Analysis of the Molecular Structure of TMC114 HIV-1 Protease Inhibitor and the HIV-1 Protease Protein

Molecular Modeling: Jterm 08

Background

HIV-1 Protease is a protein produced by the HIV virus. It cleaves viral polypeptides into functional proteins that are needed for viral assembly and activity. HIV-1 Protease is a common target in treating AIDS, because the inhibition of this protein would prevent many essential viral proteins from being produced. Therefore the inhibition of protease could significantly slow the progress of the disease. Many drugs have been made to treat AIDS, but many of them have become ineffective. This is because the HIV virus mutates very easily so that the active site or surface receptor the drug targets are no longer recognizable to the drug. If the drug cannot recognize the target protein it can no longer attach to and inhibit the protein.

Most protease inhibitors bind to the active site of the protease protein so that the active site of the protease cannot bind to the substrate (the proteins to be cleaved to produce active viral proteins). When the molecular structure of the active site mutates, the inhibitors no longer can bind to the active site. This is a problem when combatting AIDS because drugs that are produced eventually become ineffective.

The recently released (June 23, 2006) TMC114 protease inhibitor (also known as Darunavir) is unique because unlike many other inhibitors it has conformational flexibility. This means that the molecule is not stiff, the single bonds can rotate so that the molecule has many different conformations. Since the TMC114 inhibitor has so many conformations, it can still bind to the active site of the protease when it mutates.

In this project, I will be investigating the flexibility of this inhibitor. I will try to locate the important areas of the molecule that allow various conformations that can bind to protease mutants. I will also analyze the active site of the protease and determine the amino acid sequences where hydrogen bonding occurs with the inhibitor.

Hydrogen bonding and the HIV-1 Protease active site

This image shows the acidic and basic areas of the HIV-1 Protease. The TMC114inhibitor will form hydrogen bonds with acidic and basic regions when it docks onto the active site. The inhibitor is embedded within the molecule in the image.

This image shows how the inhibitor binds by hydrogen bonding in the active site of the protease.

HIV-1 Protease has many hydrogen bonding sites within the molecule that allow for direct bonding with the active site of the protease. The hydrogen bonding sites can also undergo intramolecular hydrogen bonding, allowing for more conformations of the inhibitor. The intramolecular bonding and flexibility of TMC114 allows for many conformations that can bind to mutated protease active sites. More information on intramolecular bonding will be presented in the next section. This figure highlights tha acidic and basic regions of TMC114 that have the ability to create hydrogen bonds.

The TMC114 inhibitor has a bis-tetrahydrofurane (bis-THF) group that makes it unique from similar inhibitors, such as Amprenavir. Both oxygens in this group hydrogen bond with amino acids ASP 29 and ASP 30 in the protease active site (ASP is the amino acid aspartic acid). This particular pair of hydrogen bonds is apparently essential to the unique stability the TMC114-protease complex has (more about the energetic stablity of TMC114 will be explained in later sections). It is important for an inhibitor to be stable when bound to the protein because it may otherwise fall off, and fail to prevent the protease from acting on its substrate. If this happens the protease will continue to aid in the construction of more viruses.

Intramolecular Hydrogen Bonding and its Impact on Conformations of TMC114

The figure below shows the five types (b-e) of intramolecular hydrogen bonding that occurs in TMC114 to create approximately 43 energetically stable conformations. Theoretically there are 260 conformations, but many of these are energetically unfavored conformations. Figure "a" shows the TMC114 without any intramolecular hydrogen bonding. The following energies (kcal/mol) are relative to the most stable conformation (letter f in the figure) of the 43 conformations tested by Kanda Nivesanond and others: a)7.05 b)0.27 c)3.54 d)2.19 e)6.12

The range of relative energies to most stable conformation is 0 to 8.32 kcal/mol. It is important to note that the conformation with no hydrogen bonding was much higher in energy, and was much less stable then those with intramolecular hydrogen bonding. The intramolecular hydrogen bonding obviously plays an important role in stabilizing the different conformations of the molecule.

The figure above also shows the regions on the molecule that undergo intramolecular bonding. Over half of the conformations have hydrogen bonding at the -OH and O=S site (H1 in the figure). This intramolecular hydrogen bond is probably the most common because the bonding regions are very close to each other and are in a flexible region (area with numerous single bonds) of the molecule.

Intramolecular hydrogen bonding and flexibility give TMC114 the ability to have several stable conformations. As a result TMC114 has the ability to adapt to the mutating active site of the protease, therefore making it an effective drug to combat the rapidly evolving HIV virus.

The Energetic Stability of Various TMC114 Conformations when Bound to the HIV-1 Protease

The stability of TMC114 conformations is important, but this means nothing if the complex it forms with protease is not stable. The table below shows the binding energies Nivesanond and others obtained of various TMC114 conformations bound to wild and mutant protease. It is important to note that sometimes stable conformations do not have low binding energies, such as conf17 with the wild protease. Particular conformations have lower energies depending on if the molecule is bound to the wild or mutant protease.