Current Pharmaceutical Design, Volume 8, No. 28, 2002
Contents
Protease
Inhibitors
Executive Editor: Vicki L. Nienaber
Amyloid Forming Proteases: Therapeutic
Targets for Alzheimer's Disease
Pp.2521-2531
Frauke
Schimmöller, Jeffrey N. Higaki and Barbara Cordell
Peptidic Inhibitors of the Hepatitis C Virus
Serine Protease within Non-Structural Protein 3 Pp.2533-2540
T.O.
Fischmann and P.C. Weber
Inhibitors of the Protease Domain of
Urokinase- Type Plasminogen Activator Pp.2541-2558
T.W.
Rockway, V. Nienaber and V.L. Giranda
The Determination and Use of Optimized
Protease Substrates In Drug Discovery and Development Pp.2559-2581
Paul
L. Richardson
[Back to top] Amyloid Forming Proteases: Therapeutic
Targets for Alzheimer's Disease
Frauke
Schimmöller, Jeffrey N. Higaki and Barbara Cordell
Alzheimer's disease is an age-related neurodegenerative disease which
causes global loss of cognitive function. Drug treatment for Alzheimer's
disease has been limited to agents that ameliorate behavioral symptoms but
these agents are without effect on disease progression. Rational drug design
for the treatment of Alzheimer's disease now seems possible. As a result of
major advances in medical research in this area, knowledge of the etiology of
Alzheimer's disease is rapidly being understood. This information has uncovered
relevant and novel targets for treatment. At the center of the etiological progression
of this disease is β-amyloid. A substantial body of evidence strongly
suggests that this small protein is critical to the development of Alzheimer's
disease. The β-amyloid protein is generated by two different proteolytic
cleavages. Recently, the proteases responsible for producing the β-amyloid
protein have been identified. The proteases represent viable targets for
therapeutic intervention against Alzheimer's disease. In this review, we
describe the biological characteristics of the β-amyloid-forming proteases
in the context of pharmaceutical development.
[Back to top] Peptidic Inhibitors of the Hepatitis C Virus
Serine Protease within Non-Structural Protein 3
T.O.
Fischmann and P.C. Weber
New treatments for HCV (Hepatitis C virus) infections are likely to
arise from inhibition of the essential, virally-encoded enzymes. These targets
include the serine protease required for processing of the HCV polyprotein. The
protease constitutes one functional domain of the bifunctional HCV NS3 (non-structural
protein 3). Here, insights regarding the NS3 structure and recently synthesized
NS3 inhibitors are reviewed. Interestingly, many NS3 protease inhibitors have
taken advantage of an unusual product inhibition by Nterminal products of
cleavage at the polyprotein processing sites.
[Back to top] Inhibitors of the Protease Domain of
Urokinase- Type Plasminogen Activator
T.W.
Rockway, V. Nienaber and V.L. Giranda
Human urokinase-type plasminogen activator (uPA or uPA) has been
implicated in the regulation and control of basement membrane and interstitial
protein degradation. Since Urokinase plays a role in tissue remodeling, it may
be responsible, in part, for the disease progression of cancer. Inhibitors of
urokinase may then be useful in the treatment of cancer by retarding tumor
growth and metastasis. Urokinase is a multidomain protein, two regions of the
protein are most responsible for the observed proteolytic activity in cancer
disease and progression. The N-terminal domain or ATF binds to a Urokinase
receptor (uPAR) on the cell surface and the C-terminal serine protease domain,
then, activates plasminogen to plasmin, beginning a cascade of events leading
to the progression of cancer. Investigations of urokinase inhibition has been
an area of ongoing research for the past 3 decades. It began with the discovery
of small natural and unnatural amino acid derivatives or peptide analogs which
exhibited weak inhibition of uPA. The last decade has seen the generation of
several classes of potent and selective Urokinase inhibitor directed to the
serine protease domain of the protein which have shown potential anti-cancer
effects. The availability of structural information of enzyme-inhibitor
complexes either by nuclear magnetic spectroscopy (NMR) or crystallography has
allowed a detailed analysis of inhibitor protein interactions that contribute
to observed inhibitor potency. Structural studies of specific inhibitor-uPA
complexes will be discussed as well as the contributions of specific inhibitor
protein interactions that are important for overall inhibitor potency. These
data were used to discover a class of urokinase inhibitor based on the
2-Naphthamidine template that exhibits potent urokinase inhibition and
excellent selectivity for urokinase over similar trypsin family serine
proteases.
[Back to top] The Determination and Use of Optimized
Protease Substrates In Drug Discovery and Development
Paul
L. Richardson
There is an increasing need to rapidly determine the specificity of
proteases that potentially play a role in human and animal disease. Substrates
for novel proteases can be discovered by testing standard protease substrates
such as oxidized insulin B-chain, by screening commercially available
substrates for other proteases, or by preparing derivatives of known biological
targets. The relative importance of each substrate residue can be determined
through alanine-scanning, or by preparing incremental changes at one or more
positions within the known substrate. More efficient methods such as coupled
liquid chromatography – mass spectrometry (LC-MS) or C-terminal/N-terminal
sequencing of reaction products allow the selection of improved substrates from
mixtures of peptides. In other cases mixtures of substrates can be spatially
segregated prior to protease treatment during chemical synthesis on beads or
membranes. Positional scanning libraries can be used to find substrates for
proteases with interdependent subsites, while minimizing required synthetic and
screening effort. As proteases catalyze both hydrolysis and amide bond
formation, acyl transfer from protease-substrate intermediates to mixtures of
peptide nucleophiles provide substrate sequence information. Genetic methods
including substrate phage display, retroviral display, bacterial display, and
yeast α-halo assays combine selection with the ability to propagate
selected sequences and directly deconvolute the cleaved peptide via sequencing
of substrate-coding DNA. This review describes various methods for optimizing protease substrates for biological activity and
the use of optimized substrates in pharmaceutical discovery.