Protease inhibitors [Updated 25 November 1997]

Some questions have arisen upon the subject of the new protease inhibitors
used in the treatment of AIDS.

We have pointed out in many of our updates that certain proteolytic enzymes,
specifically trypsin and chymotrypsin, are utilized by the body to control
the proliferation of trophoblast and it's pathological homolog cancer.
However, the targeted proteases are not in the same class in which the
trypsins fall.

To help clear this question up, I am appending the full text of an excellant
article which Dr. David Rasnick wrote and forwarded to me last summer on the
HIV protease inhibitors, and some of the more important ramifications of
interpretation of the results of their utilization. I should also point out that there
have been found inhibitors (search Medline for acarbose, and RBI)  that act on
the glycogen or carbohydrate digesting enzyme amylase as well, which would
play a very important negative role in the degree of success of immuno-enzyme
therapy of cancer.  It is very important that both amylase and the various proteolytic
serine enzymes act together, and not proteolytic enzymes alone. rsc.

----begin article----
Date: Tue, 13 Aug 1996 06:23:58 -0800
To: rsc@europa.com (Roger Cathey)
From: us034537@interramp.com (David W Rasnick)
Subject: Re: 2 HIV protease inhibitors

---------------------

HIV Protease and Its Inhibitors

by

David Rasnick, Ph.D.


One of DNA's main functions is to provide the instructions for making
proteins. Proteins are so versatile that they are the primary structural
elements of all living things including viruses. With the exception of a
few peculiar RNAs, all enzymes are protein. Enzymes are the biological
molecules that get things done by acting on other molecules called
substrates.

One of the largest classes of enzymes are the proteases. On the surface,
proteases perform one of the simplest of biological reactions: they clip
proteins into smaller proteins or peptides. Peptides are just short pieces
of proteins. The point at which a peptide is elevated to the exalted status
of a protein is arbitrary. Proteases that cleave primarily proteins are
called proteinases, whereas proteases that cleave primarily peptides are
called peptidases. The naming game gets quite complex so I won't bore you
with the details. For our purposes HIV protease can properly be called a
proteinase since its primary substrate is the gag-pol polyprotein coded for
by the HIV proviral DNA. More on this latter.

The 20 or so amino acids are linked together through amide bonds to make
all the proteins and peptides that exist. The amide bonds of proteins have
been given the special name of peptide bonds to signify that they belong to
proteins and peptides. Peptide bonds are very strong chemical bonds and are
difficult to break. Nonetheless, under the right conditions proteases can
easily break peptide bonds. An every day example is the very active
cysteine protease called chymopapain that comes from the papaya plant and
is the active ingredient in meat tenderizer. Chymopapain breaks the peptide
bonds of meat before it is cooked. The aspartyl protease pepsin in your
stomach and the serine proteases chymotrypsin and trypsin in your small
intestine digest the proteins you eat. It turns out that proteases are far
more complex and interesting than this simple picture implies. The vast
majority of proteases are involved in the processing and regulation of
other proteins including other enzymes.

Proteases have been divided into four main classes according to the active
site features that are common to each group: serine, cysteine, aspartyl,
and metallo. These designations have nothing to do with the substrates the
proteases cleave. HIV protease belongs to the aspartyl family of proteases.
A few examples of human aspartyl proteases are pepsin (a digestive enzyme
in the stomach), cathepsin D (located inside lysosomes within cells), and
renin (which is part of the cascade of proteases that regulates blood
pressure).

Proteases are globular proteins with an indentation or cavity called the
active site. Substrates fit into the active site of the protease where the
enzyme catalytically breaks the specific peptide bonds to be cleaved. HIV
protease doesn't clip every peptide bond in sight; it is very particular
about the sites of cleavage. Of the hundreds of possibilities, there are
only eight specific peptide bonds of the gag-pol polyprotein that HIV
protease must cleave for the virus to replicate and mature properly into
infectious particles.

Inhibitors of HIV protease are small synthetic molecules designed to stick
tightly to the active site, preventing the enzyme from processing the viral
protein (its substrate). One possible side effect of HIV protease
inhibitors is that they might inhibit the human aspartyl proteases.
Fortunately, the peculiar structure of HIV protease makes it possible to
synthesize very specific inhibitors that have little or no effect against
the known human enzymes. Nevertheless, the specificity of these inhibitors
is not absolute and it is impossible to determine the toxic effects of new
inhibitors just by looking at them. Indeed, clinical trials have shown that
HIV protease inhibitors do have toxic side effects in humans, though much
less severe than the life threatening consequences of AZT treatment.

HIV, like other retroviruses, contains genes that code for viral structural
proteins, envelop proteins as well as enzymes. During the production of
these proteins (called translation) they are all stuck together end to end
via peptide bonds to form the polyprotein. It appears that the envelop
proteins are severed from the polyprotein by a host protease and not by HIV
protease. The viral structural proteins (coded for by the gag region of the
gene) and the viral enzymes (including HIV protease, coded for by the pol
region of the gene) are connected by peptide bonds that must be processed
(cleaved) by HIV protease in a sequential manner at the right time.

The production of infectious HIV particles is dependent on proper assembly
of structural proteins into the core particle. The initial steps in
assembly involve the association of the gag and gag-pol precursor proteins
with the inner face of the membrane of the infected cell, followed by
interaction of the precursors with each other. HIV protease is part of the
larger gag-pol polyprotein and is only functional as a dimer. This
arrangement allows the precursor proteins to arrive at the membrane in a
coordinated manner and is largely successful in preventing premature
activation of HIV protease. The membrane-based association of the precursor
proteins precedes cleavage of the precursors by HIV protease. Once bound to
the membrane, HIV protease processes the precursor proteins in an ordered,
sequential manner. If all goes well, budding and release of the mature,
infectious virus particle occurs, leading to a new cycle of infection and
viral replication. Incomplete processing of the precursors by HIV protease
still leads to budding but the viral particle produced is not infectious.
In the presence of inhibitor infected cells are still capable of producing
viral particles, but the virus produced is defective and not infectious.
This explains why it is possible to detect viral proteins in patients
treated with HIV protease inhibitors. The so-called viral-load assays
detect viral proteins but give no indication as to the viability or
infectivity of the virus. Complicating the issue, defective, non-infectious
viral particles are more stable than wild-type virus and give an
erroneously high measure of "viral-load." It may turn out that the
viral-load assays are not detecting infectious virus at all-it's more
likely they're measuring viral debris.

Disrupting the proteolytic processing of HIV precursor proteins is an
excellent strategy of blocking the production of infectious virus. The
central question is will inhibiting HIV be of therapeutic benefit? Numerous
in vitro experiments have demonstrated that impaired proteolytic activity,
due either to the presence of protease inhibitors or deleterious mutations
of HIV protease, results in noninfectious HIV particles. As a consequence
of these studies and several human clinical trials, a number of HIV
protease inhibitors have recently been approved for clinical use. However,
the disappointing clinical efficacy of these inhibitors during the early
trials led to the widespread belief that the HIV protease develops
resistance to the inhibitors by mutating to less susceptible forms of the
enzyme.

In 1994 I attended a conference where HIV protease inhibitor results were
discussed. John Kay provided interesting information on the clinical trials
of Roche's HIV protease inhibitor Ro 31-8959. He made the astounding claim
that Roche had synthesized 800 tons (that's right: tons) of this compound.
When given the opportunity to change his statement he stuck to 800 tons.
The clinical trial consisted of 400 AIDS patients receiving 2 g of the
Roche inhibitor per day. After 18 months there was no clinical difference
between the group given the protease inhibitor and the controls. Kay
announced that Roche was putting an information blackout on further reports
on the HIV protease inhibitor clinical trials due the disappointing
results.

The prevailing hypothesis for the failure of the inhibitors was that HIV
protease was mutating to resistant forms. At the time no one had actually
seen any of these mutated enzymes from AIDS patients, nevertheless, the
mutation explanation was generally accepted. In his talk, Kay addressed the
mutation hypothesis. He said that mutations of HIV protease that result in
lowered sensitivity to the inhibitors from all sources were extremely rare.
However, virtually all those mutations had been produced under laboratory
conditions.

A representative from Vertex presented a poster on the consequences of four
laboratory produced mutations on the efficiency of the protease to cleave
synthetic substrates that mimicked four of the natural cleavage sites. The
activities of the mutant proteases ranged from 1.0 to 0.04 times the wild
type enzyme for any one of the four cleavages. However, some of the mutants
were as much as 400-fold less sensitive to the inhibitors than the wild
type enzyme. The author concluded that this demonstrated that the mutation
hypothesis of HIV protease was a reasonable explanation for the failure in
the clinical trials. I pointed out to the author that the activity of the
enzymes toward only one substrate cleavage did not represent the overall
effect of the mutations on the eight obligatory cleavages required for
viable viral maturation. You had to multiply the efficiencies of all the
cleavages to get the overall efficiency. My analysis of the Vertex data
showed that the overall activity of the mutant proteases turned out to be
50 to 10,000 times less efficient for the four cleavages than the wild type
enzyme, and that didn't include the effect on the remaining four cleavages!
What's more, for the mutant that was 400 times less sensitivity to a
particular protease inhibitor, its overall efficiency for the four
cleavages was 10,000 times less than the wild type enzyme.

At the discussion session the next day I argued that the data do not
support the hypothesis that mutations of the HIV protease are responsible
for the lack of clinical efficacy of the inhibitors. Other explanations are
called for. To broach the possibilities gently I first suggested a simple
bio-availability problem. I then went on to propose that the HIV protease
inhibitors may be performing as designed and that inhibiting the enzyme is
irrelevant. After making that remark, someone in the back suggested that
what was needed was a cocktail of very specific inhibitors to go after the
various mutated proteases.

During private discusions none of my colleagues found any obvious flaws
with my reasoning and even thought it was right. I left the meeting
thinking that these fellows would continue the analysis where I left off.
Well, that of course didn't happen. The HIV protease mutation hypothesis
becomes more entrenched with time. Recently I decided to do something about
that.

In March 1996 I began looking for the mathematics I needed to confirm my
original intuition as to the effects of mutations on the overall
proteolytic activity of HIV protease towards the eight obligatory
cleavages. I found just what I needed in a 1974 paper by Philip Kuchel on
the kinetics of consecutive enzyme catalyzed reactions. I wrote a paper
(Kinetics analysis of consecutive HIV protease catalyzed cleavages of the
gag-pol polyprotein) and sent it to Biochemistry.

When I applied my analysis to the published data I was able to show that
the ordered, consecutive cleavages of the gag-pol polyprotein by HIV
protease present severe limitations on viable mutations of the enzyme. I
was able to come up with a simple description of the overall kinetics of
mutant and wild-type HIV protease in the presence or absence of inhibitors.

None of the inhibitor-resistant mutant HIV proteases reported so far has
come anywhere near the minimum level of overall catalytic activity
necessary for viral viability. Even in the absence of inhibitors it is
extremely unlikely that mutations of the enzyme, substantial enough to
protect the protease against inhibition, will at the same time leave
virtually unimpaired its proteolytic activity towards all eight cleavages.
The conclusion of my analysis is that inhibitor-resistant mutant HIV
proteases are very unlikely to contribute to viral viability in vivo.
Therefore, the failure of the HIV protease inhibitors to alter the
progression of AIDS is not due to inhibitor-resistant mutations of this
enzyme.

The well publicized claims that the Abbott, Merck, and Roche HIV protease
inhibitors could prolong the lives of AIDS patients is already turning out
to be more hype than fact. At the recent AIDS conference in Vancouver less
is being made of the life-saving benefits of the HIV protease inhibitors.
The presenters are once again reverting to the surer ground of surrogate
markers (CD-4 counts and viral-load measurements) to assess the effects of
the HIV protease inhibitors. I suspect that the HIV protease inhibitors are
working remarkably well at preventing the production of infectious virus.
If I'm right, a properly conducted clinical trial using HIV protease
inhibitors could seriously test the HIV hypothesis of AIDS. There is so
much interest in HIV protease inhibitors that this test may actually
happen. And there is another, more sinister possibility.

Right now HIV protease inhibitors are approved for use only in combination
with AZT and the other nucleoside analogs. Physicians are well aware of the
severe toxic effects of AZT etc. When a patient receiving the protease
inhibitor shows signs of AZT poisoning a doctor is likely to stop the AZT
treatment. If the patient is taken off AZT early enough he will likely
improve, for a while at least. The physician may attribute the improvement
in the health of the AIDS patient to the continued use of the HIV protease
inhibitor. When enough physicians report similar benefits of mono-therapy
(that is treatment with HIV protease inhibitors alone), the NIH, CDC, and
drug companies will declare that HIV protease inhibitors are the magic
bullets they've all been looking for to treat AIDS. Of course they will
have completely missed the point that they are killing fewer patients
because they are using a less toxic drug.

This ends my short course on HIV protease and its inhibitors. If you have
specific questions that I have not covered or certain points were not made
clear please contact me at rasnick@interramp.com. I will answer as best I
can.
------------

Dave
-----end article------

As noted above by Dr. Rasnick, the trypsins belong to the serine class,
while the proteases targeted by the inhibitors in question belong to the
aspartyl class.  As pepsin is an example of the aspartyls, there are
problems inherant in this program, which indirectly affect the function of
trypsins.  Trypsins are not first-action enzymes.  Pepsin acts on
macro-proteins, which are then acted upon by the trypsins.  In the case of
the cancer glyco-proteins, these conjugated proteins are quite different
from ingested proteins, and may be viewed as appropriate substrates for
action by trypsin in conjunction with amylase.  In this situation, trypsin
and amylase are acting synergistically as first action enzymes.  However,
pepsin is also found in the serum, and may be conceived as being a
preperatory protease on the cancer cell. It is uncertain how this will
affect the action of the pancreatic enzymes in the event that Kaposi's
(pronounced Kahp' oshee) sarcoma obtains.  In terms of Kaposi's sarcoma,
however, I don't know whether it is indeed a true form of cancer or not.  If
it has hCG secretion products, we would normally interpret this as a
trophoblastic hormone. But as has been pointed out in various Medline
abstracts on the work of Dr. Acevedo (found in the cancer ascii index on
the web site), there are indications that hCG may be produced
by various parasites or pathogens such as mycoplasmas as well.

Medline search terms:

Ragi (Eleusine coracana Gaertneri) (RBI)
"the only member of the alpha-amylase/trypsin inhibitor family that inhibits
both trypsin and alpha-amylase"
[See: FEBS Lett 397: 11-16 (1996)
RBI, a one-domain alpha-amylase/trypsin inhibitor with completely
independent binding sites.
Maskos K, Huber-Wunderlich M, Glockshuber R
Institut fur Molekularbiologie und Biophysik, Eidgenossische Technische
Hochschule Honggerberg, Zurich, Switzerland. PMID: 8941704, MUID: 97096883]


acarbose [See for example:
Metabolism 45: 1368-1374 (1996)
L-arabinose selectively inhibits intestinal sucrase in an uncompetitive
manner and suppresses glycemic response after sucrose ingestion in animals.
Seri K, Sanai K, Matsuo N, Kawakubo K, Xue C, Inoue S
Pharmacological Section, GODO SHUSEI Co, Chiba, Japan.
PMID: 8931641, MUID: 97085501]

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