What does the transition state of the neuraminidase-catalyzed reaction look like?

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What does the transition state of the
neuraminidase-catalyzed reaction look like?
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• Note that a key step in the previous mechanism is
  the loss of an alkoxy (sugar) moiety from the
  position (C2) next to the carboxylate group.
• This creates a carbocation (Sn1 process), which
  is sp2 hybridized.
• The initial substrate is sp3 hybridized at C2
• Thus it was decided to try to synthesize and test
  compounds which had a double bond to C2, with the
  prospect of identifying something which bound
  tighter than the substrate and which could,
  therefore, function as an effective inhibitor.

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Chemical evolution of neuraminidase inhibitors

• Note that lower Ki values correspond to more
  active inhibitors
• The final product, Relenza (Zanamivir), has a
  positively charged guanidinium cation
  (southern end of molecule)
• Thus, it is too polar to be absorbed orally and
  must be administered by inhalation.

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Further chemical evolution of neuraminidase
inhibitors
While the C5 substituent (glycerol side chain)
bound to a polar pocket, some of the more recent
analogs have shown there is also a hydrophobic
pocket, which can bind more hydrophobic C5 side
chains, such as the tertiary amide side chain of
I.
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• Note that the actual reaction intermediate
  (lower left) involves a resonance-stabilized
  carbocation (with oxygen providing electrons to
  stabilize the C2 carbocation.
• Thus it was decided that the double bond of
  Relenza (Zanamivir) might be in a somewhat
  incorrect position for optimal binding.
• To prepare a stable compound with the double
  bond in the optimal position, they had to replace
  the oxygen of the six-membered ring with a carbon
  (called a carbon isostere).
• Note the improvement in activity as the double
  bond position is altered.

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Further chemical evolution of neuraminidase
inhibitors

• Thus a series of compounds was prepared having
  an appropriately placed CC and an adjacent
  hydrophobic group (in this case a substituted
  ether linkage).
• The most active of these (above) has a branched
  side chain (3-pentyl side chain) on the ether.
• Due to the improvement in inhibitory activity,
  it was possible to remove the highly polar
  guanidinium side chain and replace it with a
  slightly less polar amine side chain.

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Evolution leading to the final product, Tamiflu.

• The carboxylic acid of GS 4071 is still too
  polar.
• Thus, they replaced the acid with an ester,
  which can be hydrolyzed by esterases.
• Compounds related to II (above) are currently in
  clinical trials. These seem to show a further
  improvement in selectivity against viruses.

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Other Drugs to Treat Influenza
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Antiviral Drugs

• amantadine (Symmetrel) used prophylactically
  against influenza A in high-risk individuals.
• rimantidine (Flumadine) used for treatment and
  prophylaxis of influenza A.

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Mechanism of Action of Amantadine and Rimantadine

• These agents were discovered by random screening
  and are now known to interfere with a viral ion
  channel called matrix (M2) protein.
• This causes an inhibition of uncoating of the
  virus.
• At high concentrations, they also buffer the pH
  of the endosomes and prohibit the acidic
  environment needed for Hemagglutinin (HA) to fuse
  the viral membrane with that of the endosome.

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What is HSV?

• HSV Herpes Simplex Virus
• HSV-1 is the usual cause of cold sores
• HSV-2 is the usual cause of genital herpes
• Both types look the same under the microscope and
  share about 50 of their DNA.

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What is the difference between HSV-1 and HSV-2?

• Both types infect the bodys mucosal surfaces,
  usually mouth or genitals, and then establish
  latency in the nervous system.
• For both types, at least two-thirds of the
  infected people have no symptoms, or symptoms too
  mild to notice.
• However, both types can recur and spread, even
  after a period in which there were no symptoms.

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mlLinkLink
HSV-1 and HSV-2
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• HSV spends much of its time hiding in a latent
  form.
• The virus reproduces efficiently in epithelial
  cells and it hides in nerve cells.

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• HSV-1 usually establishes latency in the
  trigeminal ganglion, a collection of nerve cells
  near the ear. Then it tends to recur on the
  lower lip or face.

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HSV-2 usually establishes latency in sacral
ganglion at the base of the spine. From there,
it recurs in the genital area.
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• For a virus, the herpes genome includes an
  extremely large number of genes (at least 74
  genes, or open reading frames ORFs).
• About half of these genes code for proteins that
  are involved in evading host defenses.

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HSV-1

• One can have HSV-1 both genitally and orally.
• HSV-1 is usually mild, especially when it infects
  the lips, face, or genitals.
• However, HSV-1 can recur in the eye, causing
  ocular herpes, which can lead to blindness, and
  can even spread spontaneously to the brain,
  causing herpes encephalitis, which can lead to
  death.

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HSV-2

• 22 of adult Americans have HSV-2
• Like HSV-1, HSV-2 symptoms are usually mild, so
  mild, in fact, that two-thirds of infected people
  dont know they have it.
• HSV-2 rarely causes complications or spreads to
  other parts of the body.
• Oral HSV-2 infections are rare. But even when an
  infection does occur, recurrent oral outbreaks
  are uncommon.

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Transmission of HSV-2

• In the first year of HSV-2 infection, people shed
  the virus from the genital area about 6 to 10 of
  those days when they are asymptomatic. This
  decreases over time and can also be further
  lessened by the use of oral medication. Sex
  should be avoided in the presence of symptomatic
  lesions.

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• Having a previous HSV-1 infection seems to
  provide some immunity to an HSV-2 infection.
  This is probably the reason that oral HSV-2
  infections are rare, given the studies which show
  that a significant proportion of the population
  practices oral sex.

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How severe an infection?

• HSV is a lifelong illness
• But HSV-2 usually produces only mild symptoms or
  signs or no symptoms at all.
• However, HSV-2 can cause recurrent painful
  genital sores in many adults, and HSV-2 infection
  can be severe in people with suppressed immune
  systems.
• Another factor is how long a person has had the
  infection. It seems to decrease in severity over
  time, for reasons which are unclear.

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Symptoms

• If signs and symptoms occur during the first
  episode, they can be quite pronounced. The first
  episode usually occurs within two weeks after the
  virus is transmitted, and the sores typically
  heal within two to four weeks.
• Other signs and symptoms during the primary
  episode may include a second crop of sores, or
  flu-like symptoms, including fever and swollen
  glands.

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Is there a cure?

• There is no treatment that can cure herpes, but
  antiviral medications can shorten and prevent
  outbreaks during the period of time the person
  takes the medication.

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Vaccines?

• No vaccine for HSV-2 is currently available.

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DNA Virus

• Recall that HSV is a DNA virus (influenza was an
  RNA virus)
• In general, more drugs are available to treat DNA
  viruses than for RNA viruses (excluding those
  used to treat HIV).
• Most of the drugs available for treatment of DNA
  viruses have been developed against
  herpesviruses.
• Diseases include cold sores, genital herpes,
  chickenpox, shingles, mononucleosis, etc.

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Antiviral Chemotherapy for HSV

• There are several prescription antiviral
  medications for controlling herpes outbreaks,
  include acyclovir (Zovirax), valacyclovir
  (Valtrex), famcyclovir (Famvir), and pencyclovir.
  
• Acyclovir was the original and prototypical
  member of this class
• Valacyclovir and famcyclovir are prodrugs of
  acyclovir and pencyclovir respectively, with
  improved oral bioavailability.

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Chemotherapy for HSV
Acyclovir (Zovirax)
Valacyclovir (Valtrex),
pencyclovir
Famcyclovir (Famvir),
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Gertrude B. Elion won the Nobel prize in
Physiology or Medicine in 1988, partly for the
discovery of acyclovir.
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Drugs Discovered by Gertrude B. Elion Include

• 6-mercaptopurine (Purinethol), the first
  treatment for leukemia.
• Azathioprine (Imuran), the first
  immuno-suppressive agent, used for organ
  transplants.
• Allopurinol (Zyloprim), for gout.
• Pyrimethamine (Daraprim), for malaria.
• Trimethoprim (Septra), for meningitis,
  septicemia, and bacterial infections of the
  urinary and respiratory tracts.
• Acyclovir (Zovirax), for viral herpes.

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Mechanism of Action of Antivirals to treat HSV

• Both acyclovir and pencyclovir work by
  interfering with viral replication, effectively
  slowing the replication rate of the virus, and
  providing a greater opportunity for the immune
  response to intervene.
• All drugs in this class depend on the activity of
  the viral thymidine kinase to convert the drug to
  a monophosphate form and subsequently interfere
  with viral DNA replication.

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Acyclovir (ZOVIRAX)

• Discovered by random compound screening and
  introduced into the market in 1981.
• It was the first non-toxic herpes drug to be used
  systemically.
• It is used for the treatment of infections due to
  both HSV-1 and HSV-2.

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The Mechanism of Acyclovir

• The genome of the Herpes viridae code for the
  production of viral DNA polymerase (rather than
  using the host cell DNA polymerase).
• The drug interferes with the action of the viral
  DNA polymerase, with high selectivity over that
  of the host enzyme.
• Additionally, to be activated the drug must first
  be phosphorylated. This is achieved by viral
  thymidine kinase, which is less selective than
  cellular thymidine kinase.

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• Aciclovir interferes with DNA synthesis, but
  must first become activated.
• To become activated, Aciclovir must be
  phosphorylated (3x)
• However, Aciclovir itself is not a good
  substrate for mammalian kinases, thus it relies
  on the viral thymidine kinase to become
  phosphorylated the first time.
• This is good, since the drug cannot interfere
  with DNA synthesis in cells that are not infected
  with the virus, thus reducing the toxicity of the
  drug.
• The second and third phosphorylations, however,
  are performed by the cellular thymidylate kinase.

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• Aciclovir triphosphate is mistaken for
  deoxyguanosine triphosphate.
• However, since it lacks the 3-OH group, it
  cannot be linked to the adjacent residue in the
  usual fashion.

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ml

• The mechanism of action of Acyclovir is analogous
  to that utilized by Fred Sangers method of DNA
  sequencing.
• http//www.dnalc.org/view/15479-Sanger-method-of-D
  NA-sequencing-3D-animation-with-narration.html
• http//www.biostudio.com/demo_freeman_dna_sequenci
  ng.htm

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Valacyclovir

• Valaciclovir is an esterified version of
  aciclovir, that has greater oral bioavailability.

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Penciclovir

• Penciclovir is used primarily as a cream (it has
  poor oral bioavailability) to treat herpesvirus
  infections.
• It is the active ingredient in the cold sore
  medications Denavir and Fenistil..

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Famciclovir

• Famciclovir is a di-esterified version of
  penciclovir
• The ester groups give the drug better oral
  bioavailability
• To treat shingles, it is taken three times a day
  for seven days.
• To treat genital herpes, it is taken twice a day
  for five days.

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Major members of the herpesvirus group of the
family Herpesviridae

• Herpes simplex viruses HSV-1 and HSV-2
• Cytomegalovirus (CMV)
• Varicella-zoster virus (VZV)
• Epstein-Barr virus (EBV)

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Varicella-zoster virus (VZV)

• The Varicella zoster virus (VZV) is one of the
  eight herpes viruses known to affect humans (and
  other vertebrates). It commonly causes chickenpox
  in children and shingles later in life.

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Shingles
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Treatment of Varicella Infection

• Acyclovir has been shown to reduce fever and skin
  lesions in patients with varicella and it use is
  recommended in healthy patients over 18 years of
  age.

Acyclovir (ZOVIRAX)
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Cytomegalovirus

• Cytomegalovirus (CMV) is a common virus that
  infects most people during their life. Most
  people are infected in early childhood (under 3
  years of age) or in the teenage years. Usually
  there are no symptoms of CMV infection.

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• Since most CMV infections are mild and usually do
  not cause long-term problems, most people don't
  even know they are infected.
• However, CMV can cause problems in a developing
  baby if the mother gets the infection during
  pregnancy.

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Cytomegalovirus (CMV)

• After a person has a CMV infection, the virus
  stays in the body but is not active. The virus
  can reactivate months or years later, which
  occurs most often when a person's immune system
  is weakened.

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Cytomegalovirus (CMV)

• Anyone with a weakened immune system is at risk
  for problems with CMV infection. A weakened
  immune system can be related to infection with
  the human immunodeficiency virus (HIV) or medical
  treatments. 
• Medical treatments that can weaken the immune
  system include chemotherapy, radiation therapy,
  steroids, and stem cell or organ
  transplantation. 

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Cytomegalovirus (CMV)

• A few people will have symptoms such as sore
  throat, fever, headache and fatigue. People who
  have weakened immune systems may develop severe
  symptoms, such as pneumonia or infections of the
  eyes, liver, or intestinal tract. People with HIV
  infection should be sure to let their doctor know
  if they are having any painless blurring of their
  vision, "floaters" in the eye, light flashes,
  areas of blindness, or shortness of breath.

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Treatment of CMV Infection

• Gangiclovir, a nucleoside analog of acyclovir,
  inhibits CMV replication and reduces the severity
  of CMV syndromes, such as retinitis and
  gastrointestinal disease.

Deoxyguanosine
Acyclovir
Gangciclovir
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Ganciclovir
Ganciclovir is used to treat or prevent
cytomegalovirus
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Gangciclovir for ocular use is marketed under the
trade name Vitrasert.
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Valganciclovir
Valganciclovir is a more orally bioavailable
version of this drug.
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Epstein-Barr Virus (EBV)

• EBV is the etiologic agent of infectious
  mononucleosis
• Symptoms include fever, tender lymph nodes, sore
  throat, mental fatigue
• Treatment is usually supportive
• Acyclovir can decrease replication of EBV in
  culture, but has little impact on clinical
  illness.

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Assigned Reading

• Wasik, Mitzi Kachlic, Marlowe Djuric. A review
  of common sexually transmitted diseases.
  Formulary (2009), 44(3), 78-85.
• De Clercq, Erik Field, Hugh J. Antiviral
  prodrugs - the development of successful prodrug
  strategies for antiviral chemotherapy. British
  Journal of Pharmacology (2006), 147(1), 1-11.

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Homework Questions

• Describe the methods used for diagnostic testing
  for HSV.
• List the accepted treatment for this disease.
• From one point of view, all antiviral nucleosides
  can be considered prodrugs. Explain. What
  enzyme do they require for activation?
• What two undesirable properties of acyclovir
  (ACV) prompted the search for a prodrug?
• What protein is responsible for the improved
  bioavailability of valaciclovir?
• Gangiciclovir (GCV) has superior activity than
  acyclovir against which virus?
• Oseltamivir (Tamiflu) is also a prodrug. Which
  functionality will be transformed in vivo? Why
  was this functionality incorporated?