HIV Lecture 2

Question 1

HIV protease

Answer 1

• a proteolytic enzyme responsible for cleaving the large polyprotein precursor into biologically active protein products
• has broad substrate specificity and can cleave a variety of peptide bonds in viral polypeptides
• cleaves bonds between proline residue and aromatic residue (does not occur with mammalian protease)
• symmetrical nature of viral enzyme and its active site is NOT present in mammalian protease

Question 2

Structure of HIV protease

Answer 2

• Dimer made up of two identical protein units (homodimer)
• Two-fold rotational symmetry
• Aspartic acid residues contribute to active catalytic site (aspartyl protease)
• loops of each monomer are interlinked by 4 hydrogen bonds contributing to the stabilisation of the two subunits around the symmetry axis

Question 3

HIV Protease inhibitors

Answer 3

• are based on the transition-state mimetic approach (competitive inhibitors for the natural substrate)
• based on disrupting twofold rotational C2-symmetry axis by forming specific interactions with the residues involved in stabilising the dimer

Question 4

First generation HIV protease inhibitors

Answer 4

peptide inhibitors

Question 5

Second generation HIV protease inhibitors

Answer 5

Non-peptide inhibitors

Question 6

Mechanism of transition state inhibitors

Answer 6

• Two aspartate residues are in enzyme active site, disposed on opposite faces of the peptide bond to be cleaved
• One asp acts as a general base to active the attacking H-OH, the second asp acts as a general acid to protonate the departing amine product
• The two asps act in complementary fashion for breakdown of tetrahedral adduct
• Replacement of the scissile peptide bond of substrate with the hydroxyethylene group in the inhibitor

Question 7

Saquiniavir

Answer 7

• first inhibitor to reach market
• design started by considering a viral polypeptide substrate and identifying a region of the polypeptide which contains a phenylalanine-proline peptide link
• however it has a high MW and high peptide-like character, so bad oral bioavailability

Question 8

Ritonavir

Answer 8

• Greater selectivity over mammalian proteases
• Symmetrical molecules might be less recognisable to peptidases (improve oral bioavailability)
• similar binding pockets allow addition of two bulky groups to improve binding

Question 9

Key features of HIV-1 Protease

Answer 9

• it is a dimer with C2 symmetry
• 2 asp-s at bottom of active site
• water molecules have 2 H-bonds with the backbone of LEU and 2 H-bonds with carbonyl oxygens of the inhibitor
• these structural features were used in pharmacophore building

Question 10

how drug resistance is developed

Answer 10

• from mutations in viral genome
• mutations alter viral enzymes in a way that the drug no longer inhibits enzyme function and virus restores its free replication power

Question 11

factors contributing to HIV drug resistance

Answer 11

• high rate of replication increases risk of mutation
• poor patient compliance
• inappropriate choice of antiretroviral agents
• subtherapeutic blood levels of antiretroviral agents
• pharmacokinetic factors

Question 12

HIV mutation rates

Answer 12

• HIV lifecycle requires two copying events between virion and daughter particles
• first event is copying of RNA genome to DNA carried out by RT, which has no proof reading ability so the error rate is high
• second event is production of RNA genome by RNA pol II which is not a proof reading enzyme
• this means error rate is high with most progeny virions containing new mutations

Question 13

Approaches to overcome drug resistance

Answer 13

• switching drug class
• combination therapy, such as a protease inhibitor with two nucleotide RT inhibitors
• prodrug conjugates

Question 14

Active management of drug resistance

Answer 14

• use of multiple drugs keeps viral loads low even if one drug is beginning to fail
• low viral copy numbers reduces risk of mutation
• monitoring viral load gives early warning that regime is failing
• viral genome sequencing will say which drug is failing and which replacements will be most effective
• early switching prevents resistance mutation from becoming fixed and removing selection pressure gives rapid reversion
• the failed drug can thus often be reintroduced at a later stage having regained efficacy
• very effective in high tech settings but not possible in low tech settings, so drugs should be rotated