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

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

2. 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.

4. 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.

6. 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.

8. 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.

9. 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.

10. 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.

11. Other Drugs to Treat Influenza

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

13. 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.

15. 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.

20. What is the difference between HSV-1 and HSV-2? • Both types infect the body’s 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.

21. HSV-1 and HSV-2 http://www.healthscout.com/animation/68/21/main.htmlLinkLink

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

23. 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.

24. HSV-2 usually establishes latency in sacral ganglion at the base of the spine. From there, it recurs in the genital area.

26. 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.

27. 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.

28. 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 don’t 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.

29. 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.

30. 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.

31. 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.

32. 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.

33. 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.

34. Vaccines? • No vaccine for HSV-2 is currently available.

35. 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.

36. 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.

37. Chemotherapy for HSV Acyclovir (Zovirax) Valacyclovir (Valtrex), pencyclovir Famcyclovir (Famvir),

38. Gertrude B. Elion won the Nobel prize in Physiology or Medicine in 1988, partly for the discovery of acyclovir.

39. 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.

40. 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.

41. 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.

42. 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.

44. 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.

45. 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.

47. http://www.dnalc.org/ddnalc/resources/sangerseq.html • The mechanism of action of Acyclovir is analogous to that utilized by Fred Sanger’s method of DNA sequencing. • http://www.dnalc.org/view/15479-Sanger-method-of-DNA-sequencing-3D-animation-with-narration.html • http://www.biostudio.com/demo_freeman_dna_sequencing.htm

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

49. 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..

50. 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.