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Two different therapeutic
approaches for treating:
A. A DNA virus (e.g. Herpes)
B. An RNA virus (e.g. Influenza)
Neuramindase is an enzyme which is capable of cleaving
the sialic acid sugar moiety from selected glycoproteins and
glycolipids on the surface of infected cells.
This cleavage promotes the release of progeny virus from infected cells.
The neuraminidase enzyme
• Neuraminidase is attached to the viral surface by a single
hydrophobic sequence of 29 amino acids
• The enzyme can be easily mutated. There are two main types
corresponding to influenza A and B.
• However, the active site is located in a deep pocket and the 18
amino acids making up the active site itself are constant.
• The enzyme is critical to the infective process, particularly
including preventing viral aggregation or binding to hemaglutinin
or inactivation by respiratory mucous. It is essential for proper
liberation (shedding) of the new virus.
• Both neuraminidase (NA) and hemaglutanin (HA) act as
antigens for flu vaccines.
• However, due to the frequency with which influenza A mutates
these proteins, new flu vaccines are required each year.
Transition state analogs
• An early search for inhibitors of neuraminidase was
unsuccessful.
• Once the crystal structure was available, it was
decided to search for transition state analogs.
• Recall that the active site of the enzyme will bind and
stabilize the transition state more effectively than it
will stabilize the substrate itself, thus resulting in an
overall decrease in activation energy for the chemical
transformation.
What does the transition state of
this reaction look like?
• 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.
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.
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.
• Note that the actual reaction intermediate (lower left) involves a resonancestabilized 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.
Further chemical evolution of neuraminidase inhibitors
• Thus a series of compounds was prepared having an
appropriately placed C=C 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.
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.
Other Anti-influenza Agents
• Amantadine and Rimantadine were the
earliest antiviral drugs.
Mechanism of Action of
Amanadine and Rimantadine
• These agents were discovered by random
screening and are now known to interfere
with a viral ion channed called matriz (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 Hemaglutanin (HA)
to fuse the viral membrane with that of the
endosome.