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HIV-1 Protease and Inhibitor Complex

Kaleb Keyserling '09 and Ken Noguchi '10


I. Introduction

    The AIDS epidemic currently affects 40 million people worldwide and has caused over 22 million deaths. AIDS is caused by the retrovirus HIV which attacks the human immune system leaving the victim susceptible to disease. Due to the severity of the HIV virus, much research has been devoted to developing antiviral therapy. In recent years many reverse transcriptase and protease inhibitors have been synthesized to fight HIV (Brik and Wong, 2002). Although these inhibitors do not cure the disease, they are able to improve the quality and prolong the life of those infected. Here is a diagram of the HIV life cycle and targets where inhibition occurs. (Pomerantz and Horn, 2003) This Jmol tutorial focuses on the protease inhibitor.

    HIV-1 protease is important in HIV replication and infectious capacity because it cleaves polypeptide chains to create mature enzymes and structural components of the virus (including itself). HIV protein synthesis occurs in the host cell. Once translation of the viral mRNA occurs, the components of the HIV virus assemble in the golgi apparatus and the virion begins to bud from the host cell. As the budding occurs or shortly after the virion emerges, it matures and infects other host cells. It is during this maturation stage when the HIV-1 protease cleaves gag and pol polyprotein precursors into mature functional proteins. Research has shown that HIV viruses containing inactive protease cannot replicate and infect further cells (Wlodawer et al., 1989).

    There are currently 9 FDA approved protease inhibitors and several more in late stages of clinical development (Wu et al., 2008). Here are the structures of six of the FDA approved HIV-1 inhibitors. The AIDS virus is constantly evolving to resist inhibitors; therefore, there is a constant need for the development of new inhibitors. Many of the current HIV protease inhibitors have poor pharmacokinetic properties such as low aqueous solubility, poor membrane permeability, high protein binding, and insufficient metabolic stability. The inhibitor/protease complex shown in this tutorial was crystallized by Wu et al. in 2008. It is a novel inhibitor because it has a two carbon elongation with a tertiary-alcohol-containing transition state mimic. This inhibitor has been shown to bind HIV-1 protease in vitro, but has yet to be tested in vivo.  

II. HIV-1 Protease Structure

    HIV-1 protease is a homodimer containing 99 amino acids . It is an aspartyl protease with one active site which is C2-symmetric when ligand free . The ligand inhibitor binds to the active site preventing polyprotein cleavage . The protease contains two β-sheet flaps which are connected by glycine rich loops . These flaps are part of the active site and play an important part in ligand binding. More than 140 structures of protease (wild type and mutant) have been complexed with various inhibitors. Here is an example of HIV-1 protease complexed with an inhibitor lacking the two carbon extension (Duskova et al., 2006)

III. Active Site

    HIV-1 protease belongs to the class of aspartic proteases. The active site of HIV-1 protease is made up of two loops with the sequence Asp-Thr-Gly, the active site triad, which is conserved among proteases of this family . These active site loops are held together by a network of hydrogen bonds between the active site amino acids and the surrounding residues. The rigid structure of the active site is due to the “fireman’s grip” which is formed when each of the Thr 26 residues accept a hydrogen bond from the amino group of the Thr 26 in the opposing chain and donate a hydrogen bond to the carbonyl oxygen of the Leu 24 residue located next to each catalytic aspartate .

    Each Asp 25 residue is also bound to the backbone NH group of the opposing Gly 27. Researches have found through mutational analysis that Asp 25 is important for proteolytic activity . When these aspartic acids are replaced by other amino acids the protein becomes inactive. All aspartyl proteases have a similar mechanism for proteolytic cleavage. One of the aspartic residues, Asp 25, is always deprotonated in the physiological pH range and can act as a hydrogen acceptor. Asp 25 can activate a nearby water molecule which attacks a carbonyl carbon on the peptide chain forming a oxyanion tetrahedral intermediate . The intermediate is then protonated and broken down into its hydrolysis products. Click here to see a more detail mechanism of this aspartic protease cleavage. Here is an example of an HIV-1 protease complexed with its cleaved protein product (Das et al., 2006) .

IV. Inhibitor Structure

    A commonality in all HIV-1 protease inhibitors is that they mimic the tetrahedral transition state of the proteolytic reaction. A nonhydrolyzable hydroxyethylene or hydroxyethylamine moiety is the basic core of almost all inhibitors as well as other non-cleavable protein transition states. More than 2000 inhibitors have been synthesized, but only 9 have been FDA approved. The inhibitor in this complex (Wu et al., 2008) has the basic structure of the FDA approved Indinavir inhibitor (a hydroxyethylene inhibitor) with two main differences

    First, it has a two carbon elongation between the central alcohol and the hydrazide part of the molecule . This inhibitor also has a tertiary alcohol in the transition-state mimicking scaffold in contrast to the secondary alcohol of other inhibitors . The tertiary alcohol is shielded by a benzene ring . There is also a  bromobenzene group in the P'1 substitute

    This complex shows one inhibitor of many that Wu et al. (2008) synthesized. A list of the other inhibitor compounds along with their Ki (binding affinity) and EC50(median effective concentration) can be found here. The authors made several changes to the P'1 substitute (shown as the R group in the figure).

V. Inhibitor Complex

    Here is the active site of the HIV-1 protease in complex with the two-carbon-elongated HIV-1 protease inhibitor . The inhibitor interacts with the active site of the protease by direct hydrogen bonds and indirect hydrogen bonds through two water molecules . The elongated inhibitor only accepts hydrogen bonds from one of the Asp 25 in the active site compared to shorter inhibitors which bind weakly to both. The P'1 bromobenzene extension of the inhibitor does not make any direct contacts with the active site of the protease and is extended towards the solvent. However, the P'1 extension of many other inhibitors does interact directly with the active site.

    Wu et al. (2008) crystallized complexes for three protease inhibitors. Compound 12d is the inhibitor featured in this tutorial, compound B lacks the carbon elongation and binds weakly to both Asp 25, and compound 15 has an extra central carbonyl oxygen. All three of these inhibitors have shielded tertiary alcohols. The HIV proteases in the Wu et al. (2008) paper were cloned and inserted into E. coli cells where binding affinity was measured. The anti HIV activity (EC50) was measured in vitro in Mt40 cells. While the in vitro experiments show promise, the drugs must be tested in vivo before it can be considered as a possible drug candidate.

VI. Mutations

    While there are currently several FDA approved protease inhibitors, the HIV-1 virus is able to mutate and develop resistance to these inhibitors. 1929 isolates from patients have been shown to contain drug induced mutation of the protease. Point mutations are often found in the region of the active site (Gly 48-Ile 50) , which interacts with the P'1 group of the inhibitor.  There are also 6 common point mutations found in the HIV-1 protease that are also found in FIV (Feline immunodeficiency virus protease): K20I, V32I, I50V, N88D, L90M, Q92K . These similarities allows for FIV to be used as a model for HIV-1 resistant proteases (Brik and Wong, 2002). Due to the frequency of mutations in HIV-1 protease, it is important to continually develop new inhibitors.  

VII. References

Brik, A., Wong, C.H., 2002. HIV-1 protease: mechanism and drug discovery. Organic Biomolecular Chemistry 2003. 1:5-14.

Das, A., Prashar, V., Mahale, S., Serre, L., Ferrer, J.-L/, Hosur, M.V., 2006. Crystal structure of HIV-1 protease in situ product complex and observation of a low-barrier hydrogen bond between caralytic aspartates. Proc Natl. Acad. Sci. USA 103: 18464-18469

Dun, B.M., Goodenow, M.M., Gustchina A., Wlodawer A., 2002. Retroviral proteases. Genome Biology 2002 3(4):reviews3006.1–3006.7 253: 1001-1007.

Duskova, J., Dohnalek, J., Skaova, T., Petrokova, H., Vondrackova, E., Hradilek, M., Konvalinka, J., Soucek, M., Brynda, J., Faby, M., Sedlacek. J., Hasek J., 2006. On the role of the R configuration of the reaction-intermediate isoster 1 protease-inhibitor binding: X-ray structure as 2.0 A resolution. Acta Crystallogr Sect. D, 62:489-497.

Pomerantz R.j., Horn, D.L., 2003. Twenty years of therapy for the HIV-1 infection. Nature Medicine 9(7):867-873.

Wlodawer, A., Miller M., Mariusz, J., Sathyanarayana, B.K., Baldwin E., Weber, I.T., Selk, L.M., Clawson, L., Shneider, J., Kent S.B.H., 1989. Conserved folding in retroviral proteases: Crystal Structure of a Synthetic HIV-1 Protease. Science 245(4918):616-621.

Wu, X., Ohrngren, P., Ekegren, J.K., Unge, J., Unge, T., Wallberg, H., Samuelsson, B., Hallburg, A., Larhed, M., 2008. Two-Carbon-Elongated HIV-1 Protease Inhibitors with a Tertiary-Alcohol-Containing Transition-State Mimic. Journal of Medicinal Chemisty 51(4):1053-1057.

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