Medical Pharmacology Chapter 36:  Antiviral Drugs

• Viral Replication

  • The first step in viral replication is specific association with the virion and cell surface receptors.¹ 

    • Specific virion binding is mediated by cell surface receptors consisting of target proteins, lipids and carbohydrates.

    • Viral surface proteins may preferentially associate with particular target cell glycoproteins and glycolipids.

    • Some viruses bind to cell surface proteins whose expression is limited to particular cell types sometimes species-specific.

      • In this case of viral "tropism" is directed to particular organisms and tissues.

        • Sometimes viruses first bind to a less specific primary receptors such as a carbohydrate and then bind to a more specific cell surface protein receptor.¹

        •  These higher affinity, more specific binding sites, appear especially important for viral infection.² 

          • Furthermore, receptor binding may be assisted by interactions involving viral surface proteins and other cell-surface proteins as co-receptors that may be important in promoting virus entry.²  

            • One example of receptor + co-receptor interactions is found in the HIV envelope glycoprotein which binds first to the T cell surface CD4 protein and then subsequently interacts with a chemokine receptor, e.g. the β-chemokine receptor,  CCR5, which functions as a co-receptor.²  

            • Mechanism of HIV Viral Entry³

              Jones M HIV Membrane Fusion Panel (first two steps, see original reference for all steps). "1.  Initial interaction between gp120 and CD4. 2. Conformation changes in gp120 allows for secondary interaction with CCR5.3 http://en.wikipedia.org/wiki/HIV#mediaviewer/File:HIV_Membrane_fusion_panel.svg ; http://en.wikipedia.org/wiki/HIV

          • Similarly, Epstein-Barr virus (EBV) glycoprotein gp350 first binds to the B lymphocyte complement CD21 receptor and then uses MHC class II (Major Histocompatibility Complex) as a co-receptor.²

    • Viral-Cell Membrane Fusion:  The association of viral surface protein receptors often causes a conformational change which promotes virus in cell fusion along with formation of a pore that enables viral nucleocapsid delivery into the cell cytoplasm.²  

       

      • Viral Membrane Fusion⁸

        Attribution:  Harrison S  Part 2:  Viral membrane fusion iBiology https://www.youtube.com/watch?v=qcepGvFUM38 (3/2012) iBiology YouTube Channel:  https://www.youtube.com/channel/UCsvqEZBO-kNmwuDBbKbfL6A

      •  

        "Fusion of virus membrane with host cell membrane"⁵

        Three steps of viral-cell membrane fusion: 1.  "Receptor binding" 2. "Virus with host membrane fusion" 3. "Release of virion contents into host cytoplasm." Attribution:  Swiss Institute of Bioinformatics © ViralZone 2011 http://viralzone.expasy.org/all_by_protein/987.html

    • An alternative mechanism employed by some envelope viruses as well as certain non-envelope viruses involve endocytosis, as opposed to membrane fusion.²

    • Endosomal low pH promotes viral membrane or capsid fusion with the endocytic membrane.²

      • Influenzavirus provides an example of low pH effects on viral penetration.

      • Influenza hemagglutinin promotes viral absorption, receptor aggregation and endocytosis.

        • At low pH values endosomes favor structural changes of hemagglutinin, exposing protein domains which interact with cell membranes, thus allowing viral fusion with cell membranes.

          • The M2 membrane protein is central in viral envelope uncoating for influenza.

        • An example of the importance of viral protein fusion with cell membranes in the development of pharmacological antiviral approaches is noted in HIV infection.

          • In that case the association of the HIV gp120 protein and cellular CD4 (along with chemokine receptors) induces structural changes which allow the HIV gp41 protein to begin the cell membrane fusion process.

        •  

          "Schematic diagrams of HIV-1 and influenza virus fusion and entry processes"⁴

          "Major conformational changes in the viral envelope glycoproteins occur for fusion of both HIV-1 and influenza virus, but the requirements for fusion differ, as do the viral target cell types (not shown)." "For HIV-1, gp120 bind sequentially to its primary receptor CD4 and, after an initial conformational change, to co-receptors (chemokine receptor) CCR5 or CXCR4 (not shown) (step 1a)." "Co-receptor interaction triggers fusion of the viral and cellular membranes, initiated by the HIV-1 fusion peptide that is located in gp41 (step 2a). Fusion and entry of the HIV-1 genomic RNA and accompanying viral proteins into the target cell occurs at the cell surface at neutral pH." "Following entry, the HIV-1 RNA genomes are transcribed by reverse transcriptase into DNA (step 3a) and the HIV pre-integration complex is transported to the nucleus for integration into target cell genomic DNA to initiate chronic infection. On different cell types from those affected by HIV-1, influenza virus binds via hemagglutinin 1 (HA1) to terminal sialic acids present on glycoproteins or on glycolipids (step 1b). The virus is subsequently internalized by receptor-mediated endocytosis into a low pH compartment (endosome), triggering conformational changes that exposed the viral fusion peptide that is located in HA2 (step 2b). Subsequently, the genomic ribonucleoprotein complexes transported to the nucleus to initiate transcription and replication of the viral genome (step 3b)." Attribution:  Figure 4 from Karlsson GB Fouchier RAM Phogat S Burton DR Sodroski J Wyat RT Nature Reviews Microbiology 6, 143-155 (February 2008).4  http://www.nature.com/nrmicro/journal/v6/n2/fig_tab/nrmicro1819_F4.html

          • The drug enfuvirtide, derived from gp41, binds to gp41 in a way that prevents the structural change needed for fusion.

            •  

              Site of Enfuvirtide (Fuzeon) Anti-Viral Activity⁹

              a: The lifecycle of HIV-1, showing the step at which enfuvirtide acts, and also the steps targeted by other drugs.9  b:  "Simplified schematic of the mechanism by which enfuvirtide inhibits viral entry into host cells.9    The envelope glycoprotein of HIV-1 consists of two non-covalently associated subunits, gp120 and gp41. After attachment of HIV-1 to its target cells carrying the CD4 receptor, gp120 interacts with the CD4 receptor, which initiates a series of conformational changes in gp41 and gp120 that lead to the insertion of a region of gp41 into the membrane of the host cell, and formation of a ' pre-hairpin intermediate' (top). Further changes in the confirmation of gp41 bring the viral and cellular membranes into close enough proximity for membrane fusion (bottom left). Enfuvirtide binds to a region of gp41 that mediates this conformational change from pre-hairpin intermediate to the fusion-active structure, thereby preventing fusion and viral entry (bottom right). Abbreviations: NNRTI, non-nucleoside reverse transcriptase inhibitor; NRTI, nucleoside reverse transcriptase inhibitor; PI, protease inhibitor" Attribution:  Slight modification of Figure 1 from:  LaBonte J Lebbos J Kirkpatrick nature Reviews Drug Discovery 2, 345-346 (May 2003).9  Original figure attribution before modification:  Kilby JM Hopkins S Venetta TM DiMassima B Cloud GA Lee JY Alldredge L Hunter E Lambert D Bolognesi D Matthews T Johnson MR Nowak MA Shaw GM Saag MS Potent suppression of HIV-1 replication in humans by T-20, a peptide inhibitor of gp41-mediated virus entry. Nat. Med 4(11): 1302-1307, 1998.

             

            • Enfuviritide (Fuzeon) Pharmacology

              Enfuviritide (Fuzeon) is a synthetic peptide consisting of 36 amino acids.6    Enfuviritide (Fuzeon) Part of its amino acid sequence was modelled  based on the sequence of the transmembrane gp41 HIV-1 protein important in viral membrane fusion with the target cell membrane. Enfuviritide exhibits selectivity against HIV-1 laboratory and clinical variants; moreover, this drug is not active against HIV-2. The mechanism of anti-HIV activity was determined associated with inhibition of HIV-mediated membrane fusion. Enfuviritide exhibits a unique pharmacodynamic anti-retroviral mechanism, blocking the interaction between the N36 and C34 sequences of the gp41 glycoprotein.6   The peptide inhibition is due to association in the hydrophobic groove associated with the N36 coil. Because of its distinctive mechanism of antiretroviral action, enfuviritide may continue to exhibit antiretroviral activity in patients in which their viral isolates exhibit resistance to antiretroviral drugs associated with other classes. However, HIV-enfuvirtide resistance may develop as a result of mutations in the enfuvirtide- gp41 binding domain. Patients exhibiting virologic failure while on enfuvirtide treatment usually (94%) present with mutations in this gp41 region (in vitro). Single amino acid substitutions at one of two common mutation sites may increase resistance by 450 fold in laboratory tests. However,  clinical resistance typically presents with two or more amino acid alterations. Enfuviritide (Fuzeon) was as of 2011 the only approved antiretroviral agent requiring parenteral administration, with the bioavailability of subcutaneously administered enfuvirtide estimated at about 80% compared to the IV dose.6  The approximate elimination half-life (t½) of the parenteral drug is about four hours, thus requiring twice-daily dosing. Adverse effects:6   Injection site reactions represent the most prominent adverse effect with nearly all patients exhibiting injection site pain, erythema and induration. About 5% of patients discontinue therapy due to these local reactions. Clinical indications:6   Enfuvirtide (Fuzeon) is FDA-approved only for use in those adults exhibiting HIV replication despite ongoing antiretroviral drug treatment. Adding enfuvirtide to and optimize antiretroviral treatment plan increases the fraction of patients with undetectable plasma HIV-1 RNA concentrations evaluated after six months of treatment. The magnitude of this effect is described as an increase from about 6% of patients with undetectable plasma HIV-1 RNA to about 16%. In this consideration undetectable plasma HIV-1 RNA is defined as <50 copies/cc.   Background:  Mechanism of Drug Action7 Gp41 and gp120 constitute the envelope (Env) glycoprotein in HIV-1. Gp120 is the surface subunit binding to host cell receptors; whereas gp41 is the transmembrane unit that inserts into host cell membrane promoting membrane fusion. Gp41 exhibits two helical regions with one residing near the amino terminus (N helix) and one near the carboxyterminus (C helix). These two helices interact in an anti-parallel fashion, like a hairpin bringing the amino and carboxy termini together. The membrane fusion structure gp41 has been described as three gp41 hairpins (trimer-of-hairpins) with the N helices from three gp41 domains forming a central three-stranded coiled coil while being surrounded by three antiparallel C helices.7 Therefore, it may not be surprising that synthetic peptides derived from these regions of gp41 inhibit HIV-1 viral infection.7

          • Another drug, maraviroc, inhibits viral entry by binding to one of the chemokine HIV co-receptors, CCR5, thus inhibiting interaction with gp120 and inhibiting fusion initiation.²