In The Name of God

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In The Name of God
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Antiviral Agents
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Introduction

• Viral Structure
• Viruses range in size from 10 to 450 nanometers
  in diameter.
• A typical virus consists of nucleic acid which
  can be DNA (DNA viruses) or RNA (RNA viruses,
  retroviruses) and may be single or double
  stranded, circular or linear, fragmented or
  continuous.
• Along with the nucleic acid there is a protein
  coat known as a capsid which surrounds it. This
  capsid is highly ordered and generally consists
  of repeating protein subunits called capsomers.

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• The entire structure of the nucleic acid and
  capsid is called nucleocapsid.
• Some viruses have an extra layer of protection
  known as an envelope surrounding the capsid. The
  envelope is a lipid bilayer that is taken from a
  host cell as the virus buds off and is released.
• The most important part of the outer coat in
  terms of infection (whether protein or lipid) is
  Signaling Molecules that are important virus
  antigens.
• These glycoprotein signals are recognized by cell
  surface receptors located on host cells and tell
  the cell to admit the virus.

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• The arrangement of coat proteins defines the
  overall shape of the viruses. Spheres, rods,
  filaments, rectangles, triangles, and elongated
  tubes are some of the shapes of viruses.
• The complete infectious virus is called a virion.

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Viral Attachment and Penetration to the Host Cell

• The virus attaches to the target cell, usually
  through specific protein-protein interactions
  between capsid and cell surface receptors.
• It is then internalized via endocytosis,
  phagocytosis, or fusion. Internalization can take
  from a few minutes to several hours to complete.
• Once inside the cell, the virus is uncoated in a
  process that frees the viral genome from its
  protective protein layer. The liberated genome is
  then transported to the nucleus.

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• Hemagglutinin is the signaling protein found on
  the envelope, or coat, of the virus that together
  with an enzyme called neuraminidase helps the
  virus to lock on and invade its target cells.

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• Most viruses enter the cell via receptor-mediated
  endocytosis. However, some viruses are simply
  taken up through phagocytosis or fusion (only if
  enveloped).

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• Others inject their genome through the cell
  membrane, leaving the empty capsid outside the
  cell.

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Signaling Proteins

• Haemagglutinin
• Haemagglutinin (HA or H) is a glycoprotein
  containing either 2 of 3 glycosylation sites,
  with a molecular weight of approximately 76,000.
  It spans the lipid membrane so that the major
  part, which contains at least 5 antigenic
  domains, is presented at the outer surface.
• HA serves as a receptor by binding to sialic acid
  (N-acetyl-neuraminic acid) and induces
  penetration of the interior of the virus particle
  by membrane fusion.
• Haemagglutinin is the main influenza virus
  antigen the antigenic sites being A, B (carrying
  the receptor binding site), C, D, and E.

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• The antigenic sites are presented at the head of
  the molecule, while the feet are embedded in the
  lipid layer.
• The body of the HA molecule contains the stalk
  region and the fusiogenic domain which is needed
  for membrane fusion when the virus infects a new
  cell.
• At low pH, the fusion peptide is turned to an
  interior position. The HA forms trimers and
  several trimers form a fusion pore.

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• Neuraminidase (Sialidase)
• Like HA, neuraminidase (NA or N) is a
  glycoprotein, which is also found on the surface
  of the virus. It forms a tetrameric structure
  with an average molecular weight of 220,000. The
  NA molecule presents its main part at the outer
  surface of the cell, spans the lipid layer, and
  has a small cytoplasmic tail.
• NA acts as an enzyme, cleaving sialic acid from
  the HA molecule and from glycoproteins and
  glycolipids at the cell surface. It also serves
  as an important antigenic site, and in addition,
  seems to be necessary for the penetration of the
  virus through the mucin layer of the respiratory
  epithelium.

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Adsorption of The Virus

• The influenza virus binds to the cell surface by
  fixing the outer top of the HA to the sialic acid
  of host cell glycoproteins and glycolipids. Since
  sialic acid-presenting carbohydrates are present
  on several cells of the organism, the binding
  capacity of the HA explains why multiple cell
  types in an organism may be infected.
• Entry of the virus
• After attachment, the virus is taken up by the
  cell via endocytosis process.
• When internalised, the vesicle harbouring the
  whole virus fuses with endosomes.
• The contents of the vesicle are usually digested
  through a stepwise lowering of the pH within the
  phagosome.

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Uncoating of The Virus

• M2 protein
• M2 protein is a protein on the virus surface
  which forms a channel in it allowing the entrance
  of ions to lower the pH of the endosomal
  compartment, a process which is essential to
  induce conformational changes in the HA protein
  to permit membrane fusion.
• When the virus particle is taken up in the
  endosome, the activity of the M2 ion channel is
  increased so that ions flood into the particle,
  inducing a low pH.
• As a result of this the particle opens, the
  fusion peptide within the HA is translocated, and
  the HA fuses with the inner layer of the endosome
  membrane.
• The ribonucleoproteins are liberated into the
  cytoplasm of the cell and transported to the
  nucleus, where viral RNA synthesis is initiated.

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Replication of an RNA virus.

• Many viruses have evolved their own specific
  enzymatic mechanisms to preferentially replicate
  virus nucleic acids at the expense of cellular
  molecules.
• Once inside the cell, the virus utilizes cellular
  machinery to replicate.
• Most viruses lack a polymerase, which is needed
  to copy the genome. Also missing from a virus are
  ribosomes and the golgi apparatus, which are
  needed for protein processing.
• Cells provide all of these for the virus,
  allowing them to be extremely compact.

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The Replication of an RNA Virus
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Budding

• After the replication of DNA and RNA viruses, the
  release of viruses may occure after lysis of the
  host cell or by budding from the cell.
• The latter process is less harmful to the host
  cell.
• The time from entry to production of new virus is
  on average 6 h.

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General Approaches to Viral Drug Therapy

• Because of the unique properties of viruses and
  the need for host cell metabolic activities,
  antiviral agents have been developed to act at
  various stages in the viral replication cycle
  such as attachment, replication and release of
  virus.
• General approaches for treating viral infection
  by antiviral agents are

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• Inhibition or interference of virus attachment to
  host cell receptor, virus penetration and
  uncoating.
• Inhibition of virus-associated enzymes, such as
  DNA polymerases and others.
• Inhibition of transcription processes.
• Inhibition of translation processes.
• Interference with viral regulatory proteins.
• Interference with assembly of viral proteins.
• Interference with release of virus from cell
  surface membrane.

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Agents Inhibiting Virus Attachment, Penetration
and Early Viral Replication

• Amantadine HCl
• A symmetric tricyclic primary amine
  1-adamantanamine HCl.
• It inhibits penetration of RNA virus particles
  into the host cell.
• It also blocks the uncoating of the viral genome
  and the transfering of nucleic acid into the host
  cell.
• It is used in preventing and treating all
  straines of influenza, and to a lesser extent,
  German measles.
• CNS side effects such as nervouseness, confusion,
• headache, drowsiness, insomnia and
  deppression.

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• Amantadine blocks the pore of the M2 ion channel
  located within the virus envelope preventing
  virus uncoating.

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Rimantadine HCl

• It is structurally and pharmacologically related
  to amantadine.
• It appears more effective than amantadine HCl
  against influenza A virus with fewer CNS side
  effects.
• It interferes with virus uncoating.

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Neuraminidase Inhibitors

• NA is an enzyme involved in various aspects of
  activation of influenza viruses.
• It is involved in catalytically cleaving
  glycosidic bonds between a terminal sialic acid
  and an adjacent sugar on HA.
• This cleavage facilitates the spread of viruses.
• In the absence of this cleavage, viral binding to
  HA will occure interfering with the spread of the
  infection.

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• It is believed that the hydrolysis of sialic acid
  proceeds through an oxonium cation stabilized
  carbonium ion.
• Mimicking the transition state with novel
  carbocyclic derivatives of sialic acid has led to
  the development of transition-state-based
  inhibitors.

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Mechanism of Neuraminidase Actionin Sialic Acid
Hydrolysis
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• The first of such compounds, 2-deoxy
  2,3-dehydro-N-acetylneuraminic acid (DANA) was
  found to be an active NA inhibitor.

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• Crystallographic studies of DANA bound to NA
  defined the receptor site to which the sialic
  acid binds.
• These results suggest that substitution of the
  4-hydroxy with an amino group (oseltamivir) or
  the larger guanidino
• group (Zanamivir)
• should increase binding
• of the inhibitor to NA.

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• The 4-amino derivative (oseltamivir) was found to
  bind to a glutamic acid (Glu 119) in the receptor
  through a salt bridge.
• The guanidino group (in zanamivir) was able to
  form both a salt bridge to Glu 119 and a
  charge-charge interaction with a glutamic acid at
  position 227.
• The result of these substitution was a dramatic
  increase in binding capacity of the amino and
  guanidino derivatives to NA and effective
  competitive inhibition of the enzyme.

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Therapeutic Agents

• Zanamivir
• It is an effective antiviral agent against
  influenza A and B virus when administered via the
  nasal, intraperitoneal, and intravenous routes.
• Oseltamivir Phosohate
• It has additional site to bind to NA involving
  the C-5 acetamido carbonyl and an arginine (Arg
  152), the C-2 carboxyl and arginines as 118, 292,
  and 371, and the potential for hydrophobic
  binding to substituents at C-6 (with glutamic
  acid, alanin, arginin, and isoleucin).
• SAR studies showed the maximum binding to NA when
  C-6 was substituted with 3-pentyloxy as found in
  oseltamivir. Esterification with ethanol gave
  rise to a orally active compound.
• It is effective against influenza A and B.

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Receptor Binding for the Transition-State-Based
Carbocyclic Sialic Acid Analogues
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Agents Interfering With Viral Nucleic Acid
Replication

• Acyclovir
• Acyclic carbohydrate analogue of deoxyguanosine.
• Mechanism of action
• Conversion to monophosphate by viral thymidine
  kinase. Then further phosphorylation to di- and
  triphosphate by guanosine monophosphate kinase.
• Competitive inhibition of viral DNA polymerase,
  acyclovir triphosphate lacks the 3-OH of a
  cyclic sugar so it terminates further elongation
  of the DNA chain.
• Preferential uptake of acyclovir by
  herpes-infected
• cellsas compared to uninfected cells
  results in a
• higher concentration of it in infected
  cells.

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• Valacyclovir
• It is an amino acid (valine) ester prodrug of
  acyclovir, which exhibits antiviral activity only
  after metabolism.
• It has an increased GI absorption.
• As with acyclovir, valacyclovir is active against
  HSV-1, VZV and CMV because of its affinity for
  the enzyme thymidine kinase encoded by the virus.

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Metabolic Reactions of Valacyclovir and Acyclovir
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• Cidofovir
• A phosphorylated acyclic purine nucleotide
  analogue.
• It is additionally phosphorylated by host cell
  enzymes to its active intracellular metabolite,
  cidafovir diphosphate.
• It has antiviral activity by interfering with DNA
  synthesis. And inhibiting viral replication.
• Active against HSV-1, HSV-2, VZV, CMV and EBV.

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• Famciclovir
• A purine nucloside analogue related to guanine, a
  pro-drug of penciclovir.
• Famciclovir and its metabolite, penciclovir
  triphosphate possesses antiviral activity
  resulting in inhibition of viral DNA polymerase.
• Its pharmacologic and microbiologic activities
  are similar to acyclovir.

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• Ganciclovir
• An acyclic deoxyguanosine analogue of acyclovir
  that inhibits DNA polymerase.
• Its active form is ganciclovir triphosphate which
  inhibits viral enzyme more than the host enzyme.
• The mechanism of action is similar to that of
  acyclovir.
• Greater activity than acyclovir against CMV and
  EB.

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• Idoxuridine
• Analogue of thymidine.
• Its active form is idoxuridine triphosphate.
• Antiviral mechanism as before.
• In the treatment of HSV keratocunjunctivities and
  genital HSV infections.

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Some more nucleoside analogues interfering with
viral nucleic acid synthesis
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Antiretroviral (Anti-HIV) Agents Including RT
Inhibitors

• Replication of RNA viruses is different from that
  of the DNA viruses.
• In RNA viruses a reverse information flow exist
  RNA DNA
• The enzyme responsible for this replication is
  called reverse transcriptase (RT, RNA-directed
  DNA polymerase).
• The synthesis of viral DNA under the direction of
  RT requires availability of purine and pyrimidine
  nucleosides and nucleotides.
• A variety of chemical modifications of the
  natural nucleosides have been investigated.

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• Two such modifications have resulted in active
  drugs
• Removal of the 3-hydroxyl group of the
  deoxynucleosides has given rise to
  dideoxyadenosine (didanosine is the pro-drug for
  this drug), dideoxycytidine, and
  didehydrodideoxythymidine.
• Replacement of the 3-deoxy with an azido group
  has given
• 3-azidothymidine and 3-azidouridine.
• All of these drugs have similar mechanisms of
  action in that their incorporation into the viral
  DNA will ultimately lead to chain terminating
  blockade due to the lack of a 3-hydroxyl needed
  for the DNA elongation.
• All are changed to triphosphates before
  incorporation.

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Examples of Nucleoside and NonnucleosideRT
Inhibitors (NRTIs, NNRTIs)
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• Nevirapine binds directly to RT. Thus, it blocks
  RNA- and DNA-dependent polymerase activities by
  causing a disruption of the enzymes catalytic
  site.

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HIV Protease Inhibitors

• HIV protease is an enzyme that is essential for
  viral growth.
• It is responsible for the post-translation
  modification of core proteins into structural
  viral proteins.
• HIV protease exists as a dimer in which each
  monomer contains one of two-conserved aspartic
  residues at the active sites.
• Drugs are designed as transition-state mimetics
  to align at the active site of HIV-1 protease, as
  defined by three-dimensional crystallographic
  analysis of this molecule. The best-fit compounds
  replace amino acids near the active site.

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Some HIV Protease Inhibitors

• Saquinavir

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Saquinavir interactions with HIV protease active
site. Blue important hydrogen bond
interactions. Red interactions with
specificity sites of the protease.
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Indinavir interactions with HIV protease active
site. Blue important hydrogen bond
interactions. Red interactions with
specificity sites of the protease.
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Ritonavir interactions with HIV protease active
site. Blue important hydrogen bond
interactions. Red interactions with
specificity sites of the protease.