Proteases of Infectious Agents

302 Pages·1999·35.34 MB·English

by  Ben Dunn (Editor)

PREFACE
As the decade of the 1990s draws to a close, it is appropriate to assess the
changes that have taken place in the drug discovery process. Previously, an
infectious agent was identified and cultured for two purposes: (1) so that tests
could be established to screen compounds for inhibition of growth of the infec-
tious organism, and (2) to permit isolation of proteins that could serve as drug
targets in favorable cases where sufficient quantities could be obtained. In this
decade a new paradigm has emerged. Isolation of the genetic material of the
infectious organism, followed by sequence analysis at the DNA level by rapid,
automated methods, can reveal the entire genomic structure in a short time.
Straightforward analysis using sequence-searching algorithms can lead to the
identification of possible critical functional activities. Cloning of the specific
region of the genome can yield the target for new drug development. With the
exploding database of protein structure, and with homology modeling pro-
grams, it is now possible to predict structure and initiate the drug discovery
process while waiting for solution of the X-ray structure.
During the period following the identification of sequences within the HIV
genome that fit the template for the structure of an aspartic protease, drugs
were designed and synthesized, and the effect on viral growth was demon-
strated. Now that FDA-approved drugs have been shown to suppress viral
growth and improve the health of thousands of patients, we can conclude that
the new paradigm for drug discovery has been successful. Of course, this ac-
count should not fail to point out the continuing problems of patient noncom-
pliance, low bioavailability, and rapid development of resistant viral strains.
Nonetheless, the value of protease-directed drugs, as well as the best pathway
to finding them, is clear.
Fittingly then, this book begins with an account of the development of anti-
viral agents targeted for AIDS. Important lessons derived from this work in-
xv
1VX ecaferP
clude the demonstration, through catalytic mutation, that an active protease
was essential for viral replication. Also, the value of the early determination of
the three-dimensional structure of the enzyme and enzyme-inhibitor com-
plexes has been established. Next, the iterative design process, based on struc-
ture determination, marks the HIV-1 protease case as a defining example for
future work. Finally, the problems associated with drug metabolism and the
resistance question complete the catalog of lessons learned in this case.
Other chapters in this book summarize current knowledge regarding new
drug targets from other infectious organisms. A significant new effort is di-
rected toward a serine protease encoded by the hepatitis C virus. Due to the
relatively recent identification of this virus and the lack of a cell-culture system,
progress was very slow. In the past three years, however, expression of the pro-
tease and the solution of its structure have dramatically stimulated the search
for drugs in this area. In addition, a shift occurred when many pharmaceuti-
cal companies cut back their HIV efforts because of the success of the Roche,
Abbott, and Merck compounds. Hepatitis C was the next disease likely to have
a significant public health impact in the United States and elsewhere due to the
spread of the infection through transfusion before its discovery. As detailed in
the chapter by Urbani, De Francesco, and Steink~hler on this subject, a strategy
similar to that in the HIV case was followed.
A huge body of work, conducted largely in pharmaceutical companies, is
summarized by Qiu and Abdel-Meguid in their chapter on the human herpes-
viruses. Here again, the paradigm of discovery of a viral sequence, cloning, ex-
pression, and structure determination has proven to be the route to drug de-
sign. In this case especially, the structural insights reveal novel mechanisms of
action that could provide clues for potent and selective inhibitor design.
The Candida genus provides an example of an infectious agent that is not a
problem for a healthy human. However, in the case of a patient whose immune
system has been impacted by infectious agents such as HIV or through treat-
ment with immune-suppressing agents to avoid transplant rejection, severe sys-
temic infections can be the ultimate cause of death. In the chapter by Stewart,
Goldman, and Abad-Zapatero, several related crystal structures derived from
protease variants cloned from .C albicans are described. Unique insights that
point the way toward selective inhibitor design are derived.
Work in the picornarvirus arena has been under way for some time; how-
ever, the new information described by Bergmann and James seems likely to
stimulate this area significantly. Progress in this field will have widespread im-
pact, as the picornavirus family includes the rhinoviridae, which bring us the
common cold, as well as many others.
The chapters by Berry, on malaria, and by Cazzulo, on Chagas disease, rep-
resent the field of protozoan infectious species. Berry provides a thorough sum-
mary of current knowledge on hemoglobin degradation during the blood-borne
ecaferP xvii
stage of malaria. The parasite is somewhat unique in presenting two targets for
drug discovery: a cysteine protease (falcipain) and an aspartic protease (plas-
mepsin). The interplay of these two enzymes in the complicated process of
breakdown of the globin chain provides several strategies for attack. In the case
of .T cruze, the organism that causes Chagas disease, the major antigen is a
cysteine protease, cruzipain (or cruzain). Cazzulo describes the involved life
cycle, the properties of cruzipain, and other putative serine proteases of the
organism.
Successful infection by foreign agents frequently requires some function of
the host. In the chapter by Kido, Chen, Murakami, Beppu, and Towatari, the
role of cellular proteases in infection by influenza A and Sendai viruses in the
respiratory tract and tryptase TL2 in T lymphocytes is described. The involve-
ment of a cellular enzyme in the entry of HIV-1 into cells is also discussed. The
implications for future therapeutic intervention are clear, but the complica-
tions of attempting to alter the function of a normal cellular enzyme are also
significant.
While it has been clearly established that polyprotein processing in the case
of viruses is an essential feature of their life cycle, the world of bacteria is not
so straightforward. One point of attack is the necessity for processing newly
synthesized proteins targeted by the bacteria for export. Lively discusses the
bacterial signal peptidases in a chapter that precedes structure determination.
Nonetheless, the ground is well-prepared for the anticipated structural infor-
mation. In this case as in the others, the differences between the bacterial en-
zyme and the related human enzyme will be critical to development of selective
inhibitors.
The world of plants is represented by the chapter of Garcia, Fernandez-
Fern~indez, and L6pez-Moya describing plant viruses. This area is unique in
that a wide range of protease mechanisms are found, lacking only the metallo-
proteases. Given the huge impact of plant viruses on crop production, this is a
field with a large potential for future growth.
While this compilation is not encyclopedic, the chapters presented cover a
wide range of infectious agents and mechanisms. There also are obvious differ-
ences in the state of knowledge of the different proteases described. This is
appropriate, as efforts in the area of proteases will continue to expand into re-
search in new diseases and infectious agents as they are discovered. The task
before us is clear: Find the critical protease and develop a potent and selective
inhibitor.
Ben M. Dunn