Drug Strategies to Target HIV:
Enzyme Kinetics and Enzyme Inhibitors

Chemical Kinetics Experiment

Authors: Rachel Casiday and Regina Frey
Department of Chemistry, Washington University
St. Louis, MO 63130


Key Concepts:


The Deadly HIV Virus

HIV (human immunodeficiency virus), the virus that causes AIDS, is one of the hottest areas of medical research today. It is estimated that 30.6 million people throughout the world were infected with HIV by the end of 1997. After a person has been infected with HIV, the virus usually remains dormant for long periods of time. Then the virus begins a cycle of attacking cells of the immune system by incorporating its genetic material into the cells, using the immune cells' machinery to make more viruses from the incorporated genetic material, and then breaking the cells apart (killing them) so that the new viruses can infect more cells. In this manner, the immune system is weakened, so that the body can no longer defend itself against the pathogens that it encounters every day. Sadly, patients typically die within a few years of showing symptoms of AIDS (i.e., signs of drastically decreased immunity due to the virus's attack on the cells of the immune system). Because so many people are affected by HIV, and because the virus is so deadly, much research has been devoted to finding ways to fight this epidemic. Researchers are seeking treatments for HIV-infected individuals, cures, and vaccines. So far, the most promising findings have been treatments to lessen the damage to the immune system from HIV; several treatments that have been developed have dramatically improved the outlook for many HIV patients. However, these treatments have many severe side effects, and in most cases are too costly to be administered widely in many parts of the world. Thus, more research is needed to improve the available treatments, making them more tolerable to patients and more accessible, and to continue the search for a cure for HIV and a vaccine, which would prevent the infection in the first place.

This tutorial describes two of the most successful treatment options available today: reverse-transcriptase inhibitors (e.g., AZT) and protease inhibitors. These treatments interfere with enzymes that are needed for HIV to make copies of itself, a key step in the virus's attack on the cells of the immune system. In order to understand how these treatments help, we must first discuss the immune cells that come under attack, and the mechanism by which the virus kills these cells if left untreated.

HIV Attacks Helper T Cells

Our body's immune system contains many different types of cells, but only one of these cell types, known as helper T cells, is attacked by HIV. Helper T cells are necessary to stimulate the activation of other immune cells that attack infectious particles (antigens) in the body. When these cells come under attack by HIV, the immune system can no longer function effectively, and the body is incapable of combating many types of foreign infections and cancers. A more detailed description of normal immune function, and why helper T cells are critical to the immune system, is given in the peach box, below.

The Immune System: The Body's Defense Against Infection

The immune system is a collection of cells found in the blood and in tissues throughout the body that help to protect the body from infection. These cells perform a variety of nonspecific and specific functions to defend the body against harmful particles (e.g., bacteria, viruses, toxins, and cancerous cells). Nonspecific responses do not require that the cell recognize a particular type of infectious agent; such responses include inflammation (which destroys or inactivates invading particles and prepares a site for tissue repair) and phagocytosis (which engulfs the harmful particles using large, specialized immune cells). Specific immune responses are directed against a particular infectious agent, and involve the recognition and attack of particular particles, known as antigens.

Types of Immune Cells

The most numerous cells of the immune system are the leukocytes (white blood cells), which are produced in the bone marrow and travel through the blood to other tissues, where they do their infection-fighting work. Other immune cells include plasma cells (which make and secrete antibodies), macrophages (large cells that engulf invading particles), and mast cells (which secrete locally-acting chemicals involved in inflammation). The classification of the cells of the immune system is summarized in Figure 1, below. Because the leukocytes are the most numerous immune cells, and also include the type of cells attacked by HIV, we shall focus on the different types of leukocytes, and how they protect the body from infections.

Figure 1

This diagram summarizes the classification and functions of the major types of cells in the immune system. Note that the helper T cells, a specific type of leukocyte, are the target for HIV infection.

There are five major classes of leukocytes: neutrophils, basophils, eosinophils, monocytes, and lymphocytes. The neutrophils, basophils, eosinophils, and monocytes, like many of the other immune-cell types described above, participate in nonspecific immune defenses, including stimulating inflammation, engulfing particles, and making the body more sensitive to invading particles. The lymphocytes, which is the class of leukocytes that the HIV virus targets, are a far more complex class of leukocytes that participate in specific immune defenses, i.e., they must recognize the specific material being attacked. A particle that triggers a specific immune response, such as a toxic molecule or a special protein bound to the outside of a bacterium or infected cell of the body, is known as an antigen.

Lymphocytes' Attack on Antigens

How do the lymphocytes recognize and attack antigens in the body? First, a lymphocyte must encounter and recognize the antigen. Each lymphocyte in the body has a receptor (Figure 2) that can bind to a specific antigen; the body contains millions of lymphocytes with different receptors to recognize a huge number of different antigens that the body might encounter. The receptors are proteins with a specific amino acid sequence at the binding site, giving the binding site a shape that will allow it to bind to a specific antigen (depending on the shape and polarity of the antigen). When a lymphocyte encounters the antigen for which it has a receptor, the receptor binds to the antigen.

Figure 2

This figure shows how the receptor on a lymphocyte recognizes and binds to a specific antigen. Here, a cytotoxic T cell (turquoise) recognizes a cell that has been infected by a virus (mauve), because the T cell's receptor binds to a viral protein (yellow) on the outside of the cell. The particle that binds to the receptor (i.e., the viral protein on the outside of the cell) is the antigen.

Once a lymphoctye has bound to an antigen, the lymphocyte then becomes "activated". The lymphocyte undergoes a series of cell divisions to produce many cells that are identical to the one that first recognized the antigen. Finally, the activated lymphocytes attack the antigen, and all antigens of the same kind that are found throughout the body.

To understand the activation and attack processes, we must differentiate between the different types of lymphocytes. The principal types of lymphocytes are called B cells, cytotoxic T cells, helper T cells, and NK cells. (The designation "B" and "T" refer to where the cells mature. Although all lympocytes originate in the bone marrow, the T cells then travel to the thymus ("T" for "thymus") to mature; B cells mature in the bone marrow ("B" for "bone"); NK stands for "natural killer", and it is uncertain where these cells mature.)  B cells recognize foreign antigens, such as bacteria, viruses, and toxins. Cytotoxic T cells, on the other hand, recognize as antigens the body's own cells that have become cancerous or infected by a virus.

B cells, cytotoxic T cells, and helper T cells all contain receptors that bind to antigens and become activated. Once activated, helper T cells do not attack the antigen themselves, but help to activate B cells and cytotoxic T cells by secreting special chemical signals. With very few exceptions, these signals from helper T cells are required for the activation of the B cells and cytotoxic T cells. Helper T cells are the cells in the body that are attacked by HIV. Hence, HIV disrupts the immune system by destroying the cells needed for B cells and cytotoxic T cells to become activated and thus attack antigens in the body.

With the chemical signal from helper T cells, the B cells and cytotoxic T cells that have recognized and bound to an antigen can then become activated and begin their attack. The two types of lymphocytes differ in the types of antigens they recognize and attack, and in their mode of attack. B cells, which recognize foreign antigens, do not attack the antigen directly. Instead, they release particles known as antibodies, which guide other cells (e.g., macrophages and NK cells) to the antigen to engulf or neutralize the antigen. Cytotoxic T cells, which recognize the body's own cells that are cancerous or infected as antigens, directly attack their antigens, killing the cancerous or infected cells.

Overview of HIV's Attack on the Immune System

How does HIV invade and kill helper T cells, thereby depleting our immune system? To answer this question, we must first clarify what we mean by the term virus. Then we shall outline the major steps in the life cycle of HIV, and how these steps lead to the destruction of helper T cells. As you read this description, note that many of these steps are made possible by particular enzymes, biological catalysts that change the mechanism (and therefore the kinetics) of a biochemical reaction in order to enable the reaction to proceed with a smaller activation energy. (A more in-depth explanation of how enzymes work is given in the section below, "Enzymes as Biological Catalysts".)

A virus is often classified as a living thing, although it is not made up of cells, like other organisms. Viruses consist of proteins surrounding genetic material. Depending on the virus, this genetic material may be either DNA (the form in which our cells' chromosomes contain genetic material) or RNA (the form used for the expression of genetic information in cells, and the form in which some viruses carry their genetic information). Of the two types of viruses, DNA viruses and RNA viruses, HIV represents the second. A virus containing RNA is known as a retrovirus. In order to reproduce, a retrovirus must attach to a cell of the infected organism, insert its RNA into the cell, and make a DNA copy of the RNA. This DNA copy then incorporates into the cell's own chromosomes (which are made of DNA), and uses the cell's biochemical machinery to replicate the viral DNA (along with the host cell's DNA), make viral proteins from the DNA that is replicated, and assemble new viral particles from these proteins.

The steps by which HIV infects and kills helper T cells are described below, and can be viewed in Figure 3.

Figure 3

This schematic shows the major steps in HIV's attack on the human immune system. The numbers correspond to the numbered steps in the description of the infection cycle, below.

HIV Infection Cycle

  1. The first step in HIV's attack on helper T cells is attaching to the cell. Helper T cells contain proteins called CD4 proteins in their cell membrane that extend outside of the cell. Normally, these proteins help the cells to bind to antigens (infectious particles) in order to stimulate activation of the helper T cells, and they are also required for normal T cell development. Unfortunately, however, CD4 proteins also function as receptors for HIV, allowing the virus to attach itself to the cell and thereby gain access to the cell's biochemical machinery.
  2. Once the virus has attached to a helper T cell, it injects its genetic information (as RNA) into the cell, along with the enzyme reverse transcriptase.
  3. Reverse transcriptase catalyzes the production of DNA from the viral RNA, making a DNA copy of the virus's genetic material. This DNA copy is capable of incorporating itself into the cell's genetic material, because it is now in the same form as the cell's chromosomes. Hence, the step catalyzed by reverse transcriptase is one of the most important steps in the infection cycle.
  4. The viral DNA copy then enters the nucleus of the infected helper T cell, where it is incorporated into the cell's genetic material (i.e., the chromosomes).
  5. Using the cell's own DNA-replication mechanisms, the viral DNA replicates.
  6. Using the cell's mechanisms for producing proteins from the genetic information contained in DNA, many copies of the proteins needed by the virus are made from the replicated HIV DNA. As part of this step, RNA copies of the viral DNA are made.
  7. When they are first synthesized, the proteins are too long (containing extra fragments) to be assembled into new viruses. They must be cut to their proper size. The HIV enzyme protease, which is produced by the cell's biochemical machinery from the viral DNA incorporated into the cell's chromosomes, catalyzes the cutting of these proteins to their proper size.
  8. New HIV particles (viruses) are assembled inside the cell from the cut viral proteins and the viral RNA copies.
  9. Once assembled, the new viruses then burst out of the host cell (killing it) and invade new cells, continuing the infection.

As you can see from the steps outlined above, enzymes play a vital role in HIV's attack on helper T cells. The generation of a DNA copy of the viral genome by reverse transcriptase (Step 3) and the cleavage of viral proteins (Step 7) by protease are two important processes catalyzed by enzymes. Therefore, the enzymes reverse transcriptase and protease are major target sites for HIV-fighting drugs. Before we can explain how these drug treatments work to combat HIV, we must first discuss how enzymes work to catalyze biochemical reactions.


Questions on HIV's Attack on the Immune System


Enzymes as Biological Catalysts

Many biological reactions, such as most normal reactions of the cells, and the reactions employed by HIV to replicate itself, are thermodynamically favorable (DG<0) or can be made thermodynamically favorable by coupling with other reactions. Recall that thermodynamics tells us whether a reaction (or process) is spontaneous under a specified set of conditions. However, thermodynamics does not tell us how fast (or the rate with which) a reaction will proceed. Rates of reactions are the realm of chemical kinetics and are determined experimentally. From Arrhenius Theory, the rate of a reaction is dependent on the activation energy (Ea), which is the minimum amount of energy needed for a reaction to occur (See Figure 4, Path A). Hence, even though a reaction is thermodynamically favorable, it still can not occur unless there is enough energy available (an amount greater than or equal to the activation energy) to initiate the reaction. If sufficient activation energy cannot be supplied (i.e., the rate of the reaction is too slow), then a catalyst may be used. Recall from the introduction to the Experiment that a catalyst is a substance that increases the rate of a reaction without being consumed. A catalyst influences the mechanism (pathway) of a reaction, but does not affect the thermodynamics (DG). A catalyst allows the reaction to proceed by an alternate mechanism that has a lower activation barrier (Figure 4, Path B). Often biological reactions require catalysts called enzymes to change the pathway (mechanism) of the reaction, and thus lower the activation energy. When a catalyst changes the reaction pathway to lower the activation energy, the reaction rate is increased, but the thermodynamics of the reaction are not changed. That is, a catalyst cannot form products that are not allowed by thermodynamics (DG); it does increase the rate of forming the products that are allowed by thermodynamics. Enzymes are molecular catalysts in biological systems, and are usually proteins. Virtually all biological reactions are catalyzed by enzymes.

Figure 4

The schematic on the left, Path A, (blue) shows the high activation energy associated with an uncatalyzed reaction, and the schematic on the right, Path B, (red) shows the lower activation energy associated with the same reaction in the presence of a catalyst.

How Enzymes Work

Enzymes are very specific to the substrates (reactants) and reactions that they can catalyze. Enzymes work by binding the substrate into a favorable orientation in an enzyme-substrate complex (an intermediate in the reaction), which promotes the making and breaking of chemical bonds. The substrate is bound to the active site (a specific region of the enzyme), is converted to the product, and then released from the active site. The active site is a three-dimensional crevice, and is usually only a small portion of the total volume of the enzyme (Figure 5).

Figure 5

This is a schematic diagram showing the active site of an enzyme (green)

The substrate is "bound" to amino acids in the active site of the enzyme by multiple intermolecular interactions, including charge-charge interactions (electrostatics), hydrogen bonding, and van der Waals forces. Enzymes are often very sensitive to changes in pH and temperature, in part because these changes can affect the shape and charge of the active site, thus changing the interaction with a substrate. If the optimal conformation of the enzyme is lost, the enzyme becomes nonfunctional (i.e., the substrate can no longer bind to active site).

Enzyme Kinetics

The study of the kinetics of a reaction that is catalyzed by an enzyme is essentially like the kinetics studies that you perform in lab, but involves a few additional, specialized concepts. The model for understanding the kinetic properties of most enzymes is known as the Michaelis-Menten model. This model proposes the following mechanism for enzyme catalysis. First, the enzyme (E) and substrate (S) come together to form an enzyme-substrate complex (ES), as shown in Equation 1, below. The reaction occurs, and the substrate is converted to the product of the reaction. Then, the enzyme-substrate complex is broken apart, yielding enzyme (E) plus product (P), as shown in Equation 2, below.

Mechanism:

(1)

(2)

Overall reaction:

(3)

The Michaelis-Menten model assumes that only a negligible amount of enzyme-substrate complex reverts to reactants(i.e., k-1 << k1 in Equation 1). The rate of formation of product (shown below in Equation 4) can be determined from Equation 2 in the mechanism written above

Rate of formation of Product = k2[ES]

(4)

and the rate of formation of the intermediate ES (shown in Equation 5) can be determined from Equations 1 and 2 in the mechanism written above

Rate of formation of ES = k1[E][S] - (k2 + k-1)[ES]

(5)

Using the steady-state approximation (i.e., the assumption that the concentrations of intermediates (in this example, ES) stay constant while the concentrations of reactants and products change), and making several substitutions (which are not shown here), we can form an equation for the rate of formation of the product (Equation 6).

(6)

where [E]o is the initial concentration of free enzyme, [S] is the substrate concentration, and Km is a constant specific to a given enzyme known as the Michaelis-Menten constant. The value of Km relates to the rate constants shown in Equations 1 and 2, as given by Equation 7:

(7)

The Michaelis-Menten constant (Km) is very important, because it can be determined experimentally and describes the catalytic power of an enzyme. Km can also be used to predict the rate of a reaction catalyzed by an enzyme, given the starting conditions.

Enzyme Inhibition

Enzyme function may be hampered by the addition of molecules or ions called inhibitors. Many drugs work by inhibiting (slowing or stopping) various enzymes. Inhibitors function by forming an enzyme-inhibitor complex, which impedes the ability of the enzyme to convert substrate to product.

Inhibitors can be reversible or irreversible. Irreversible inhibitors are tightly bound to the enzyme, often through covalent bonds.  Hence, the enzyme-inhibitor complex does not dissociate (or it dissociates very slowly). Reversible inhibitors bind through electrostatic interactions (e.g., dipole-dipole interactions).  These weaker interactions form an enzyme-inhibitor complex that dissociates very quickly. Reversible inhibitors are generally classified as competitive or noncompetitive.  (Irreversible inhibitors do not dissociate; therefore, they cannot be classified as competitive or noncompetitive.)

A competitive inhibitor often binds to an enzyme's active site (i.e., competes with the normal substrate for the enzyme's active site), thus preventing substrate molecules from binding to the enzyme and reacting.  Note that both the substrate and the inhibitor bind loosely enough to the enzyme that the enzyme-substrate and enzyme-inhibitor complex dissociate rapidly.  In a typical scenario, a single molecule of enzyme collides with a substrate molecule and binds it  for a short time.  Either the substrate is rapidly converted to product, or it quickly "falls off" the enzyme unchanged.  The enzyme will then collide with and bind another molecule, perhaps an inhibitor this time.  While the inhibitor is bound, the enzyme does not convert any substrate to product, because the competitive inhibitor and substrate do not bind simultaneously.  After a short period, the inhibitor dissociates, and another collision between the enzyme and either substrate or inhibitor results in a new binding event.  Note that competitive inhibitors prevent the substrate from binding. 

Noncompetitive inhibitors usually bind to a different site on the enzyme (i.e., not the enzyme's active site for the normal substrate). When a noncompetitive inhibitor binds to an enzyme, the shape of the enzyme may be changed. This changes the mechanism of the enzyme reaction and slows the rate at which the substrate is converted to product.  Note that the presence of a noncompetitive inhibitor does not completely prevent the substrate from binding, it just slows down the rate at which the substrate is converted to product.

The most common method of determining whether a reversible inhibitor is competitive or noncompetitive is to see what effect the inhibitor has on the rate at which product is formed using different concentrations of the substrate.  Recall, the enzyme randomly binds either a competitive inhibitor or substrate molecule, but not both, depending on which it collides with first.  A competitive inhibitor will be more effective at low substrate concentrations because the enzyme is more likely to collide with and bind the inhibitor if there are fewer substrate molecules in the vicinity.  A noncompetitive inhibitor will be equally effective at low and high substrate concentrations.  Recall, noncompetitive inhibitors bind along with substrate, not instead of it.  Hence, adding more substrate does not influence whether the enzyme-inhibitor complex forms.  In other words, to determine whether an inhibitor is competitive or noncompetitive, one compares the measured value of Km in the presence and absence of inhibitor.  For a competitive inhibitor, the measured value of Km increases in the presence of inhibitor. For a noncompetitive inhibitor, the measured value of Km remains the same. As will be discussed below, the most successful treatments for HIV have resulted from using inhibitors for the two enzymes reverse-transcriptase and protease.


Questions on Enzymes as Biological Catalysts

  1. Can a competitive inhibitor be overcome by saturating the reaction with substrate? Briefly, explain your answer.
  2. Can a noncompetitive inhibitor be overcome by saturating the reaction with substrate? Briefly, explain your answer.

Enzyme-Targeting Drugs to Fight HIV

We have described the role of two critical enzymes, reverse transcriptase and protease, in HIV's ability to infect helper T cells, make copies of itself, and ultimately destroy the cells, depleting the body's immune system. We have also seen how enzymes catalyze biochemical reactions. Now, we turn our focus to two of the most successful treatments for HIV, reverse-transcriptase inhibitors and protease inhibitors. As their names imply, these drugs inhibit enzymes that are necessary for HIV to replicate itself inside helper T cells and deplete the body's immune system.

Reverse-Transcriptase Inhibitors: AZT

Recall from Figure 3, above, that reverse transcriptase catalyzes the formation of a DNA copy of the virus's RNA genetic information. Without the reverse transcriptase enzyme, the virus HIV cannot make DNA copies of its genetic material (i.e., the RNA), because the activation energy for the uncatalyzed reaction is too large. The DNA copy is essential for the virus to take over the infected cell's machinery and produce new copies of itself. Therefore, reverse transcriptase has long been an obvious target in the scientific battle against HIV and AIDS.

How does reverse transcriptase lower the activation energy to enable the virus to make the DNA copy of its RNA? The reverse-transcriptase enzyme (Figure 6) is a protein consisting of two peptide subunits (amino-acid chains). At the interface of the two subunits is an active site where a strand of viral RNA, together with the newly-emerging strand of DNA formed from the RNA template, can fit. A portion of the larger subunit functions as a ribonuclease, digesting the RNA once the DNA copy has been made. Except for the ribonuclease region, the two subunits are identical.

Figure 6

This is a three-dimensional CPK (space-filled) representation of HIV Reverse Transcriptase, complexed with the viral RNA (yellow) and the newly-forming strand of DNA (pink). The enzyme consists of two subunits, known as p51 (green) and p66 (blue). A portion of the p66 subunit functions as a ribonuclease and is shown in red.

Note: The coordinates for the model were determined from x-ray crystallographic data, and the image was rendered using SwissPDB Viewer and POV-Ray (see References).

Note: To view the reverse-transcriptase enzyme interactively, please use Jmol, and click on the button to the left.

To more fully understand how reverse transcriptase enables the virus to make DNA copies of its RNA, we must know something about the structure of DNA, and the mechanism by which a strand of DNA is generated. The concepts are described in the blue box, below.

Structure of DNA

DNA and RNA, like proteins, consist of chains of smaller building-block molecules. The building blocks for DNA and RNA are called nucleotides (Figure 7). There are four different nucleotides in DNA, and the sequence of these nucleotides in a DNA strand determines the sequence of amino acids in the protein that will be made from the genetic information contained in the DNA. RNA consists of a single strand of nucleotides, and DNA consists of two strands of nucleotides that are connected by hydrogen bonds along the length of the chain. Each nucleotide has a complementary nucleotide with which it is paired in the opposite chain. For instance, the nucleotide adenine is always situated across from a thymidine nucleotide in a double-stranded piece of DNA. As shown in Figure 7, each nucleotide consists of a single- or double-ring structure (known as a pyrimidine or a purine, respectively), attached to a sugar molecule. The sugar molecule contains two -OH groups, known as the 5' -OH group and the 3' -OH group. At the 5' -OH group, a phosphate (PO43-) group is attached to complete the nucleotide.

Figure 7

This figure shows the important features of a nucleotide: the pyrimidine or purine (blue), the sugar (black), and the phosphate group (green). The pyrimidine or purine determines the identity of the nucleotide. The oxygens in the 3' and 5' OH groups are shown in red. The hydrogen from the 5' -OH group is removed in order to form the bond with the phosphate group.

Making a DNA Copy of RNA Genetic Material

To make DNA from RNA, a short piece of RNA, known as a primer, that contains the proper sequence of amino acids to form complementary pairs with a segment of the RNA chain to be copied (the "template"), is aligned opposite the RNA strand to be copied and forms hydrogen bonds with it. The 3' -OH group of the primer can now form a covalent bond with the phosphate group of another nucleotide that complements the next nucleotide on the template RNA strand. The new nucleotide will hydrogen bond with the complementary nucleotide on the template strand, as shown in Figure 8. Additional nucleotides are added in the same manner, until the strand is complete.

Figure 8

This figure shows a strand of DNA being synthesized, using an RNA strand (orange) as the template. The part of the DNA strand that has already been synthesized is at the bottom of the right-hand side of the image. The hydrogen bonds that form between complementary purines and pyrimidines are shown as dotted magenta lines. A new nucleotide (pink background) is added by forming a covalent bond between the 3' -OH group (red) of the last nucleotide on the existing strand, and the phosphate group (green) of the new nucleotide. The site of this bond is shown with yellow arrows. Note that the new nucleotide actually has three phosphate groups attached at its 5' -OH position; two of these will be removed when the bond between the phosphate and the existing strand is formed.

How does reverse transcriptase catalyze the formation of a DNA copy of HIV's RNA genetic material (the reaction described in the blue box, above)? To synthesize DNA, reverse transcriptase first positions the RNA strand (called the "template") into the active site, together with an additional, special piece of viral RNA that serves as the primer, or first piece, of the new strand. The primer RNA lines up opposite the template RNA strand, forming hydrogen bonds between the complementary purines and pyrimidines, as shown in Figure 8, above. Then, reverse transcriptase positions a new nucleotide such that a covalent bond can form between the 3' -OH group of the last nucleotide in the primer and the phosphate group of the new nucleotide (see Figures 7 and 8). Reverse transcriptase lowers the activation energy for this reaction by bringing the molecules in close proximity to one another in the active site.

It is possible to inhibit the action of reverse transcriptase using drugs. Currently, six drugs that act as inhibitors of reverse transcriptase are on the market for treating HIV-infected patients. Zidovudine, or AZT, is one of the earliest and best known of these drugs; it is marketed under the brand name Retrovir. AZT (Figure 9) functions as an analog for thymidine (Figure 10), one of the nucleotide building blocks of DNA. This means that AZT has the same shape as thymidine, and therefore it can be incorporated into the developing nucleic acid in place of a thymidine molecule. The phosphate group attached to thymidine or AZT forms a bond with the 3' -OH group of the preceding nucleotide in the developing DNA chain. When thymidine is incorporated into the DNA chain, its 3' -OH becomes the binding site for the next nucleotide's phosphate group. However, AZT lacks the -OH functional group that is necessary to form a bond with the next nucleotide; in its place is an azido (-N3) group. Because the azido group cannot form a bond with a phosphate group, no additional nucleotides can be added once AZT is incorporated into the DNA chain. Hence, reverse transcription stops after AZT is incorporated.

Figure 9

This is a two-dimensional representation of AZT with a phosphate group attached to the 5' -OH group (i.e., the -OH group attached to the 5' carbon of the sugar). AZT stands for "azidothymidine" because it resembles thymidine but has an azido (-N3) group in the 3' position (i.e., attached to the 3' carbon) of the sugar portion of the molecule. This azido group terminates the nucleic acid chain because it cannot bond to another nucleotide.

Figure 10

The normal phosphorylated thymidine molecule can be phosphorylated (addition of a phosphate group) to become one of the nucleotide building blocks of a DNA strand. The 3' -OH group (i.e., the -OH group attached to the 3' carbon of the sugar) allows thymidine to bond to another nucleotide via the phosphate linkage, continuing the nucleic acid chain.

A major problem with AZT is that the HIV virus quickly mutates, and strains that are resistant to the drug may arise in patients who have been taking AZT for extended periods of time. One strategy that doctors use to get around this problem is "multiple-drug therapy." AZT is administered in combination with other reverse transcriptase inhibitors or, increasingly, with one or more protease inhibitors (see next section below). Thus, mutants that evolve with resistance to any one of the drugs are still likely to be killed by the other drugs in the therapeutic regimen.

Protease Inhibitors: A New Line of Attack

In December 1995, the FDA approved a new type of drug for combating HIV. This class of drug acts by inhibiting protease, the enzyme required by HIV to cut its protein into the proper segments to assemble new viral particles. Protease inhibitors, used in combination with two reverse transcriptase inhibitors, have proven to be quite successful. In 80 to 90 percent of patients, this combination treatment reduces the amount of HIV in the blood to an undetectable level.

As shown in Figure 3, the proteins produced from HIV's genetic material are larger than the proteins needed to form new viral particles. In fact, a large "polyprotein" is formed that contains several viral proteins joined together. The polyprotein must be cleaved into the individual functional proteins. This cleavage is catalyzed by protease (Figures 11 and 12). (Cells also contain proteases of their own; the protease described in this section, however, refers to the protease that is made from HIV's genetic information and is used to cleave HIV proteins.) This enzyme is a symmetric homodimer, or a protein consisting of two identical peptide subunits. Like the active site of reverse transcriptase, the protease active site lies at the interface of its two subunits. The mechanism by which protease cleaves the HIV polyprotein will not be discussed in this tutorial. Protease inhibitors reversibly bind to the protease enzyme and, while bound, prevent the enzyme from cutting the viral protein molecules down to their proper sizes.

Figure 11

This is a CPK representation of protease with a protein substrate (gold) occupying the active site. The active site lies at the interface of the two identical subunits (green and purple).

Note: The coordinates for Figures 11 and 12 were determined from x-ray crystallographic data, and the images were rendered using SwissPDB Viewer and POV-Ray (see References).

Note: To view the uninhibited protease enzyme interactively, please use Jmol, and click on the button above.

Figure 12

This is a CPK representation of the inhibited protease. The compound DMP323 (blue), although not approved for use in humans, is a potent inhibitor of protease and is used in scientific investigations to understand how protease inhibitors work, and how the virus might mutate to gain resistance to this class of drugs.

Note: To view the inhibited protease enzyme interactively, please use Jmol, and click on the button above.

Questions on Enzyme-Targeting Drugs to Fight HIV

  1. Is AZT a competitive or a noncompetitive inhibitor?
  2. Does AZT change the value of Km for reverse transcriptase?

Thymidine Kinase + Substrate + Phosphate ---> Thymidine Kinase + Substrate-Monophosphate

where Substrate = Thymidine, ADRT, or AZT

Chen et al. found that the Km value for the thymidine kinase reaction with thymidine as a substrate is 0.7 mM, but the Km value for the thymidine kinase reaction with ADRT as a substrate is 8.3 mM. Using the definition of Km (Equation 7), together with your understanding of competitive enzyme inhibition, predict whether ADRT will be a good inhibitor or a poor inhibitor of thymidine kinase. Briefly, explain your reasoning.


Conclusion

To reproduce, HIV infects an immune cell called the helper T cell. (T cells help control the body's response to many types of infections.) The HIV then uses the machinery of the helper T cell to make copies of the HIV virus. The major reproductive steps in HIV's infection of the helper T cell are (1) attaching to the cell, (2) injecting RNA into the cell, (3) making a DNA copy of the genetic information contained in HIV's RNA, (4) incorporating the viral DNA into the cell's chromosomes, (5) replication of the viral DNA using the cell's machinery, (6) producing proteins from the viral genetic information, using the cells machinery, (7) cutting the proteins to proper size, (8) assembling new viral particles, and (9) bursting out of the host cell to continue the infection.

As discussed in the tutorial, most of these steps are accomplished by using enzymes, which, in biological systems, are proteins that catalyze reactions. Enzymes form an enzyme-substrate complex with the normal reaction substrates. The formation of the enzyme-substrate complex helps to lower the activation energy of the reaction. The treatment to help HIV-infected people has been based on developing drugs that inhibit these major reproductive steps. Inhibitors are drugs that work by forming an enzyme-inhibitor complex, which impedes the ability of the enzyme-substrate complex to form. The most successful treatments for HIV have resulted from using inhibitors for the two enzymes reverse transcriptase (step 3 above) and protease (step 7 above). These two types of inhibitors are described in the tutorial.

Since the discovery of HIV virus in 1984, much research has been conducted and resulted in an increased understanding of the virus, and the development of drugs that have been successful in the treatment (but not cure) of HIV. However, more research is needed in order to effectively combat the global epidemic of HIV. Research is continuing to develop new drug treatments for the disease. In addition to the inhibitors described in the tutorial, current research is focusing on inhibitors that will block the attachment of HIV to helper T cells (step 1 above), the integration of HIV's DNA into the cell's genetic material (step 4 above), and the assembly of new viral particles (step 8 above). An inhibitor for the enzyme integrase (used to incorporate viral DNA into the cell's chromosomes in step 4 above) has been developed and is currently in clinical trials. The development of the other new inhibitors is still in its infancy.

Of course, researchers would ultimately like to find a cure and a vaccine for the virus, rather than rely forever on treatments that only limit the extent of the infection. Researchers are focusing on the attachment of HIV (in step 1) for developing vaccines, and many researchers are hopeful that an effective vaccine will be found within the next ten years.


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