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Revision as of 05:10, 29 April 2011

HIV-1 PROTEASE

INTRODUCTION

HIV-1 Protease, Homodimer of two identical monomers, 99 amino acids each,1hsg

Acquired immune deficiency syndrome (AIDS) is arguably of the most prominent and deleterious pandemics of the modern era. AIDS is a condition in humans that causes progressive failure of the immune system, leaving individuals susceptible to opportunistic infections and cancers. The failure of the immune system results from viral assault on the human body’s white blood cells. Since the middle of the 20th century, the disease has spread rapidly in a near ubiquitous fashion, and now the world is on the brink of the fourth decade of the AIDS epidemic.

In the United States, the Center for Disease Control (CDC) first recognized AIDS during the summer of 1981 (Gallo, 2002). Subsequently in 1983, the discovery of the sinister “AIDS virus” coined it as the human immunodeficiency virus (HIV). According to the 2010 UNAIDS Global Report, in 2009, there were 33.3 million people (adults and children) living with HIV, and 1.8 million AIDS related deaths that year, which leads to a deplorable grand total of roughly 30 million deaths due to AIDS since the HIV virus began ravaging the world.

VIROLOGY

The HIV is classified in the Retroviridae family, and more specifically belongs to the genus Lentivirus – slow viruses characterized by a long incubation period, which typically result in long-duration illnesses (Lévy, 1993). Retroviruses (retro is the Latin prefix for “backward”) uniquely possess an RNA genome along with an RNA-dependent DNA polymerase, reverse transcriptase, which is an enzyme that can remarkably direct the synthesis of DNA from RNA (Nelson and Cox, 2008).

The HIV retrovirus has a fairly simple life cycle. The infection begins with the fusion of the viral and host membranes of essential immune cells, typically T-cells and macrophages. Reverse transcriptase then produces DNA from the virus RNA genome, which is then inserted into the host's chromosome by another key enzyme, integrase. At this point the viral genome can become dormant indefinitely. Alternatively, it undergoes transcription and translation into proteins and RNA, required for the new virus. Much of the viral genes are translated initially into precursor polyproteins, which are ultimately cleaved by a final key enzyme of the virus, HIV protease (HIV-1 protease is specific to the HIV-1 class) (Figure 2). The cleavage of the polyproteins into individual proteins may occur during assembly and/or after budding of the immature virus from the host cell (Gelderblom, 1997). These final steps completed by HIV protease are absolutely critical to the maturation of the new virus.

Since the HIV retrovirus contains enzymes imperative for its life cycle that are unique from humans, these are potential inhibitory drug targets to treat infection and to prevent the progression to AIDS. The usual, most effective treatment for HIV infection is a cocktail of drugs that inhibit fusion of the virus and the key enzymes reverse transcriptase, integrase, and protease.

STRUCTURE AND ACTIVE SITE OF HIV-1 PROTEASE

The first structures of HIV-1 protease were reported in 1989 revealing its homodimeric structure consisting of two identical monomers, each made up of 99 amino acids residues, related by a two-fold axis of symmetry (Spinelli et al., 1991). The secondary structure of each monomer contains antiparallel beta-sheets and a single alpha-helix (Figure 3).

Flexible flaps form from an extended turn of a beta sheet (beta hairpin loop) that covers the active site (Figure 3) (Louis et al., 2007). Incidentally, the active site tunnel is normally too narrow for the polyprotein to fit, as depicted in Figure 4. However, the solution is the two flexible protein flaps (amino acid resides 45-55 on each monomer) that can move to allow the peptide to enter the tunnel active site (Toth and Borics, 2006). These flaps undergo a dramatic conformational change from open to closed states to bind the substrate in the proper conformation for catalytic cleavage. The highly flexible tips of the flaps are glycine rich, which curl inside the cleft as the tunnel expands, burying many hydrophobic residues and exposing electronegative active site, while widening the tunnel enough for the substrate to enter (Toth and Borics, 2006).

ASPARTYL PROTEASE MECHANISM

HIV-1 protease is an aspartyl protease, similar to pepsin, which contains two essential aspartate (Asp25) residues, one from each monomer, that play a key role in the enzyme’s catalytic function at the active site (Figure 5). Like all aspartyl protease, the active site of HIV contains a highly conserved catalytic triad of three amino acid residues, Asp25-Thr26-Gly27 (Aspartate-Threonine-Glycine) (Castro et al., 2011). Each monomer contributes a single Asp-Thr-Gly triad to the active site, amounting to six amino acids in the active site of HIV-1 protease. The active site triads are located in a loop whose structures are stabilized by a network of hydrogen bonds. It has been hypothesized that the role of the Thr26 residues is to stabilize the conformational state of the active site through hydrogen bonding forces with one another, also known as “fireman’s grip” (Castro et al., 2011). Moreover, the Gly27 residues function to accommodate and bind the substrate for subsequent attack by the Asp residues (Mager, 2001). The Asp25 residues are nearly coplanar and interactive directly with the substrate. Each Asp25, being negatively charged amino acids, holds a water molecule, crucial for catalysis, through hydrogen bonding (Mager, 2001). The Asp25 residues and water together cleave the substrate through a general acid/base hydrolysis reaction. The active site aspartyl residues assume opposite roles during catalysis (Castro et al., 2011). One Asp25 residue behaves like an acid, donating a proton to the carbonyl oxygen of the substrate, as the other Asp25 behaves like a base, accepting a proton from water. This promotes the nucleophilic attack by the water on the substrate, cleaving the peptide bond of the polyprotein (Prashnar et al., 2009).

DRUG INHIBITORS AS TREATMENTS

The active site of HIV-1 protease has been one of the key targets for fighting HIV/AIDS. The drug inhibitors are highly stable mimickers of the polyproteins that cannot be cleaved by HIV-1 protease. They are able to bind with higher affinity than the polyprotein substrate. These drugs function as reversible inhibitors that compete with the usual substrate for the enzyme’s active site through competitive inhibition (Louis et al., 2007).

Presently, nine protease inhibitors have been approved for clinical treatment of HIV infection: saquinavir, indinavir, ritonavir, nelfinavir, amprenavir, lopinavir, atazanavir, darunavir, and tipranavir (Castro et al., 2011). These inhibitors are smaller in size to the intended peptide substrates. They contain a central hydroxyl group that mimics the tetrahedral reaction intermediate, interacting with the carboxyl groups of the Asp25 residues of the active site, increasing their affinity for protease. Additionally, all the inhibitors contain polar groups that create hydrogen bonding interactions with numerous other residues within the active site tunnel, which are mediated by a conserved water molecule (Louis et al., 2007). Figure 6 shows an inhibitor, Indinavir (PDB 1HSG), tightly bound to the active site of HIV-1 protease.

Protease inhibitors have been fundamental components of treatment therapies for HIV/AIDS since 1995, when antiviral protease inhibitors were first approved for clinical use. The integration of protease inhibitors in drug therapy has been associated with successful therapeutic treatment of HIV/AIDS, significantly reducing AIDS mortality and morbidity.