Book: Don't Touch That Doorknob!
Background
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).
Book: Don't Touch That Doorknob!