A recent, highly interesting article written by Michael Balter which appeared in the 20 December, 1996 issue of the journal Science concerned the selection of Protease Inhibitors as a new treatment for AIDS as the "Breakthrough of the Year." [New Hope in HIV Disease, (1997), Science, 274: 1988-1989]. This article describes the use of protease inhibitor formulations in concert with at least two reverse transcriptase inhibitor preparations to effectively treat HIV infection. In many instances, such combined therapy has led to the inability to any longer detect the virus within treated patients. And, while it is certainly recognized that such treatment does not offer a cure for the disease, the treatment does for the first time offer significant hope that HIV infections might at least be managed sufficiently to allow a reasonable life for many who suffer from this disease . Further, the research which led to the use of protease inhibitors for treatment of HIV infection has provided further information as to the mechanism of HIV infection, and may perhaps lead to other treatment therapies. OK - let's get started.......
What is a Protein?
A protein is a substance made of many individual amino acids (20 different chemicals which can appear in any combination), each connected one after the other (linearly) via a covalent bond called a peptide bond. Another name for a protein is, polypeptide, since any protein will normally have many amino acids, and therefore many peptide bond connections within the protein itself. The overwhelming majority of genes within our chromosomes contain information which is eventually translated into the formation of thousands of the different and uniquely functional proteins upon which a cell's life depends (You may wish to read, What the Heck is a Gene?).
The unique total number and combination of amino acids within any given protein determines what the protein's final shape and function will be, e.g., a heart muscle protein, or perhaps a protein which transports something through a cellular membrane, or, perhaps a protein which has catalytic activity - an enzyme. If you don't know what the word, enzyme, means, you may wish to access: What The Heck is an Enzyme.
What is a protease?
The term, protease, refers to a specific kind of enzyme - an enzyme which displays a unique, highly specific catalytic activity against a given protein or similar proteins. The commonly accepted terminology for anything which is an enzyme requires that the last three letters of the word end in -ase - therefore, prote-ase. The word itself is a shortcut word commonly used to define any naturally physiologic entity which is capable of breaking chemical bonds between certain side-by-side amino acids within proteins (peptide bonds which connect individual amino acids within the protein). The more descriptive terminology would be: proteolytic enzyme. Other terminology examples are: ligase - an enzyme which "ligates" (ties) things together; synthetase - an enzyme which is involved in the synthesis of something. Since enzymes "recognize" unique molecular shapes, a given protease can act on only certain proteins - those which happen to have the unique molecular shape present within a particular protein.
What is a Protease Inhibitor?
A protease inhibitor would be any substance which partially or completely blocks the ability of a proteolytic enzyme to carry out its activity. Therefore, protease inhibitors inhibit the ability of a particular protease to break the peptide bonds within a given protein. The inhibitor must necessarily have a shape which can bind to the protease at the place within the structure of the protease within which bond formation/breakage occurs, e.g., the catalytic site (active site), or, at some place on the protease's structure which prevents the active site from functioning properly. Otherwise, the inhibitor would not work. Since the ability to work as an inhibitor requires such specificity, a given inhibitor will not work for all enzymes, just for certain ones - or - maybe only one type.
What Do Protease Inhibitors Have To Do With HIV?
Well, we now need to cover some additional territory. Continuous research over the years snce HIV was first identified has led to significant progess in understanding how HIV actually infects a cell, reproduces, and leaves the cell to infect other cells. However, it has been recognized for some time that the precise requirements necessary for HIV to infect a cell are not yet completely understood. Consequently, the research into this particular virus's infection mechanism continues. The information presented below is a generalized synopsis of what most investigators agree upon with respect to HIV.
How does HIV Infect a Cell?
For many years it has been known that HIV infection primarily occurs when HIV binds specifically to a protein in the cellular membrane of lymphocytes called T-helper cells (although, very recent information now shows other important requirements for cellular entrance - see below - additional information - last paragraph). These cells express and are identifiable by this expression of a protein within their cellular membrane called CD4 (Cluster of Differentiation antigen number 4). Normally, this particular T-helper cell membrane-associated protein is required for the ability of the T-helper cell to participate effectively in an immune response (to help other important lymphocytes express _their_ activity, e.g., B-lymphocytes and antibody production, and cytotoxic T-cells' ability to kill virally infected cells). Therefore, HIV infects the very cell required to help other immune system cells to protect us from any foreign thing which enters our body.
What Happens When HIV Enters the Cell?
The first thing that happens is the uncoating of the virus which exposes the virus's genetic material - RNA. At this point, an HIV enzyme (tightly bound to the RNA) begins to act. This enzyme, named reverse transcriptase is responsible for synthesizing a complementary base-pair copy of the RNA - but - the copy consists of DNA, not RNA (hence the name, reverse transcriptase - the enzyme transcribes the RNA into DNA). You may wish to take a look at: What the Heck is a Gene. Then, this very same enzyme makes a complementary base-pair copy of the DNA to form a double-sranded DNA molecule - which enters the nucleus of the infected cell and integrates into one of the cell's chromosomes... becomes an integral stable part of one of the chromosomes. Then, the viral genes are expressed, and many different viral proteins result. One of these proteins is a protease - HIV protease. The viral protease acts on a long newly-synthesized precursor viral protein and clips this protein into several differently-sized smaller proteins. These smaller proteins are necessary for proper viral assembly and for the ability of newly-formed virus particles to be infectious after they are released from the infected cell.
How Do Protease Inhibitors Work?
They work by specifically binding within the active site of HIV protease - and therefore prevent the active site from acting on the long precursor HIV protein produced during viral infection. Consequently, the necessary smaller-sized viral proteins cannot be made, and therefore, complete, proper viral assembly cannot occur.... thus, effectively "killing" the virus and preventing spread of the virus from cell to cell.
How In The World Was This Feat Accomplished?
The HIV protease gene has been identified, isolated, genetically cloned and the gene product - the protease protein itself - produced in large quantities within bacteria ( a particular laboratory strain of E. coli, and the three-dimensional structure precisely defined - took _years_ of very difficult work. Using the atom-for-atom three-dimensional structure of the protease, the catalytic site (active site) of the protease was identified. Then, computer-driven design software which has been generated to allow design of three-dimensional chemical structures were applied to design a chemical which would fit precisely within the active site of HIV protease and effectively block the site - and therefore effectively inhibit the ability of the protease to function. Further, as each new potential inhibitor appeared, all kinds of tests had to be done to assure that the inhibitor inhibited only HIV protease, and not one of the many different proteases necessary for our cells to normally live. Then, clinical trials had to be perfomed to test whether or not other unexpected dangerous side effects might occur through use of the drug. Once these trials were completed, treatments started - with the success as noted.
How Are These Protease Inhibitors Used?
They are most commonly used in combination with at least two different reverse transcriptase inhibitors (one of which is AZT). The names of these protease inhibitors are: saquinavir, ritonavir, and indinavir. The total treatment is estimated to cost each patient approximately $12,000 per year.
It has been recognized that simple association of HIV with CD4 is not sufficient to allow HIV to enter the cell.
What Additional Requrement(s) for HIV
Infection is Known?
Over the years, accumulated information led to the discovery of several different individuals who had HIV present within their body, but who never showed signs of illness and never progressed to AIDS. Sort of like the fact that while most of us harbor the Epstein-Barr virus throughout our lifetime, only a small percentage of us ever get sick with the disease called mononucleosis. Consequently, researchers have been looking long and hard for the "additional" requirement for HIV infection to lead to such devastating consequences in most, but not all individuals. The answer appears to be associated with certain cell membrane receptors for a relatively newly identified class of molecules involved with inflammation named chemokines. As it so happens, there are some individuals with a mutated form of one of these receptors, namely CCR5, which cannot be infected with HIV. Apparently, HIV requires the additional association of a viral protein with one of these chemokine receptors to effectively enter and infect the target cell... even in the presence of CD4. Normally, during an inflammatory response due to the presence of a foreign invader, certain immune system cells will express such receptors - involved with cellular activation.... Consequently, there is now an enormous effort underway to identify the precise interactions of HIV with these particular chemokine receptors. Hopefully, this research will lead to the design of yet other substances which can block the ability of HIV to bind and enter target cells. For further information about chemokines and the cellular receptors for them, please access either: Chemokine Family, or, Chemokines - the latter of which is Horst Ibelgaufts' page of information on Chemokines (of the Laboratory for Molecular Biology, in the Ludwig-Maximilians-University of Munich).