Antiviral Chemotherapy 2 (DONE)

Question 1

HIV replication

Answer 1

Fusion of HIV to the host cell surface
HIV RNA, reverse transcriptase, integrase and other viral proteins enter the host cell
Viral DNA is formed by reverse transcription
Viral DNA is transported across the nucleus and integrates into the host DNA
New viral RNA is used as genomic RNA and to make viral proteins
New viral RNA and proteins move to the cell surface and a new, immature HIV forms
Virus is released, viral protease cleaves new polyproteins to create mature, infectious virus

Question 2

NNRTIs

Answer 2

NNRTIs target allosteric site to NTPs, causing conformational change, while NTPS target catalytic site
The structures of the NNRTIs are very diverse, but are all in the shape of U or a butterfly
Tend to be lipophilic to bind allosteric pocket of enzyme

Question 3

TSAOs

Answer 3

Discovered by accident: the spiro ring at C3’ was not the intended product, and the silyl groups are just temporary protecting groups- just there for the chemistry
But: if the silyl groups are removed, all NNRTI activity is lost. Like most NNRTIs, the TSAOs are very lipophilic

Question 4

HEPTs

Answer 4

Again, these were designed as nucleoside analogues, intended to act (after 5’ phosphorylation) as NRTIs
But: they act as NNRTIs, not as nucleosides, despite their structural resemblance to nucleosides

Question 5

How could we establish that such a compound (HEPT) were acting as an NNRTI, not a NRTI?

Answer 5

Would observe direct inhibition if incubated with HIV RT (nucleosides are inactive, until 5’-phosphorylated)
Activity is retained on (chemical) removal of 5’-OH
Would be HIV-1 specific (NNRTIs don’t work on HIV-2)

Question 6

Limitations with NRTIs

Answer 6

Emergence of resistant virus
Cross-resistance possible
No activity in resting cells
Clinical toxicities common/can be severe

Question 7

Limitations with NNRTIs

Answer 7

Rapid emergence of resistant virus
Cross-resistance to several NNRTIs common
Clinical efficacy alone is limited
No activity in resting cells
Entirely HIV-1 specific

Question 8

Viral protease inhibitors

Answer 8

Most viruses translate their genome into a large polyprotein precursor that needs to be cleaved into individual (functional) viral proteins
This crucial cleavage step is often carried out by viral proteases

Question 9

HIV protease role

Answer 9

The viral genome is incorporated into the human genome (by the integrase)
The pro-viral DNA is transcripted into mRNA
HIV genes are expressed to give large polyproteins
HIV protease cleaves these polypeptides into individual functional proteins

Question 10

HIV protease

Answer 10

An essential enzyme for the HIV life cycle
Cleaves specific amide bond of polypeptides at at least nine different cleavage sites
Belongs to the aspartic acid protease family
Structurally related to human proteases, such as renin and pepsin

Question 11

HIV protease structure

Answer 11

The HIV protease has been cloned and over-expressed in E. coli
It has been crystallised and its structure has been solved
The crystal structure and genetic data have been used to design inhibitors

Question 12

HIV protease inhibitors: drug design

Answer 12

Initial design of inhibitors focused on isosteres of the Phe-Pro or Tyr-Pro amide bond, in particular of the high energy tetrahedral intermediate formed during the cleavage of the amide bond
It has been postulated that enzymes in general have higher affinity for these intermediates than they have for either the substrates or the products
Inhibitors based on these transition states (TS inhibitors) should therefore be particularly potent

Question 13

HIV protease inhibitors conclusions

Answer 13

The hydroxyethylene isostere is a good mimic
These compounds act as competitive inhibitors of the enzyme
The stereochemistry at the hydroxyl is crucial for the activity (S active, R inactive)
However, such peptide structures have very poor PK

Question 14

Issues with HIV protease inhibitors

Answer 14

Viral resistance
Common side effects- increase in blood sugar, change in fat distribution, increase of fat in the blood, liver toxicity
Poor bioavailability
Short half life- dosing two to three times a day
Complex chemical synthesis

Question 15

HCV proteases

Answer 15

Hepatitis C RNA encodes for a large polyprotein precursor 3000 amino acids long
This polyprotein precursor is cleaved by viral and host proteases to form 10 structural and non-structural proteins
The non-structural proteins are further processed by two viral proteases into their mature forms

Question 16

HCV NS3 protease inhibitors

Answer 16

Two types:
Non-covalent product based inhibitors- bind to the active site of the NS3 serine protease, blocking its action and shutting down viral replication e.g. carboxylic acid containing inhibitors, acid sulphonamide containing inhibitors
Covalent reversible inhibitors- form a reversible covalent bond with serine in the active site. Acting as a serine trap e.g. boceprevir

Question 17

Issues with HCV protease inhibitors

Answer 17

Emergence of viral resistance- not suitable as monotherapy
Side effects- rash, anaemia
Short half life, requiring frequent dosing (2-3 times per day)
Complex chemical synthesis

Question 18

HIV integrase

Answer 18

Critical enzyme that incorporates the HIV genome into the human genome. This step is essential for the viral replication. Two classes of integrase inhibitors have been developed against HIV:
Integrase strand transfer inhibitors (INSTIs), in clinical use, interfere with strand transfer reaction
Integrase binding inhibitors (INBIs), under clinical evaluation, inhibit binding of the integrase to the viral DNA

Question 19

CD4 analogues

Answer 19

HIV entry requires an interaction between gp120 on the virus and CD4 on the target cell
Shortened soluble versions of the CD4 polypeptide (rsCD4) appear to have some protective anti-HIV effect in-vitro, presumably by binding to and deactivating free virus
One problem with these therapies is the very short plasma half life of the rsCD4 (<5 min). Other non-peptide analogues are under study

Question 20

Fuzeon (Enfuvirtide)

Answer 20

A 36- amino acid peptide that links to HIV gp41 and prevents entry, now approved for use
Has to be dose IV due to its peptide structure
Twice daily dosing

Question 21

CCR5

Answer 21

HIV entry requires docking onto CD4 using either CCR5 or CXCR4
Approved in 2007, maraviroc is an entry inhibitor that works by blocking the CCR5 attachment

Question 22

HIV penetration/absorption inhibitors

Answer 22

HIV absorption/penetration may be blocked by a wide variety of poly-anionic compounds. The original materials show little antiviral selectivity.
Later compounds included heparin sulphates. Their activity varies with molecular weight and charge. Some appear to have good antiviral selectivity, but they suffer from very poor bio-availability

Question 23

HIV glucosidase inhibitors

Answer 23

HIV maturation requires post-translational glucosidation of viral polypeptides
Specific inhibitors of HIV glucosidation have been sought and to some extent found. Alpha-glucosidase 1 appears to be the best target for achieving highest anti-HIV specificity. Most inhibitors of this system are mimics of natural sugars (notably glucose).