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ChemWiki: The Dynamic Chemistry E-textbook > Biological Chemistry > Enzymes > HIV

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HIV

Table of Contents

Introduction

Human immunodeficiency virus (HIV) is a retrovirus, which is a class of viruses that carry genetic information in RNA.There are two types of HIV, HIV-1 and HIV-2, with HIV-1 being the most predominant, it is commonly called just HIV.  Both types of HIV damage a person’s body by destroying specific blood cells, called CD4+ T cells, which are crucial to helping the body fight diseases in the immune system. This can lead to immune deficiency, which is when the infection with the virus progressively deteriorates the immune system and is considered deficient when it no longer works to help fight infection and disease.2

According to the 2006 Morbidity and Mortality Weekly Report, published by the Center for Disease Control there were approximately 1.1 million United State Citizens are affected by HIV.3 There was an estimated number of 56, 300 people that newly contracted HIV.4 Although the annual incidence for HIV has remained constant throughout recent years, the prevalence has increased each year.  These drugs developed for HIV treatment are based on the mechanism HIV uses, including proteases, to infect its host.7 The HIV-1 protease is synthesized from the gag and pol genes along with other proteins.8 Retroviruses, such as HIV-1, are able to reverse transcribe because of the reverse transcriptase which is transcribed by the pol gene.9 HIV-1 RNA contains many genes, specifically gag and pol, that encode for many proteins.10 The open reading frames of gag and pol genes overlap in HIV-1.11 Studies have found that the initial cleavages are made by the immature protease dimer in the membrane of the infected cell during virus budding, or replication. Once these intramolecular cleavages are made a more active gag-pol processing intermediate is released, which becomes the active protease.8

Structure

The mechanism of HIV-1 protease is still yet to be fully understood.  The main way the mechanism are studied is through the use of mimicry substrates and simulations.  HIV-1  protease has been studied intensely using various inhibitors, observing partial steps of the process.  Since the main target of these inhibitors is to bind to the Asp-25 of the catalytic triad, each inhibitor would vary in its mechanism to accomplish this.14  Further investigation would then take place of the various proposed mechanisms in attempts to synthesize new drugs that would act in a similar fashion.

The first part of the mechanism begins with the substrate binding onto the protease.  Figure 2 accents the key amino acids in HIV-1 protease that assists in substrate binding.  It is predicted that a substrate first binds via a hydrogen bond to aspartic acid 30 on one chain.  Once this initial bond is made, the binding is then further stabilized by bondage to the glycine rich region in the flap of the same monomer.  A salt bridge is then formed from the substrate to glutamic acid 35 of the other monomer.  This completes binding of the substrate to the protease.15  At this point, waters molecules that are found at the tips of the flaps at isoleucine 50 on each monomer dissociates from the protease.15,16  The release of the water molecule results in a structural conformation change of the protease, changing it from semi-open to closed, tightening the space between the protease and substrate.15

HIV protease has variable states that it exists in, such as the two states mentioned above--the semi-open and closed state. These states depend on whether a substrate is bound to it.  In its unbound state, the protease’s glycine rich flaps (shown in grey in Figure 1) are in a semi open state.  Figure 1 depicts the protease in a closed flap state, which occurs when a ligand is bound to it (ligand not shown). An open state is thought to be the least frequent of the three states.17

Once in the tightened state, aspartic acid 25 and 25’ hydrogen binds to their adjacent glycine, and then becomes supported by the following threonine. Originally, there is a water molecule bound between the aspartic acids. One of the aspartic acid exists in a deprotonated state and the other one is protonated. The water molecule stabilizes the aspartates in this form.When the substrate binds to the protease, it causes conformational changes that brings the substrate to the position of the water molecule, and the water molecule acts as a nucleophile to the substrate. The oxygen of the water attacks the carbonyl group of the substrate peptide bond that is by the active site as the nitrogen picks up the hydrogen of the protonated aspartic acid.  What results is an hydroxl group is added to the carbonyl group as an amine is formed on other side of the peptide bond, leaving a hydrogen atom behind to stablize the two aspartates. This is proposed to occur in a concerted fashion. This mechanism is outlined in Figure 3.1,18




Figure 2. HIV-1 Protease with Accented Substrate Binding Assistant Amino Acids.13,15  Aspartic acid 30 is shown in pink; Glycine 48, 49, and 51 are shown in white; Isoleucine 50 and 50’ are shown in yellow; and glutamic acid 35’ is shown in green. (Primes distinguish amino acids from each monomer.)

 



Figure 3. Proposed Proteolytic Mechanism.1,18, Boxed molecules in “a” are showing the target peptide bond of the substrate and the water molecule hydrogen bonding between the two aspartic acids.  The rest are the catalytic aspartic acids in the active site. “b” shows the proposed concerted mechanism between the water, peptide bond and aspartic acids. “c” shows the end products.

Treatment

With a disease this prevalent, medication is key in trying to extend the afflicted’s life.  As mentions above, since a mutation to the aspartic acid in the active site of HIV-1 protease renders pro-viruses that are unable to form completely and infect other sites, the protease has been one of the targets for therapy.  These drugs are referred to as protease inhibitors.14 A current Food and Drug Administration approved drug against this HIV-1 protease is nelfinavir mesylate, 2-[2-Hydroxy-3-(3-hydroxy-2-methyl-benzoylamino)-4-phenyl sulfanyl-butyl]-decahydro-isoquinoline-3-carboxylic acid tert-butylamide; C32H45N3 O4S (“Viracept”R). Figure 3 shows the drug fitting into the protease.  At the center of the drug, a hydroxy group binds with the catalytic aspartic acid (boxed in red), while the other four groups (boxed in white) stabilizes the drug to the protease, making its bond more favorable than its natural substrate. This compounds accomplishes this by making various hydrophobic interactions and hydrogen bonds.19

This drug has a high drug efficiency.  In order to prevent 50 percent of the HIV-1 infected cells from becoming necrotic, a dosage of 14nM is required.19  Although it is a high potent drug, there also a few side effects that come along with it.  Side effects include fever, back pain, rash sweating, vomiting, and diarrhea based on a study of 62 HIV infected children ages 3 months to 13 years. Fourteen out of the 62 had diarrhea as a side effect and less than 6% of the study group had the other side effects.20

Due to the high mutation rate of HIV-1, often, multiple drugs are combined as a treatment in attempts to retard its spread as much as possible. A commonly seen drug paired with protease inhibitors is reverse transcriptase inhibitor.  Protease inhibitors prevents the protease transcribed by the gag-pol gene and reverse transcriptase inhibitors prevents the reverse transcriptase transcribed by the pol gene.  This combination targets two essential proteins that have been shown to stop HIV-1’s life cycle if these genes have been mutated. By targeting both proteins, HIV-1 activity is seen to decrease more than just one. An example of a reverse transcriptase inhibitor is Efavirenz. Efavirenz, in combination with nelfinavir mesylate has shown to increase the immune cell count and decrease the seen HIV-1 molecules in the blood plasma. The side effect of this drug are similar to those of nelfinavir mesylate.21  The effects of these developed drugs are the main reason HIV-1 infected people can live on life longer than they would have been able to in the past.




Figure 3. HIV-1 Protease with nelfinavir mesylate.22 This is a top down view of the protease showing how the drug fits into the protease.  Light blue molecules are carbons, red molecules are oxygens, blue molecules are nitrogens and yellow molecules are sulfurs.  White boxed areas show the four main pockets the inhibitor lays in and the red boxed area shows the binding to the catalytic aspartic acid.

References

  1. Brik, A.; Wong, C.H. HIV-1 protease: mechanism and drug discovery. The Royal Society of Chemistry, OBC.2003, 1, 5-14.
  2. Kilmarx P. Acquired immunodeficiency syndrome. Control of communicable diseases manual2008, 9.
  3. HIV Prevalence Estimates -- United States, 2006. CDC MMWR2008, 57.39, 1073-1076.
  4. Subpopulation Estimates from the HIV Incidence Surveillance System --- United States, 2006. CDC MMWR 2008, 57.39, 985-989.
  5. Seelmeier, S.; Schmidt, H.; Turk, V.;and Von Der Helm, K. Human immunodeficiency virus has an aspartic-type protease that can be inhibited by pepstatin A. PNAS 1988, 85, 6612-6616.
  6. Debouck, C.; Gorniak, J.; Strickler, J.; Meek, T.; Metcalf, B.; Rosenberg, M. Human immunodeficiency virus protease expressed in Escherichia coli exhibits autoprocessing and specific maturation of the gag precursor. PNAS1987, 84, 8903-8906.
  7. Lillehoj, E.P.; Salazar, F.H.R.; Mervis, R.J.; Raum, M.G.; Chan, H.W.; Ahmad, N.; Venkatesan, S. Purification and Structural Characterization of the Putative gag-pol Protease of Human Immunodeficiency Virus. Journal of Virology 1988, 62.8, 3053-3058.
  8. Pettit, S.C.; Everitt, L.E.; Choudhury, S.; Dunn, B.M.; Kaplan, A.H. Initial Cleavage of the Human Immunodeficiency Virus Type 1 GagPol Precursor by Its Activated Protease Occurs by an Intramolecular Mechanism. Journal of Virology 2004, 78.16, 8477-8485.
  9. Lightfooote, M.M.; Coligan, J.E.; Folks, T.M.; Fauci, A.S.; Martin, M.A.; Venkatesan, S. Structural Characterization of Reverse Trascriptase and Endonuclease Polypeptides of the Acquired Immunodeficiency Syndrome Retrovirus. Journal of Virology 1986, 60.2, 771-775.
  10. Veronese, F. D. M.; Copland, T.D.; DeVico, A.L.; Rahman, R.; Oroszlan, S.; Gallo, R.C.; Sarngadharan, M.G. Characterization of Highly Immunogenic p66/p51 as the Reverse transcriptase of HTLV-III/LAV. Science 1986, 231, 1289-1291.
  11. Henderson, L.E.; Benveniste, R.E.; Sowder, R.; Copeland, T.D.; Schultz, A.M.; Oroszlan, S. Molecular Characterization of gag Proteins from Simian Immunodeficiency Virus (SIVMne). Journal of Virology 1988, 62.8, 2587-2595.
  12. Adkins, J.C.; Noble, S. Efavirenz. Drugs1998,56(6),1055-1064.
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