18, 19 - Antiviral Flashcards
Do viruses contain DNA and RNA?
Contain DNA or RNA surrounded by uncomplicated protein coat (serves as cell membrane)
Do viruses have a lipid bilayer?
More complex viruses do
Can viruses replicate on their own?
- No, they require living cells to do so
- Exist at interface of living and non-living organisms
Do viruses conduct their own metabolic processes?
No, dependent on host cell
What happens to viruses during replication in host cells?
Lose organized structure
What are the shapes of viruses?
- Helical
- Polyhedral
- Spherical
What happens when DNA virus enters a host cell?
- Enters nucleus of host cell, where viral DNA is transcribed into mRNA by host cell RNA polymerase
- mRNA is translated into virus-specific proteins that facilitate assembly, maturation, and release of newly formed virus into surroundings
What is the difference between DNA and RNA viruses?
- DNA viruses rely on host cell to synthesize mRNA
- RNA viruses rely on enzymes in the virus itself to synthesize mRNA
What features are used to characterize viruses?
- Nucleic acid content (DNA or RNA)
- Viral morphology (helical, icosahedral)
- Site of replication in cell (cytoplasm or nucleus)
- Coating (enveloped or non-enveloped)
- Serological typing (antigenic signatures)
- Cell types infected (B or T lymphocytes, monocytes)
Estimated that viruses cause __% of infectious diseases in developing countries
60%
Why is development of antivirals difficult?
- Viruses won’t grow in simple culture media
- Need mammalian-derived cell cultures, making drug screening techniques very difficult
- Viruses have much simpler biochemistry than bacteria, meaning fewer potential drug targets
- Viral infections don’t appear until well established, making tx more difficult
What is an antiviral?
Chemical compound that may inhibit various biochemical targets or arrest biochemical events
What are the 7 stages of viral infection?
- Absorption (attachment to specific receptors)
- Penetration (entry of virus into cell)
- Uncoating (release of viral nucleic acid from coat)
- Transcription (production of viral mRNA)
- Translation (synthesis of viral proteins and nucleic acids, using host cell processes)
- Assembly (of viral particles)
- Release (of virus from cell by budding and rupture)
What is chemoprophylaxis?
Administration of medication for prevention of disease or infection
Which drugs are adamantanamines? What is their mechanism?
- Amantadine and rimantadine
- Interfere w/ penetration of host cells by viruses and block early stage replication (uncoating); also affect a later step involved in viral assembly
What are adamantanamines used for?
Prevention and treatment of influenza type A
What are neuraminidase inhibitors?
- Enzymes that function in early activation steps of the virus
- Important in enhancing penetration of viruses into host cells
Which drugs are neuraminidase inhibitors? What does each drug do?
- Zanamivir – inhibits neuraminidase by binding to active site of sialic acid-sugar bond cleavage (guanidino group key for competitive inhibition)
- Oseltamivir – orally active competitive inhibitor (prodrug, ethyl ester allows for oral bioavailability; non-polar 3-pentyl group key for max binding)
What is the function of neuraminidase?
- Found on surface of virus
- Cleaves sialic acid containing receptor
- Neuraminidase specifically catalyzes breakdown of glycosides containing neuraminic acid
- Allows for new virions to be released
What are interferons?
- Extremely potent cytokines that possess antiviral, immunomodulating, and anti-proliferative actions
- Synthesized by host cells in response to various inducers, and elicit anti-viral activity
What happens when interferons bind to cellular receptors?
Induce synthesis of a cascade of antiviral proteins
What do interferons inhibit to have anti-viral effects?
- Viral penetration or uncoating
- mRNA synthesis
- Translation of viral proteins
- Viral assembly and release
Interferons predominantly inhibit ____ synthesis
Protein
Which interferons are used clinically? For what purposes?
- Interferon alpha used in recombinant form
- Interferon beta used for tx of MS
Which drugs are nucleoside antimetabolites?
Acyclovir and valacyclovir
What dosage forms is acyclovir available as? What is it used for?
- Oral, injection, and ointment
- Used for acute tx of herpes simplex (cold sores) and herpex zoster (chicken pox/shingles); and tx of initial episodes of herpes genitalis
- Reduces viral shedding in shingles; also used for herpes simplex encephalitis
- Doesn’t cure but decreases severity and length of outbreak
What is the difference between valacyclovir and acyclovir?
Valacyclovir is a pro-drug of acyclovir
What is the MOA of acyclovir?
- Conversion to active monophosphate by viral thymidine kinase occurs faster by herpes virus infected cells than normal cells
- Competitive inhibitor of viral DNA polymerase, and is incorporated into viral DNA during DNA synthesis; due to lack of “sugar” it terminates DNA elongation
- Preferential uptake in herpes-infected cells
What is adefovir dipivoxil used for? What is its MOA?
- Tx of chronic hepatitis B
- Competitive inhibitor for hepatitis B virus reverse transcriptase, acting as a chain terminator
- Adefovir dipivoxil = prodrug that is converted into adefovir
Which drugs are nucleoside antimetabolites? What is their general function?
- Ganciclovir and penciclovir
- Cidofovir
- Cytarabine and idoxuridine
- Ribavirin
- Trifluorothymidine
- Vidarabine
- Sofosbuvir
- Inhibit viral replication
Nucleoside antimetabolites are analogs of _____ and the modification ______
- Analogs of acyclovir
- Modification maintains activity against HSV, but increases activity against cytomegalovirus (CMV) infections (ganciclovir)
Where are ganciclovir and penciclovir phosphorylated? What happens after this?
- Ganciclovir = phosphorlyated inside host cell; ganciclovir triphosphate incorporated into DNA and stops elongation
- Penciclovir = phosphorlyated by viral thymidine kinase and competitively inhibits DNA elongation
What is cidofovir? What is its active form? What does it do?
- Acyclic pyrimidine nucleotide analog of cytosine
- Metabolized in vivo to active diphosphate
- Inhibits viral replication via DNA synthesis interference
What are cytarabine and idoxuridine? What are their active forms? What do they do?
- Synthetic pyrimidine analogs of cytidine and uridine respectively
- Metabolically converted into active triphosphates
- Incorporated into DNA during replication
- Cytarabine blocks utilization of deoxycytidine
- Idoxuribine replaces thymidine => faulty viral proteins
What is ribavirin? What is its active form? What does it do?
- Guanosine analog w/ broad spectrum antiviral activity (both DNA and RNA)
- Phosphorylated by adenosine kinase
- Inhibits viral RNA polymerase
What is trifluorothymidine? What is its active form? What does it do?
- Fluorinated analog of thymidine
- Triphosphate is incorporated into viral DNA in place of thymidine
- Causes faulty viral mRNA proteins (HSV-1)
What is vidarabine? What is its active form? What does it do?
- Adenosine nucleoside analog
- Triphosphate is active form
- Triphosphate interferes w/ viral nucleic acid replication due to arabinose sugar
What is sofosbuvir? What is it used for?
- Uridine nucleotide analog
- Used in combination w/ other drugs for tx of hepatitis C virus infection
What are the common complications of hepatitis C that lead to death?
Cirrhosis and liver cancer
Which drug is sofosbuvir combined w/ for tx of hepatitis C? Does it treat the virus? How long is the tx?
- Combined w/ ledipasvir
- Provides essentially 100% cure rate
- 12 weeks of tx
Sofosbuvir is a substrate of _____
P-glycoprotein
What is the MOA of the sofosbuvir/ledipasvir combination?
Inhibits NS5A (viral phosphoprotein)
What is the active form of sofosbuvir? What does this do?
- Triphosphate is active
- Serves as a defective substrate for viral RNA polymerase (inhibits RNA polymerase)
What is important about sofosbuvir when comparing it to the nucleoside parent compound?
- Sofosbuvir is masked to neutralize the charge so that it can be taken up into the cell
- Cellular enzymes convert sofosbuvir to the monophosphate much faster than it converts parent nucleoside to monophosphate (so parent isn’t potent)
What is the function of reverse transcriptase enzyme?
Used to generate complementary DNA from an RNA template
What are the activities of retroviral reverse transcriptase?
- RNA-dependent DNA polymerase
- Ribonuclease H (enzyme that catalyzes cleavage of RNA)
- DNA-dependent DNA polymerase
Which drugs are used to control HIV infections?
Reverse transcriptase inhibitors
What is required for the function of reverse transcriptase inhibitors?
Availability of purine and pyrimidine nucleosides and nucleotides
What is special about azidothymidine?
Nucleoside reverse transcriptase inhibitor whose azido group increases lipophilic nature, making it easier to cross cell membrane
What is an additional method to inhibit viral DNA synthesis?
Replace 3’-OH moiety of ribose sugar, which terminates elongation
What is truvada? What is it used for?
- Combination of tenofivir disoproxil and emtricitabine
- Used to prevent HIV infection in high risk individuals (reduces risk of infection by 50-100%)
What happens to tenofovir disoproxil?
Prodrug that is hydrolyzed by esterases to yield monophosphate, which is phosphorylated to active triphosphate
What is the MOA of tenofovir?
- Tenofovir needs to be phosphorylated in 1 or 2 steps to diphosphate form
- It then interferes, as a chain terminator, w/ reverse transcriptase reaction
What is the model used to describe HIV reverse transcriptase?
Hand model
- Has a palm, 3 fingers, thumb, and extra finger (RNAseH)
How can a viral cell develop resistance against nucleoside reverse transcriptase inhibitors?
- Mutation that leads to discrimination or excision of an incorporated NRTI
- Discrimination mutation = amino acid change that reduces selectivity of an NRTI over the correct nucleotide during DNA polymerization
What is involved in the triple therapy for HIV?
- Nucleoside reverse transcriptase inhibitor
- Non-nucleoside reverse transcriptase inhibitor
- Integrase inhibitor or protease inhibitor
What is the benefit to non-nucleoside RTIs vs. nucleoside RTIs?
- NNRTIs don’t require bioactivation by kinases to give phosphate
- Bind to allosteric site of reverse transcriptase
- Exhibit classical non-competitive inhibition pattern
Why do non-nucleoside RTIs bind to allosteric sites of HIV-1 reverse transcriptase?
- HIV-1 reverse transcriptase undergoes a series of conformational changes during viral replication
- Intrinsic flexibility provides novel allosteric sites for inhibition
What does allosteric binding cause?
Distorts enzyme so it can’t form the enzyme-substrate complex at a normal rate
What is the newest method to identify potential allosteric inhibitors?
- X-ray crystallographic fragment screening
- Take crystals of reverse transcriptase and incubate w/ drug fragments using x-ray crystallography, identify binders
Describe the fragment screening for non-nucleoside RTI anti-HIV drugs?
1) Binding site comprised of 3 binding pockets
2) Crystallographic screening locates molecule fragments that bind 1, 2, or all 3 pockets
3) Lead compound is designed by organizing all 3 fragments around a core template
4) Growing out of a single fragment
How does the nucleic acid pass through the HIV reverse transcriptase hand model?
- Nucleic acid template passes between the thumb and finger domains where active site of DNA polymerisation is located
- Extra finger represents RNAseH domain that removes RNA from hybridized DNA:RNA molecules during reverse transcription
Do non-nucleoside RTIs inhibit reverse transcriptase of all retroviruses?
No, selectively inhibits HIV reverse transcriptase
Do non-nucleoside RTIs inhibit mammalian DNA polymerases?
No
Can NRTIs and NNRTIs be used together?
- Yes, produce a synergistic effect
- NRTIs mimic the normal nucleoside bases, so are technically incorporated into any enzyme that is functioning, no matter how slow; NNRTIs slow down the enzyme
What is the main problem w/ NNRTIs? How can this be prevented?
- Rapid emergence of resistance among HIV isolates, resulting from point mutations in gene coding of the enzyme
- Prevented if NNRTI and NRTI are used together
Which drugs are NNRTIs? Which ones must be used w/ at least 2 other anti-retroviral agents?
- Nevirapine
- Delavirdine *used w/ other agents
- Efavirenz *used w/ other agents
How are NNRTIs inactivated?
Hydroxylation
Where do mutations in HIV-1 reverse transcriptase?
- Residues in reverse transcriptase polymerase domain that are important in binding and incorporation of nucleotides
- Side chains of residues that interact w/ next incoming nucleotide
- Residues which interact w/ template strand to position it for base pairing w/ nucleotide; causes reduced incorporation of analogs
What does HIV protease do?
Cleaves propeptides into enzymes that function in maturation and propagation of new virus
What does HIV protease look like?
Symmetric dimer of 2 identical 99 amino acid subunits
What are HIV protease inhibitors designed to mimic?
- Transition state of hydrolysis at the active site
- Called analog inhibitors
What is special about analog inhibitors?
- Normal hydrolysis of peptides bonds proceed through an sp3-tetrahedral transition state
- Analog inhibitors possess a pre-existing sp3 hybridized center that is drawn into active site and won’t be cleaved by the enzyme
What is the goal of HIV protease inhibitors?
Arrest replication of virus at maturation step to prevent spread of infection
What are side effects of protease inhibitors?
- Dyslipidemia
- Facial atrophy
- Breast enlargement
- Hyperglycemia
Which drugs are first gen HIV protease inhibitors?
- Saquinavir
- Indinavir
- Ritonavir, amprenavir, nelfinavir
What is sp3 center for all the HIV protease inhibitors?
Phenylalanine-hydroxyl
Which HIV protease inhibitors must be used w/ at least 2 other agents?
Ritonavir, amprenavir, and nelfinavir
Why were second generation HIV protease inhibitors developed?
To inhibit HIV protease species that were resistant to first gen inhibitors (point mutations)
Which drugs are second gen HIV protease inhibitors?
- Lopinavir
- Atazanivir
- Tipranavir
- Darunavir
Which gen HIV protease inhibitors are used together? Why?
- Lopinavir (2nd gen) and ritonavir (1st gen)
- Ritonavir inhibits lopinavir’s metabolism by CYP 3A4
What is atazanivir always used w/?
Reverse transcriptase inhibitor
What is special about tipranavir? What is the benefit to this?
- Not a peptidomimetic (mimics peptides), but binds in active site like peptide-PI
- Benefit = cross-resistance doesn’t develop
What is the important portion of darunavir?
Bis-tetrahydrofuranyl moiety to form crucial hydrogen bond interactions in active site
Does second gen HIV protease inhibitors still contain phenylalanine-hydroxyl sp3 center?
Yes, except tipranavir b/c not peptidomimetic
What is the importance of the sp3 center for HIV protease inhibitors?
Acts as the intermediate for hydrolysis reaction
What is the function of retroviral integrase? What do integrase inhibitors do?
- Integrase enables its genetic material to be integrated into DNA of the infected cell
- Inhibitors block strand transfer catalyzes by retroviral integrase enzyme
Which drugs are integrase inhibitors?
- Raltegravir
- Elvitegravir
What is the primary metabolism of raltegravir?
Glucuronidation
What does raltegravir do?
Binds to viral integrase and prevents DNA transfer function that inserts viral DNA into host DNA
What must integrase inhibitors contain and why?
- A dicarbonyl functional group for activity, metal binding, and strand transfer
- A benzyl moiety to bind hydrophobic pocket
How does HIV enter a host cell?
- Glycoprotein gp120 mediates virus attachment to 2 membrane receptors (CD4 and chemokine co-receptor)
- Bivalent interaction induces a conformational change in gp41 acting as an anchor, fusing viral envelope w/ host cell membrane
- CCR5 (chemokine receptor 5) protein on surface of WBCs is chemokine co-receptor used by HIV for entry
Which drug is a CCR5 receptor antagonist? What does it do? What test must be done first?
- Maraviroc
- Drug binds to CCR5, blocking HIV gp120 association and entry
- HIV tropism test must be done to see if drug will be effective
What are the other cell entry inhibitors?
Only maraviroc currently
What is enfuvirtide? What does it do?
- HIV fusion inhibitor
- Binds to gp41 preventing creation of entry pore for capsid of the virus
- Biomimetic peptide designed to mimic components of HIV-1 fusion machinery and displace them, preventing normal fusion
When is enfuvirtide used?
For px where all other treatments have failed
