Pharm 6: Anti-Viral

Question 1

How do viruses replicate?

Answer 1

Replicate by co-opting the host cell’s metabolic machinery. As a result, there are fewer differences between viruses and their human hosts to exploit for drug development than between bacteria and humans.

Question 2

T or F. It is more difficult to develop agents that are active against a broad spectrum of viruses than it is against bacteria.

Answer 2

T. This difficulty arises because viruses are a heterogeneous group of infectious agents, whereas most bacteria share a common cell wall structure and distinct transcriptional and translational machinery

Question 3

So how can we target viruses then?

Answer 3

Despite these obstacles, all viruses encode proteins that are substantially different from their human counterparts. Additionally, certain host proteins are more important for viral replication than they are for human health. In principle, antiviral drugs could target many of these proteins. In practice, however, relatively few viral proteins and even fewer host proteins have thus far served as useful targets for therapy.

Question 4

Most antivirals are active against only one or a few viruses, whereas most antibacterial drugs target multiple bacterial species

Answer 4

Most antivirals are active against only one or a few viruses, whereas most antibacterial drugs target multiple bacterial species

Question 5

What are the basic steps of DNA virus entry and replication in a host cell?

Answer 5

1) Virus binding and entry
2) Uncoating
3) Genome replication
4) RNA and Protein synthesis
5) Assembly and Maturation
6) Egression and release from host cell

It should be noted that the details of viral replication differ for each type of virus, often presenting unique targets for pharmacologic intervention and drug development.

Question 6

Which antiviral drugs target DNA viral bonding and entry into host cells?

Answer 6

Maraviroc and Enfuviritide

Question 7

Which antiviral drugs target DNA uncoating into host cells (via ion channel blockers)?

Answer 7

Amantadine and Rimantadine

Question 8

Which antiviral drugs target DNA viral replication (i.e. polymerase inhibitors)?

Answer 8

Acyclovir, Zidovudine, and Efavirenz

Question 9

Which antiviral drugs target DNA viral replication (i.e. integrate inhibitors)?

Answer 9

Raltegravir

Question 10

Which antiviral drugs target DNA protein assembly and maturation (i.e. protease inhibitors)?

Answer 10

Saquinavir and Ritonavir

Question 11

Which antiviral drugs target DNA protein eggression and release?

Answer 11

Zanamivir and Oseltamivir

Question 12

What are the basic stages of HIV infection on CD4 host cells?

Answer 12

1. Virus attachment is dependent on binding interactions between viral gp41 and gp120 proteins and host cell CD4 and certain chemokine receptors.
2. Fusion of the viral membrane (envelope) with the host cell plasma membrane allows the HIV genome complexed with certain virion proteins to enter the host cell.
3. Uncoating permits the single-stranded RNA (ssRNA) HIV genome to be copied by reverse transcriptase into double-stranded DNA.
4. The HIV DNA is integrated into the host cell genome, in a reaction that depends on HIV-encoded integrase.
5. Gene transcription and post-transcriptional processing by host cell enzymes produce genomic HIV RNA and viral mRNA.
6. The viral mRNA is translated into proteins on host cell ribosomes.
7. The proteins assemble into immature virions that bud from the host cell membrane.
8. The virions undergo proteolytic cleavage, maturing into fully infective virions.

Question 13

T or F. The development of drug resistance can be significantly retarded by using combinations of drugs that target a single stage (e.g., two or more inhibitors of reverse transcription)

Answer 13

T, or more than one stage in the HIV life cycle (e.g., reverse transcriptase inhibitors and protease inhibitors).

Question 14

Describe the replicative pathway of Influenza A virus.

Answer 14

1) The influenza A virus protein hemagglutinin binds to sialylated glycoprotein receptors on the host-cell surface, and the virus enters the cell by receptor-mediated endocytosis.
2) Following internalization and endosomal acidification, which permits fusion of the host and viral membranes by altering the conformation of hemagglutinin (i.e. the ‘uncoating’ process, mediated by M2 ion channels), viral ribonucleoproteins are released in the cytoplasm.
3) In the nucleus of infected cells, the viral RNAs are transcribed into mRNAs and replicated by the viral RNA–dependent RNA polymerase complex.
4) The newly synthesized viral RNPs are exported into the cytoplasm and, after assembly, mature virions bud from the cell surface.

Question 15

What are the current targets of antiviral drugs for Influenza A?

Answer 15

Currently, the viral M2 ion channel protein and neuraminidase are the only two targets of influenza antiviral drugs licensed by the US Food and Drug Administration.

Adamantane drugs, which include amantadine and rimantadine, block the action of the viral M2 protein during uncoating of the virus.

Zanamivir and Oseltamivir target neuraminidase, which is required for release of progeny virus from the cell surface.

Question 16

Another limitation of antiviral drugs is that they are relatively ineffective because many cycles of viral replication occur during the incubation period when the patient is well. By the time the patient has a recognizable systemic viral disease, the virus has spread throughout the body and it is too late to interdict it.

Furthermore, some viruses (e.g., herpesviruses) become latent within cells, and no current antiviral drug can eradicate them.

Answer 16

Another limitation of antiviral drugs is that they are relatively ineffective because many cycles of viral replication occur during the incubation period when the patient is well. By the time the patient has a recognizable systemic viral disease, the virus has spread throughout the body and it is too late to interdict it.

Furthermore, some viruses (e.g., herpesviruses) become latent within cells, and no current antiviral drug can eradicate them.

Question 17

Which antivirals blocks nucleic acid synthesis by the herpes virus?

Answer 17

Acyclovir and Ganciclovir

and Valacyclovir and Valganiclovir

Question 18

Which antivirals blocks nucleic acid synthesis by HIV (NRTIs)?

Answer 18

Abacavir, Lamivudine (3-TC), Tenofovir, Disoproxil, Foscarnet (already activated), Zidovudine (AZT), Emtricitabine, didanosine (ddl), stavudine

Question 19

Which antivirals blocks nucleic acid synthesis by HIV (NNRTIs)?

Answer 19

Efavirenz and Nevirapine

Question 20

Which antiviral drugs inhibit nucleic acid synthesis by hepatitis B virus (HBV)?

Answer 20

Adefovir, Entecavir, Lamivudine, and Telbivudine

Question 21

Nucleic acid synthesis inhibitors of other viruses?

Answer 21

Ribavirin and Trifluridine

Question 22

Which antiviral drugs inhibit the integrates that integrates HIV DNA into cellular DNA?

Answer 22

Raltegravir

Question 23

Which antiviral drugs inhibit cleavage (protease) of precursor viral polypeptides following transcription/translation in HIV-1?

Answer 23

Atazanavir, Ritonavir, Amprenavir, Indinavir, Lopinavir, Nelfinavir, and Saquinavir

Question 24

Which antiviral drugs inhibit cleavage of viral precursor polypeptides of Hepatitis C virus (HCV)?

Answer 24

Boceprevir and Telaprevir

Question 25

Which antiviral drugs inhibit protein synthesis directed by viral mRNA?

Answer 25

Peginterferon alfa

Question 26

Which antiviral drugs inhibit release of influenza virus from infected cells?

Answer 26

Zanamivir and Oseltamivir

Question 27

What are the specific steps of HIV infection of host cells starting with attachment and entry?

Answer 27

A. HIV glycoproteins exist in trimeric form in the viral membrane (envelope). Each gp120 molecule is depicted as a ball attached non-covalently to gp41.

B. The binding of gp120 to CD4 and certain chemokine receptors in the host cell plasma membrane causes a conformational change in gp41 that exposes the fusion peptide, heptad-repeat region 1 (HR1) and heptad-repeat region 2 (HR2). The fusion peptide inserts into the host cell plasma membrane.

C. gp41 undergoes further conformational changes, characterized mainly by unfolding and refolding of the HR2 repeats.

D. Completed refolding of the HR regions creates a hemifusion stalk, in which the outer leaflets of the viral and host cell membranes are fused.

E. Formation of a complete fusion pore allows viral entry into the host cell.

Question 28

How does Enfuvirtide (T-20) work?

Answer 28

Enfuvirtide (T-20) is a synthetic peptide drug that mimics HR2, binds to HR1, and prevents the HR2–HR1 interaction.
Therefore, the drug traps the virus–host cell interaction at the attachment stage, preventing membrane fusion and viral entry.

Question 29

How does Maraviroc work?

Answer 29

Maraviroc is a small-molecule antagonist of the CCR5 chemokine receptor; the drug blocks cellular infection of HIV strains that use CCR5 for attachment and entry

Some HIV strains use different chemokine receptors; maraviroc is thus ineffective against these infections.

These act outside the cell. Note that tropism is the ability of different HIV strains to use different cell receptors (CCR5-R5 tropic or CXCR4-X4 tropic) to gain into a cell. So maraviroc wouldn’t work against X4 trophic HIV

Question 30

What are the specific steps of Influenza A infection starting with cellular adhesion and integration into the cell?

Answer 30

Influenza virus enters host cells by receptor-mediated endocytosis and is contained within an early endosome.

The early endosome contains an H+-ATPase that acidifies the endosome by pumping protons from the cytosol into the endosome.

A low pH-dependent conformational change in the viral envelope hemagglutinin (HA) protein triggers fusion of the viral membrane with the endosomal membrane. Fusion alone is not sufficient to cause viral uncoating, however.

In addition, protons from the low-pH endosome must enter the virus through M2, a pH-gated proton channel in the viral envelope that opens in response to acidification. The entry of protons through the viral envelope causes dissociation of matrix protein from the influenza virus ribonucleoprotein (RNP), releasing RNP and thus the genetic material of the virus into the host cell cytosol.

Question 31

What do the antiviral drugs Amantadine and rimantadine do?

Answer 31

block M2 ion channel function and thereby inhibit acidification of the interior of the virion, dissociation of matrix protein, and uncoating.

Note that the drug is shown as “plugging” the channel; however, there is also evidence that the drug may bind to the outside of the channel instead

Question 32

Since viruses rely upon polymerases for genomic replication, these enzymes represent an excellent drug target. Drugs acting against this target can be categorized as belonging to one of 2 subgroups:

Answer 32

nucleoside inhibitors and non-nucleoside inhibitors.

Question 33

T or F. Nucleoside analogues require metabolic activation, usually to a triphosphate form, before incorporation of the deoxyribonucleoside triphosphate into the growing DNA chain

Answer 33

T. Nucleoside analogues require metabolic activation, usually to a triphosphate form, before incorporation of the deoxyribonucleoside triphosphate into the growing DNA chain

Here, their incorporation usually leads to termination of chain elongation.

Question 34

What are some qualifications of nucleoside analogue inhibitors?

Answer 34

Selectivity of drug action is thus dependent upon the differential toxicity of the drug towards viral enzymes, versus those in the host cell. Drug structures must be sufficiently similar to natural nucleosides but sufficiently dissimilar to avoid inhibition of mammalian cellular processes.

Nucleoside analogues used primarily in the treatment of herpes infections and for HIV infection.

Question 35

ACYCLOVIR, GANCICLOVIR, valacyclovir, & valganciclovir are used in treatment of what?

Answer 35

herpes simplex viruses 1 & 2 and varicella zoster virus and cytomegalovirus (GAN).

As you might discern from the spelling of the names, both valacyclovir and valganciclovir are orally absorbed pro-drugs that rapidly liberate the respective active agent (acyclovir & ganciclovir) upon entry intro the circulation. This avoids the poor oral bioavailability of the parental agents.

Question 36

How do drugs like ACYCLOVIR, GANCICLOVIR, valacyclovir, & valganciclovir work?

Answer 36

The activated triphosphate form:

1. acts as a competitive inhibitor of dGTP (pppdG) binding; 2. becomes incorporated into the growing DNA chain, causing chain termination
2. traps the polymerase on the ACV-terminated DNA chain when the next deoxyribo-nucleoside triphosphate (shown here as dCTP, or pppdC) binds.

Question 37

Which antivirals blocks nucleic acid synthesis by HIV (NRTIs)?

Answer 37

Abacavir, Lamivudine (3-TC), Tenofovir, Disoproxil, Zidovudine (AZT), Emtricitabine, didanosine (ddl), stavudine

Question 38

How do drugs like Abacavir, Lamivudine (3-TC), Tenofovir, Disoproxil, Zidovudine (AZT), Emtricitabine, didanosine (ddl), stavudine work? These are NRTIs

Answer 38

These are competitive inhibitors

These drugs are used in the treatment of HIV (some also used in the treatment of HBV), a retrovirus that contains within its capsid a number of enzymes including reverse transcriptase and protease. Reverse transcriptase is a DNA polymerase that can copy both RNA and DNA. It is this enzyme that is targeted by the nucleoside reverse transcriptase inhibitors (NRTIs). When incorporated into viral DNA, NRTIs cause termination of the DNA chain, and inhibition of viral replication.

Note that binding of these drugs only INDIRECTLY inhibits the polymerase; they do NOT have the specificity of effect against HIV provided by the non-nucleoside reverse transcriptase inhibitors (NNRTIs). In consequence, NRTIs also inhibit host cell mitochondrial DNA polymerase, leading to depletion of mitochondrial DNA and subsequent depletion of mitochondrial RNA and peptides involved in oxidative phosphorylation. This leads to mitochondrial dysfunction, and this is believed to be the cause of several of the important toxicities associated with drugs of this class.

Cross-resistance to NRTIs is associated with mutations at a number of reverse transcriptase codons. These mutations are referred to as thymidine analog mutations (TAMs) because of their ability to confer cross-resistance to thymidine analogs. Or via unregulated repair that can remove the drug moiety

Impact mitochondrial DNA the most

Question 39

How do drugs like EFAVIRENZ, NEVIRAPINE: Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) work?

Answer 39

These are allosteric inhibitors

NNRTIs have a mechanism of action that is DISTINCT from the NRTIs. They bind to a hydrophobic pocket in the p66 subunit of the HIV-1 reverse transcriptase well away from the enzyme active site where the NRTIs interact (i.e., they are non-competitive inhibitors-allosteric).

In so doing, they alter the 3D structure of the enzyme and significantly impede its activity. Unlike nucleoside and nucleotide reverse transcriptase inhibitors, these compounds do not require intracellular phosphorylation to attain activity. Because the binding site for NNRTIs is virus-strain-specific, the approved agents are active against HIV-1 but not HIV-2 or other retroviruses.

Resistance arises from altered binding sites

Question 40

How do Adafovir, Enetcavir, Lamivudine, and Telbivudine work?

Answer 40

These are prodrugs used to treat chronic HBV by conversion by cellular enzymes to diphosphate (adefovir) or triphosphate (lamivudine, entecavir, ribavirin) form. The activated form acts as a competitive inhibitor of viral DNA polymerases and reverse transcriptases with respect to deoxyadenosine triphosphate (Telbivudine is with respect to Thymidine 5”-triphosphate). Serve as chain terminator of viral DNA synthesis.

Question 41

How does Ribavirin work?

Answer 41

Incompletely understood mechanism.
Converted by cellular enzymes to the triphosphate form.
Competitively inhibits GTP-dependent 5′ capping of viral messenger RNA and specifically influenza virus transcriptase activity.
Multiple sites of interacting action on GTP-dependent enzymatic processes.
Some of these (e.g., inhibition of GTP synthesis) may potentiate others
May enhance viral (lethal) mutagenesis.

Question 42

How does Trifluridine work?

Answer 42

A fluorinated pyrimidine nucleoside that irreversibly inhibits thymidylate synthase and specific DNA polymerases, thus inhibiting DNA synthesis.

To a lesser extent, trifluridine is also incorporated into cellular DNA and is capable of decreasing DNA synthesis in both infected and uninfected cells.
Viral resistance to trifluridine is rare.
Used to treat HSV-induced keratitis and keratoconjunctivitis.

Question 43

How does RALTEGRAVIR work?

Answer 43

Raltegravir blocks the catalytic activity of the HIV-1 & 2-encoded integrase by binding to it, thus preventing integration of viral DNA into the host chromosome

Although raltegravir retains activity against viruses that have become resistant to antiretroviral agents of other classes, because of its unique mechanism of action, the virus can become resistant to this agent through primary mutations in the integrase gene.

Question 44

How do HIV-1 PROTEASE INHIBITORS: Inhibitors Of Precursor Polypeptide Cleavage work?

Answer 44

ATAZANAVIR, RITONAVIR, amprenavir, indinavir, lopinavir, nelfinavir, saquinavir

These peptide-like drugs are specific inhibitors of the virus aspartyl protease enzyme that is responsible for proteolytic cleavage of HIV gag and pol precursor polypeptides that include essential structural (p17, p24, p9, and p7) and enzymatic (reverse transcriptase, protease, and integrase) components of the virus. This prevents the meta-morphosis of HIV virus particles into their mature infectious form.

Question 45

Protease inhibitors are a common component of HIV drug regimens for treatment-experienced patients. There are a number of caveats to their use. These include the ability of some of these agents (e.g., most notably RITONAVIR) to inhibit CYP3A4 metabolic capacity whereas others (e.g., ATAZANAVIR) do NOT possess such an effect. Clinically, advantage of the potent effects of ritonavir can be used to therapeutic advantage by combining a low dose of ritonavir with other agents to prolong their persistence in the plasma. All of the protease inhibitors are substrates for P-gp, the energy dependent pump found in the GI wall, liver, and several other important places in the body. Indeed the presence of P-gp in the capillary endothelial cells of the blood brain barrier, together with strong plasma protein binding, serves to limit access of the PIs across the blood-brain-barrier (BBB).

Resistance to protease inhibitors occurs initially in the active site of the enzyme, with secondary mutations sometimes arising at distant sites on the enzyme.

Answer 45

Protease inhibitors are a common component of HIV drug regimens for treatment-experienced patients. There are a number of caveats to their use. These include the ability of some of these agents (e.g., most notably RITONAVIR) to inhibit CYP3A4 metabolic capacity whereas others (e.g., ATAZANAVIR) do NOT possess such an effect. Clinically, advantage of the potent effects of ritonavir can be used to therapeutic advantage by combining a low dose of ritonavir with other agents to prolong their persistence in the plasma. All of the protease inhibitors are substrates for P-gp, the energy dependent pump found in the GI wall, liver, and several other important places in the body. Indeed the presence of P-gp in the capillary endothelial cells of the blood brain barrier, together with strong plasma protein binding, serves to limit access of the PIs across the blood-brain-barrier (BBB).

Resistance to protease inhibitors occurs initially in the active site of the enzyme, with secondary mutations sometimes arising at distant sites on the enzyme.

Question 46

How do BOCEPREVIR, TELAPREVIR work?

Answer 46

These drugs have gained recent approval for the treatment of hepatitis C virus (HCV).

Following infection in the host, the single stranded HCV RNA is translated into a single polyprotein that is subsequently cleaved into 10 proteins by the enzymes signal peptidase, NS2/3 protease, and NS3/4A serine protease.

The small molecule inhibitors prevent formation of several of the critical nonstructural proteins. Unfortunately HCV is a rapidly proliferating infection, meaning that mutations in the viral genome become expanded rapidly; thus drug resistance can occur in relatively short order. A number of mutations in the serine protease confer resistance to both of these drugs.

Question 47

How does Peginterferon alfa work?

Answer 47

Interferon-Mediated Antiviral Activity Occurs via Multiple Mechanisms.

PEGINTERFERON is simply a long-lasting formulation of interferon that is conjugated with polyethylene glycol, thereby increasing the durability in circulation of active drug.

The binding of IFN to specific cell surface receptor molecules signals the cell to produce a series of antiviral proteins. The stages of viral replication that are inhibited by various IFN-induced antiviral proteins are shown. Most of these act to inhibit the translation of viral proteins (mechanism 2), but other steps in viral replication also are affected (inhibition of transcription, maturation, etc.). The roles of these mechanisms in the other actions of IFNs are under study.

Question 48

How do INHIBITORS OF RELEASE OF INFLUENZA VIRUS FROM INFECTED CELL

ZANAMIVIR, OSELTAMIVIR
work?

Answer 48

These drugs are potent and selective sialic acid analogue inhibitors that produce conformational changes in the active site of influenza A and B neuraminidases. These viral enzymes are essential for destruction of terminal sialic acid residues, thus destroying the receptors recognized by viral hemagglutinin that are present on the cell surface, in progeny virions, and in respiratory secretions. Such an event would permit release of virus from infected cells. Use of the drugs leads to viral aggregation at the cell surface and reduced virus spread within the respiratory tract.

Influenza variants selected in vitro for resistance to oseltamivir carboxylate or zanamivir contain hemagglutinin and/or neuraminidase mutations.

Question 49

PRINCIPLES OF HIV CHEMOTHERAPY

Answer 49

1. All aspects of the disease are derived from viral effects upon CD4+ T-lymphocytes.
2. Long-term suppression of HIV (undetectable plasma HIV RNA levels) and replenishment of CD4+ cells is clinically beneficial.
3. Current HIV drugs require actively replicating virus to be effective and it is unlikely that drug treatment will eradicate all HIV infected cells, especially if the genetic material is harbored in long-lived quiescent T-cells.
4. Estimates are that some of these T-cells will likely survive for decades, possibly for the life of the patient.
5. Drug therapy does NOT cause the mutations; rather it provides selective pressure to promote growth of the naturally occurring mutant viruses.

Question 50

Roles of HIV combination therapy.

Answer 50

1. Use at least 3 drugs simultaneously.

a. Initial treatment will reduce (plasma HIV RNA

Question 51

PRINCIPLES OF HBV & HCV CHEMOTHERAPY

Answer 51

Tenofovir disoproxil fumarate & Entecavir are the preferred agents for HBV polymerase; other orally active agents include Telbivudine, Adefovir dipivoxil, Lamivudine, & Emtricitabine.

Treatment for HCV is defined by the genotype of the infective virus.

Genotype 1 Is treated with Peginterferon-alfa + Ribavirin + Telaprevir or Boceprevir HCV NS3/4A protease inhibitors.

For genotypes 2 & 3, only Peginterferon-alfa + Ribavirin are used.

Question 52

ANTI-INFLUENZA CHEMOTHERAPY

Answer 52

In the 2008–2009 influenza season, virtually all strains of influenza A H1N1 were resistant to the neuraminidase inhibitor oseltamivir. This has arisen as a consequence of overuse of these drugs, including in veterinary applications.

Fortunately the majority of 2009 H1N1 “swine” viruses were susceptible to oseltamivir; however, sporadic cases of oseltamivir resistance have been detected even with this strain. All such viruses tested to date were resistant to the adamantine antivirals, amantadine and rimantadine.

Development of resistance to these drugs, and the spread of resistant viruses, is a major challenge in the chemotherapy and chemoprophylaxis of influenza and are likely to drive future recommendations for use of these drugs in global populations.