Reference no: EM132683517

CHT353 Drug Targets - Cardiff University

Herpes simplex

1. Herpes simplex virus uses a linear molecule of DNA to store its genetic information. When the virus infects a cell, human RNA polymerase makes mRNA encoding the enzymes needed to replicate the viral DNA. These enzymes include a DNA polymerase, a primase (which makes short sequences of RNA) and a protein to separate the DNA strands during replication. Briefly explain why the primase is needed to initiate DNA replication.

2. Write a curly arrow mechanism for the reaction catalysed by the viral DNA polymerase and hence explain why the synthesis of one new DNA strand is slower than the other.

3. The prodrug acyclovir (shown below) is an effective treatment of Herpes simplex infections. Draw the active form of the drug and explain how it prevents the replication of the virus.

4. Acyclovir can be taken as a pill by patients. Briefly explain why you might expect that the casing of the pill needs to be resistant to acid and to break down under basic conditions.

Human immunodeficiency virus (protease)

5. Briefly explain why the HIV-encoded protease is essential for the replication of the virus.

6. The development of HIV protease inhibitors requires the development of an assay that gives information on the rate at which the enzyme cleaves peptide bonds. Early work on the enzyme used the following heptapeptide as a substrate:

Ac-SAAYPVV-NH₂

In the assay, HIV protease (8 ng) was incubated with 3 mM of the nonapeptide (shown above) in 10 μL of buffer at 37 ^oC for 30 min. The reaction was then stopped by adding trichloroacetic acid to precipitate the enzyme. What technique woulld you use to obtain the amounts of the cleaved peptide products in the final solution? State one advantage and one disadvantage of your method.

7. The steady-state parameters (KM and kcat) for HIV protease-catalysed cleavage of the heptapeptide substrate have values of 9 ± 2 mM and 70 ± 2 s-1, respectively. In order to be useful drugs, inhibitors need to exhibit nanomolar Ki values. Briefly comment on the challenges of using the heptapeptide substrate as a template for obtaining potent competitive inhibitors of HIV protease.

8. The compound shown below was found to be a competitive inhibitor of the heptapeptide, exhibiting a Ki value of 0.018 μM. Calculate the concentration of heptapeptide required for the initial velocity to proceed at 0.5Vmax when the inhibitor is present at 180 nM concentration.

9. Identify which amide bond in the heptapeptide is cleaved by HIV protease, and hence explain why the competitive inhibitor is so potent. (Use a clear structural diagram)