5 & 6 - HIV Protease Inhibitors

Question 1

What class of viruses does HIV belong to?

Answer 1

Retroviruses

Question 2

How does HIV infect host T cells?

Answer 2

HIV infects host T cells that have the CD4 antigen on their surface

Viral DNA enters host cell nucleus
- it is integrated into the genetic material by an Integrase enzyme

Activation of host cell then results in transcription of viral DNA into messenger RNA
- which is then translated into viral proteins

Question 3

What’re the steps of the HIV life cycle?

Answer 3

Question 4

After HIV mRNA has been translated into viral proteins what key step happens next?

Answer 4

Viral proteins are cleaved to active forms by HIV PROTEASE
- crucial for virion development

Question 5

What’s an issue when treating HIV proteases with inhibitors?

Answer 5

Like reverse transcriptase inhibitors, resistance is quickly developed to protease inhibitors

Requires combination therapy for treating HIV infections

Question 6

What enzymes can be targeted by med chemists for HIV

Answer 6

Reverse transcriptase

Integrase

Transcriptase

HIV protease

Question 7

How do protease inhibitors differ to reverse transcriptase inhibitors?

Answer 7

Protease inhibitors are not prodrugs and do not require activation

Question 8

What family is the HIV protease enzyme a part of?

Answer 8

It is an example of an enzyme family called the:

Aspartyl proteases-enzymes
- they contain aspartic acid in the active site
- crucial for catalytic mechanism

Question 9

What do apartyl protease-enzymes do?

Answer 9

They catalyse the cleavage of peptide bonds

Question 10

What is the structure of the HIV protease enzyme?

Answer 10

It is a dimer made up of 2 identical protein units, each consisting of 99 amino acids

Question 11

In the sense of assays and analysis, why is HIV protease a good drug target?

Answer 11

It is relatively small can can be obtained by synthesis

It’s can be cloned and expressed in fast-growing cells

It is easily crystallised with & without inhibitor bound

Question 12

What amino acids lie on the floor of the active site of HIV protease?

Answer 12

Aspa-25

Thr-26

Gly-27

Question 13

What makes the structure of HIV protease a good target?

Answer 13

The active site is at the interface between the protein units

This means that drugs can interact on all sides = very high affinities

Question 14

How many binding sites are there in HIV protease enzyme?

Answer 14

8
- 4 on each protein unit located on either side of the catalytic region

Question 15

What do the sub-sites of HIV protease accept for binding?

Answer 15

The sub-sites accept the amino acid residues of the substrate

Question 16

What’re the key interactions seen in the HIV protease active site?

Answer 16

Asp-25 & Asp-25’ are involved in the catalytic mechanism with a bridging water molecule

Question 17

What is the cleavage mechanism of HIV Protease?

Answer 17

Tetrahedral intermediate is key

Question 18

How do HIV Protease-1 and HIV Protease-2 differ?

Answer 18

They share 50% sequence identity to each other
- greatest variation is seen outside of the active site

Inhibitors are found to bind similarly to both variants of HIV Protease

Question 19

What’s the advantage of designing transition state inhibitors?

Answer 19

The advantage is that the transition transition state is likely to be bound to the active site more strongly than either the substrate or the product

Hence, inhibitors mimicking this are also likely to bind more strongly

Question 20

What is an issue (& its solution) when designing TS inhibitors of HIV protease?

Answer 20

The intermediate is inherently unstable

Hence, an inhibitor must contain a TS isostere which has a tetrahedral centre to mimic the TS.
- it also must be stable to hydrolyis

Question 21

Which functional group shows the most success in mimicking the TS?

Answer 21

Question 22

What’s the problem with designing inhibitors based on the natural substrates of HIV Protease, fitting all 8 sub-sites?

Answer 22

It makes sense to deign inhibitors that bind to all 8 sub-sites, allowing stronger interactions

However, this gives structures with high MW and consequently poor oral bioavailability

Question 23

What do early HIV Protease inhibitors, like saquinavir, suffer from?

Answer 23

They have amino acid residues at P2 and P2’ leading to high MW and high peptide character
- poor pharmacokinetic properties

Question 24

What is the best approach to designing transition inhibitors for HIV protease?

Answer 24

Begin with a core unit spanning S1 to S1’ sub-sites, then grow the molecule outwards (either end) to fit into S2/S3 and S2’/S3’ subsites.

Question 25

How did the design of saquinavir begin?

Answer 25

A pentapeptide sequence was identified and the amide link between Phe—Pro was replaced by the hydroxyethylamine isostere

Question 26

What is the key binding site in HIV protease, how was this found?

Answer 26

Inclusion of asparagine at P2 gave 40 fold increase in activity Shows that S2/S2’ is key binding site

Question 27

Is the S3 site hydrophilic or hydrophobic?

Answer 27

Hydrophobic

Question 28

Why was the bicyclic ring system added?

Answer 28

Because it occupies the S1’ site more fully The R-configuration is CRITICAL

Question 29

Why was the ester substituted with an amide?

Answer 29

Due to metabolic stability - amides are more stable than esters to metabolism

Question 30

What is the important about the placement of the carbonyl groups either side of the hydroxyethylamine?

Answer 30

The carbonyl groups can act as H-bond acceptors to the bridging water molecule found in the transition state in HIV Protease active site

Question 31

What amino acids does the bridging water molecule bind to in the flaps of the active site?

Answer 31

Isoleucine - Ile-50 & Ile-50’

Question 32

Where does the OH of the isostere replacement bind to in the active site?

Answer 32

It forms hydrogen bonds to both aspartic acids in the floor of the active site - Asp-25 & Asp-25’

Question 33

What the significance of the position of the t-Bu amine in saquinavir?

Answer 33

It allows for further substituents on N are capable of reaching S3’.

Question 34

What about the structure of HIV protease makes it an ideal target?

Answer 34

Because of its 2-fold (C2) symmetry Gives rise to selectivity for viral protease over mammalian aspartate proteases - as the latter do not have C2 symmetry

Question 35

What’s the point of adding Valine here?

Answer 35

It extends the molecules to S2 and S2’ pockets to be occupied Gave large increase to inhibitory activity

Question 36

What’s the point of adding Z-protecting groups to valine’s amine?

Answer 36

Aside from a major increase in potency, it improved the drugs ability to cross membranes

Question 37

Why were polar groups introduced at the ends of the molecule here?

Answer 37

Because, it was found that the terminal portions fo the molecule were exposed to solvation This meant that more polar groups could be added at those positions without affecting binding

Question 38

What’s a problem within the body with adding these polar groups?

Answer 38

The pyridine groups were extensively metabolised to N-oxides that are excreted into bile

Question 39

Why was the S-OH removed?

Answer 39

Because it only formed 1 H-bond R-OH forms 2 H-bonds with enzyme Removing it may enhance solubility

Question 40

Why was the pyridine ring replaced with a thiazole ring system?

Answer 40

Done to improve pharmacokinetics - replacement of pyridine with a more metabolically stable ring system

Question 41

Describe and explain the changes made to Ritonavir after resistance rose

Answer 41

The isopropyl group, at S3, made important hydrophobic interactions with enzyme - resistant viral strains reduced this interaction In Lopinavir, the S3 residue is removed and cyclic urea has been included to maximise interactions at S2 site Lopinavir is active against ritonavir resistant viruses

Question 42

Why are Lopinavir and Ritonavir administered in combination?

Answer 42

200mg Lopinavir and 50mg ritonavir Ritonavir acts as a CYP450 inhibitor to increase the supply of Lopinavir in blood - boosting activity

Question 43

Give the mechanism for the synthesis of 5 from 6

Answer 43