Clinically significant infections can be caused by drug resistant HSV and VZV.

CIDOFOVIR

• It is converted to the diphosphate form by cellular kinases. Initial monophosphorylation by viral TK or other kinases not needed.

• Acts as an alternate substrate of dCTP and an inhibitor of DNA polymerase.

FOSCARNET

• Foscarnet (trisodium phosphonoformate) blocks, through non-competitive inhibition, of the pyrophosphate binding site of viral DNA polymerase, thereby preventing the cleavage of pyrophosphate from deoxynucleoside triphosphate and elongation of the viral DNA chain.

• Foscarnet does not require viral/cellular thymidine kinase for activation

Foscarnet

• Foscarnet selectively inhibits viral polymerase; inhibition of mammalian DNA polymerase requires a 100-fold greater concentration of foscarnet than that required to block viral replication.

Foscarnet

• Since foscarnet, unlike acyclovir and ganciclovir, does not require intracellular phosphorylation for antiviral activity, thymidine kinase mutations in HSV or VZV and UL97 phosphotransferase mutations in CMV do not confer resistance.

• Foscarnet resistance in CMV has been associated with mutations in the viral DNA polymerase gene in patients receiving prolonged therapy for AIDS-associated CMV retinitis.

CLINICAL USES

ACYCLOVIR: Intravenous therapy for HSV encephalitis,

neonatal herpes, severe VZV infection Prophylaxis for HSV and VZV in

transplant patients. Oral agent for Primary or recurrent HSV 1

and 2, and uncomplicated VZV infections.

Clinical Uses

• Ganciclovir is the first line agent against CMV infections in immunocompromised.

• Clinically, foscarnet is employed almost exclusively to treat infections with CMV, particularly when ganciclovir resistant, and acyclovir-resistant HSV and VZV.

• Cidofovir is also used for CMV infections (administration every 1 or 2 weeks).

B. Agents for Respiratory Virus: Influenza Virus

• Amantadine

• Rimantadine

• Zanamivir

• Oseltamivir

• Ribavirin

Amantadine and Rimantadine• Tricyclic amines• Active against influenza A only at clinically achievable

concentrations• Mechanism of action: • Viral M2 protein acts as an ion channel which

normally facilitate the hydrogen ion mediated dissociation of the matrix protein from the nucleocapsid.

– Interference with function of viral M2 proteinThis prevents viral uncoating and release of viral RNA into

the cytoplasm and transport of ribonucleoprotein complex into the nucleus.

• Resistance mediated by mutations in M2 coding region: single AA substitution within the M2 transmembrane domain.

Amantadine and Rimantadine

• Useful for treatment and prophylaxis of influenza A infections

– Should not be used when amantadine resistant strains are in circulation

Cross resistance.• Can reduce severity & duration of illness if

started within 48 hrs of onset of symptoms

Zanamivir, Oseltamivir

• Mechanism of actionViral neuraminidase catalyzes cleavage of

terminal sialic acid residues attached to glycoproteins and glycolipids, a process necessary for release of virus from host cell surfaces

Neuraminidase inhibitors thus prevent release of virions from infected cell

NA Inhibitors

• These agents are Sialic acid analogues, and they block the active sites of the enzyme NA. So sialic acid cannot be cleaved and progeny viruses are not released from infected cells.

• NA mutation results in an enzyme which is less inhibited by the drug.

• No cross rersistance.

Oseltamivir, Zanamivir

• Indications

– Treament of influenza A and B within 24-48 hrs of symptom onset

– Prophylaxis

– N.B.: Neither drug interferes with antibody response to influenza vaccination

More than 300 cases of oseltamivir-resistant (H1N1) carrying the H275Y mutation have been detected, up to October 2010.

C. Hepatitis Virus: Hepatitis B Virus

• Interferon-alpha (pegylated)• Lamivudine : – Nucleoside analog first developed for HIV – Lower dose used for HBV (100 mg/day)• Adefovir dipivoxil – Nucleotide analog first developed for HIV but nephrotoxic at higher doses – Approved for HBV at lower dose (10 mg/day)• Entecavir – Nucleoside analog with activity originally thought limited to HBV but has anti-HIV-1 activity. – Approved for use at dose of 0.5-1.0 mg/day• Telbivudine – Nucleoside (thymidine) analog with activity against HBV but not HIV – Recently approved at a dose of 600 mg/day

Lemivudine,Telbivudine

• Dideoxy analogue of cytidine.

• Phosphorylated intracellularly to the triphosphate form.

• Competitive inhibitor of dCTP

• Incorporation of the triphosphate form into viral DNA by HBV DNA Polymerase results in chain termination.

• Resistance: Polymerase substitution.

Hepatitis VirusHepatitis C

• Approved

– Interferon-alpha (pegylated)

– Ribavirine

• In development

– Protease inhibitors

– Polymerase inhibitors

RIBAVIRINE

• Synthetic nucleoside analog • Active against broad range of RNA and DNA viruses – Flavi-, paramyxo-, bunya-, arena-, retro-, herpes-,

adeno-, and poxviruses• Mechanism of action complex – Triphosphorylated by host cell enzymes• For influenza – Ribavirin-TP interferes with capping and elongation of

mRNA and may inhibit viral RNA polymerase• For other agents – Ribavirin-MP inhibits inosine-5’-monophosphate

dehydrogenase depleting intracellular nucleotide pools, particularly GTP.

Ribavirine

• Indications

– Aerosol treatment of RSV in children

Effectiveness debated

– Oral treatment of HCV (in combination with pegylated IFN alpha).

Interferons

• Types

– Alpha/Beta (leukocyte/fibroblast)

• Coding genes located on chromosome 9

• At least 24 subtypes of alpha, 1 of beta

– Gamma

• Coding gene located on chromosome 12

• 1 subtype

Interferons: MOA

• Act by inducing an antiviral state within cells• Bind to specific receptors on cell surface• Receptor associated tyrosine kinases activated – Tyk2 and JAK 1 for alpha and beta – JAK1 and JAK2 for gamma• Induction of a phosphodiesterase with inhibition of

peptide chain elongation• Synthesis of MxA protein which can bind to cytoskeletal proteins and inhibit viral transcriptases• Induction of nitric oxide by gamma IFN in macrophages• Viral penetration, uncoating, viral mRNA transcription,

viral protein synthesis, replication of viral genome, assembly and release of progeny virus are inhibited.

Interferons: Use

• Antiviral indications

– IFN-alpha 2b (pegylated) for HCV (in combination with ribavirin)

– Intralesional for condyloma acuminata.

Antiviral Drug Susceptibility Tests.

• For optimal patient management.

• Used mainly for HSV, VZV, CMV, Influenza virus, and HIV.

• Phenotypic , Genotypic.Uses: 1. Defining mechanism of resistance

2. Determine the frequency with which drug resistant viral mutants emerge.

3. To test for cross resistance

4. Evaluation of a new agent.

Phenotypic Test

• Directly measure the effect of antiviral drug in viral growth.

• Can be measured by infectivity, viral antigen production, or viral nucleic acid production.

• Labour intensive, slow.• Difficult to standardize• Result is method dependent.• Herpes group, Influenza, HIV-

1.

Genotypic Tests

• Test for genetic basis(mutation) for resistant phenotype.

• RFLP, differential hybridization assays, or sequencing.

• Useful only if the genetic basis or resistance is known.

• Most useful if there are only limited number of genetic changes.

• CMV, HIV-1

Variables Affecting the Testing No existing standards regarding• Cell line

• Viral inoculum titre• Incubation time• Concentration range for antivirals• Reference strains• Assay method• End point criteria• Calculation and interpretation of end point

PHENOTYPIC TESTS

PLAQUE REDUCTION ASSAY: Considered the “gold standard” CLSI has developed a standard for PRA

testing of HSV. Inhibition of viral plaque formation in the

presence of antiviral agent. The concentration of the agent which

inhibits plaque formation by 50% is IC50.

PRA

• Cell lines are grown on wells or plates.

• A standardized viral stock is inoculated into multiple identical cell cultures.

• The viral titre of the stock is previously determined and adjusted to yield countable number of plaques in each well ( 50-100PFU/60 mm wide tissue culture plates)

PRA

• A solidifying agent (agarose) is added to the cultures to minimize spread of virus through media.

• Increasing concentration of the test drug is incorporated into a series of wells.

• IC50 : Lowest concn. of drug that results in a 50% decrease in the number of plaques compared with the control well with no antiviral drug.

Dye Uptake Assay

• Used mainly for HSV

• The vital dye Neutral red is taken up by viable cells , but not by nonviable cells.

• The extent of viral lytic activity is measured by the relative amount of the dye bound to viable cells after infection with HSV compared with the amount bound to uninfected cells.

DU Test• The dye bound by viable cells is eluted by

ethanol and measured in a colorimeter.

• Drug concentration which inhibits viral lytic activity by 50% is the IC50.

• It gives IC50 of acyclovir 3-5 times greater than that given by PRA

− Higher inoculum ( 500PFU/ml) used

− Use of a liquid overlay which allows drug resistant viruses to ‘amplify’ resulting in more sensitive detection of small amounts of drug resistant viruses.

DU Test

Advantages: Semi-automated Efficient testing of large number of isolates Ability to detect small number of resistant virus

Disadvantages: Relatively high cost of automated equipment Overseeding of cells into culture wells Precipitation of neutral red onto the monolayer.

OTHER PHENOTYPIC TESTS• EIA: Concentration of antiviral agent

which reduces the absorbance to 50% of the control.

• Flow Cytometry

• NI Assay: Assay for Neuraminidase inhibitors of influenza virus.

• Yield Reduction Assay: Ability of the agent to inhibit the production of infectious virus

GENOTYPIC TESTS

• It has been used to screen CMV isolates for mutation associated with ganciclovir resistance.

• UL 97(Phosphotransferase) and UL54 ( DNA Polymerase) mutations can be detected.

• PCR amplification of short fragment of UL97 followed by restriction endonuclease digestion to detect mutations.

Genotypic Tests• Can detect 78% of Ganciclovir resistant

isolates.

• Can recognize mutant virus when present at 10% of the total virus population.

• Every mutation is not a cause of resistance. MARKER TRANSFER EXPERIMENT must be performed to definitely define that a particular mutation is the cause of resistance.

Genotypic Tests

• Marker Transfer Resistance: PCR amplified UL 97 and UL54 fragments containing the resistance mutation are co- transfected with CMV strain AD169 (Drug Susceptible). The resulting recombinant plaques are assayed for antiviral susceptibility by PRA.

Genotypic Tests

Influenza Antiviral Susceptibility: H275Y mutation causes oseltamivir resistance in H1N1 viruses.

1. Searching for specific mutations by real-time PCR or pyrosequencing technology.

2. Full length neuraminidase gene sequencing .