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 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.

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.