![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 304, Issue 1, 433-440, January 2003
Departments of Lead Discovery, Neuroscience, and Chemistry, AstraZeneca Pharmaceuticals, Wilmington, Delaware
 |
Abstract |
|---|
 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Caspase-3 is an intracellular cysteine protease, activated as part of the apoptotic response to cell injury. Its interest as a therapeutic target has led many to pursue the development of inhibitors. To date, only one series of nonpeptidic inhibitors have been described, and these have limited selectivity within the caspase family. Here we report the properties of a series of anilinoquinazolines (AQZs) as potent small molecule inhibitors of caspase-3. The AQZs inhibit human caspase-3 with Ki values in the 90 to 800 nM range. A subset of AQZs are equipotent against caspase-6, although most lack activity against this isoform and caspase-1, -2, -7, and -8. The AQZs inhibit endogenous caspase-3 activity toward a cell permeable, exogenously added substrate in staurosporine-treated SH-SY5Y cells. The AQZs reduce biochemical and cellular features of apoptosis that are thought to be a consequence of caspase-3 activation including DNA fragmentation, TUNEL staining, and the various morphological features that define the terminal stages of apoptotic cell death. Moreover, the AQZs also inhibit apoptosis induced by nerve growth factor withdrawal from differentiated PC12 cells. Thus, the AQZs represent a new and structurally novel class of inhibitors, some of which selectively inhibit caspase-3 and will thereby allow evaluation of the role of caspase-3 activity in various cellular models of apoptosis.
| |
Introduction |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Apoptosis
is an important cellular response to severe stress, resulting in the
death of the cell. In some clinical conditions, dysregulation of
apoptosis is thought to be an important contributor to the pathogenesis
of disease. The molecular mechanisms that drive the apoptotic response
have been clarified in recent years (Green and Reed, 1998
; Strasser et
al., 2000; Zornig et al., 2001). These studies have led to the
identification of distinct biochemical pathways, pharmacological
perturbation of which could block the apoptotic response and possibly
reverse the functional decline of affected cells. Indeed, some of these
biochemical pathways contain molecular targets that may be amenable to
therapeutic intervention. One such target is caspase-3, a cysteine
protease activated in response to numerous cellular insults (Nicholson, 2000).
Caspase-3 belongs to a family of cysteine proteases that consists of at
least 14 members. These caspases can be subdivided based on structural
homology, substrate preference, or biological function (Nicholson,
1999). For example, several caspases have been shown to be important
contributors to the inflammatory response by processing cytokines
(caspase-1, -4, -5, and -13) whereas others are activated either as an
early step in the initiation of apoptosis (caspase-6, -8, -9, and -10)
or in the execution of the apoptotic response (caspase-2, -3, and -7).
Caspase-3 has received particular attention from a therapeutic
perspective because it is expressed in almost all tissues and at
relatively high levels (versus other caspases) and has high catalytic
activity compared with the other executioner caspases (Margolin et al.,
1997; Garcia-Calvo et al., 1999; Stennicke et al., 2000). Furthermore,
genetic knockout studies have shown that caspase-3 plays a critical
role in apoptosis during neuronal development (Kuida et al., 1996; Woo
et al., 1998), whereas other studies support a role in
neurodegenerative diseases (Yakovlev et al., 1997; Gervais et al.,
1999; Xu et al., 1999; Beer et al., 2000; Rigamonti et al., 2000;
Wellington et al., 2000).
Although structure-based design of inhibitors for caspase-3 have
successfully produced potent, peptide and peptidomimetic compounds
(Margolin et al., 1997; Garcia-Calvo et al., 1998; Karanewsky et al.,
1998), these inhibitors typically have pharmacokinetic and
physicochemical properties that limit their utility to intravenous applications for acute disease treatments. The development of nonpeptidic inhibitors could overcome these limitations, but
unfortunately such compounds have been difficult to identify and
optimize. To date, the only examples published include a series of
isatin sulfonamides, which, although potent inhibitors of caspase-3,
have limited selectivity versus the other executioner caspases (Lee et
al., 2000, 2001; Nuttall et al., 2001). In this report we describe the
biochemical and cellular profile of a new series of potent and
selective, nonpeptide caspase-3 inhibitors.
| |
Materials and Methods |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Materials.
(Z-DEVD)2-R110 and Hoechst
33342 stain were purchased from Molecular Probes (Eugene, OR). PC12
cells and SH-SY5Y cells were purchased from American Type Culture
Collection (Manassas, VA). Staurosporine, NGF, and anti-NGF antisera
were obtained from Sigma-Aldrich (St. Louis, MO). Caspase-1,-2, -6, -7, -8, substrates Ac-YVAD-amc, Ac-LEHD-amc, and Ac-IETD-amc, and
inhibitors Ac-YVAD-CHO, Ac-LEHD-CHO, Ac-IETD-CHO, and Ac-DEVD-CHO were
obtained from Biomol Research Laboratories, Inc. (Plymouth Meeting,
PA). Boc-Asp-fmk and Z-DEVD-fmk were purchased from Enzyme Systems
Products (Livermore, CA). Caspase-6 was obtained from BD Biosciences
(San Jose, CA). Recombinant human caspase-3 was expressed in
Escherichia coli and purified from exclusion bodies using
published procedures (Rotonda et.al.,1996). Substrate Ac-DEVD-amc and
the inhibitor Ac-DEVD-CHO were synthesized at AstraZeneca
Pharmaceuticals LP (Wilmington, DE). In situ cell death detection kit
(TUNEL staining) was purchased from Roche Diagnostics (Indianapolis, IN).
Inhibitor Synthesis.
The anilinoquinazolines (AQZs) were
prepared as described (Jacobs et al., 2001). The isatin sulfonamide
named compound 33 was synthesized and purified using published
procedures (Lee et al., 2001).
Enzyme Assays.
The enzymatic activity of caspase-1, -2, -3, -6, -7, and -8 (40, 180, 100, 5, 1, and 10 ng, respectively) was
determined from the initial rate of hydrolysis of their respective
substrates (10-50 µM), by measuring at room temperature the
accumulation of a fluorogenic product over time. The enzymes and
substrates were diluted in assay buffer containing 150 mM NaCl, 50 mM
HEPES, 5 mM EDTA, 1 mM dithiothreitol, 0.1% CHAPS (caspase-6
and -8), 10% glycerol, pH 7.0. The stock solutions for AQZs were
prepared in dimethyl sulfoxide. Formation of the product,
aminomethyl-coumarin, was detected by the increase in sample
fluorescence (
ex = 360 nm,
em = 460 nm) using a Victor plate reader
(PerkinElmer Life Sciences, Boston, MA), taking sample readings every 1 to 2 min for up to 1 h.
Intracellular DEVDase Assay. SH-SY5Y cells were maintained in Dulbecco's modified Eagle's medium plus 10% FCS. The cells were plated in 96-well plates at a density of 60,000 cells/well overnight and then the medium was replaced with PBS plus 10% FCS. The cells were incubated with (Z-DEVD)2-R110 (50 µM) with or without staurosporine (1 µM) and inhibitor for 4 h. The fluorescence intensity of each well was then measured using a microplate reader (Applied Biosystems, Foster City, CA) with excitation/emission wavelengths of 496 nm/520 nm. The cleaved product was also visualized at the single cell level by fluorescence microscopy using an Olympus IX70 inverted fluorescence microscope (20× magnification) and WIB filters.
Cell Apoptosis Assays.
The PC12 cell NGF withdrawal assay
was performed as described previously (Bhat et al., 2000). Briefly,
PC12 cells were differentiated for 9 to 12 days in RPMI 1640 medium
containing 1% FCS and 50 ng/ml NGF. The cells were extensively washed
with NGF-free media, incubated with anti-NGF antibodies (1:400) plus
inhibitors for 4 h, and then processed for DNA fragmentation.
Cell Staining. SY5Y cells were plated at 1.5E-5 cells per well in a 48-well plate. Cells were treated with staurosporine plus drugs as described for 4 h. Samples were fixed with 2% paraformaldehyde for 10 to 30 min at 4°C, washed once with PBS, then treated briefly with Hoechst 33342 stain (1:500). Cells were again washed with PBS. The cells were permeabilized using 0.1% Triton X-100 for the TUNEL staining, which was performed as recommended by the manufacturer. Fluorescence microscopy evaluation was visualized directly in the wells using a Nikon Diaphot microscope.
Data Analysis. Data are expressed as mean ± S.E. from at least three independent experiments. Nonlinear regression analysis of concentration response curves was performed using GraphPad Prism (GraphPad Software Inc., San Diego, CA). Statistical analyses were performed using the Student's t test. p < 0.05 was considered statistically significant.
| |
Results |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Enzyme Assays.
Random screening for inhibitors of recombinant
human caspase-3 revealed a series of compounds termed
anilinoquinazolines, or AQZs (Fig. 1).
These compounds inhibited the rate of product formation in the
fluorogenic enzyme assay in a concentration-dependent manner (Fig.
2A). A Dixon plot of the
concentration-response data for AQZ-3 yielded a
Ki = 589 nM. Additional AQZs were
synthesized, primarily by varying the substituent at position R8, with
several demonstrating similar or greater potency for caspase-3 (Table 1). In addition to modifications at R8,
replacing the dichloroanilide moiety with a fluoroanilide resulted in a
6-fold increase in potency (compare compound AQZ-3 with compound
AQZ-2). Compound AQZ-2 is the most potent inhibitor within the series,
with a Ki = 88 nM.
|
|
|
Whole Cell Assays.
A number of assays have been developed to
monitor caspase-3 activity in cell lysates and the consequences of
caspase-3 activation in intact cells, but methods to directly measure
caspase-3 catalytic activity in intact cells are limited. Such an assay
would help in the development and analysis of cell permeable caspase-3
inhibitors. For this reason, a new cellular assay was developed and
used to determine whether intracellular caspase-3 activity was blocked by the AQZs and whether this correlated with inhibition of other features of apoptosis. (Z-DEVD)2-R110, a
rhodamine-conjugated peptide substrate for caspase-3 that was reported
to enter cells (Liu et al., 1999), was used as substrate. This peptide
is an internally quenched substrate; cleavage of the Z-DEVD blocking groups relieves the quench and allows fluorescence detection using appropriate excitation and emission wavelengths. SH-SY5Y cells were
used for these studies, as their apoptotic response to a number of
toxicants has been well studied (Posmantur et al., 1997; Bijur et al.,
2000; Lopez and Ferrer, 2000).
|
|
,
2001) and dithiocarbamates such as disulfiram (Nobel et al., 1997). The
isatin sulfonamides are direct inhibitors of caspase-3 whereas the
dithiocarbamates have been reported to block the processing/activation
of the caspase-3 proenzyme. Selective examples of these compounds were
also tested in this assay to determine whether structurally divergent
caspase-3 inhibitors are effective in blocking the staurosporine
response. Indeed, both the isatin sulfonamide and disulfiram were able
to completely inhibit intracellular DEVDase activity and did so with
IC50 values of 9.5 ± 3.2 µM and
3.7 ± 0.2 µM, respectfully.
Compounds that inhibit caspase-3 activity should block the downstream
consequences of this enzyme's effects. One end-stage biochemical event
in apoptosis is the cleavage of chromosomal DNA into internucleosomal
fragments. Caspase-3 promotes DNA degradation because it cleaves and
inactivates ICAD, an inhibitor of the endonuclease responsible for
degrading DNA (Green and Reed, 1998). Since these DNA fragments are a
hallmark feature of apoptosis, experiments were performed to determine
whether the AQZs would inhibit staurosporine-induced DNA fragmentation.
As shown in Fig. 5, both AQZ-3 and AQZ-6
block DNA degradation in a dose-dependent fashion. The
IC50 values for AQZ-3 and AQZ-6 were 8.2 and 3.0 µM, respectively, which is consistent with that required for
inhibition of intracellular DEVDase activity.
|
|
; Kim et al., 1999). As shown in Fig.
7, AQZs reduced the DNA fragmentation
that occurs with NGF withdrawal. This inhibitory effect is also
observed with the isatin sulfonamide and with boc-Asp-fmk, an
irreversible and nonselective caspase inhibitor. The AQZs did not
affect the level of DNA fragmentation seen in the presence of NGF.
Thus, the AQZs are effective in preventing apoptosis induced by
multiple cellular insults via a caspase-3-dependent mechanism.
|
| |
Discussion |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
This study describes the biochemical and cellular properties of a
new series of small molecule inhibitors of caspase-3. The AQZs are
structurally distinct from the other known class of nonpeptide caspase-3 inhibitors, the isatin sulfonamides. However, both series share an electrophilic carbonyl that presumably represents the site of
nucleophilic attack by the active site cysteine thiolate. This has been
verified for the isatin sulfonamides using X-ray crystallography data
(Lee et al., 2000). Although not formally proven, molecular modeling
studies with the AQZs are consistent with this same mechanism of action.
The caspases chosen for selectivity assessment include members of each
of the three major subgroups that are defined by substrate specificity
at the P4 site (Nicholson, 1999): caspase-1 (group I), caspase-2 and -7 (group II, which includes caspase-3), and caspase-6 and -8 (group III).
The AQZs display selectivity within the caspase family, with caspase-6
being the only other isoform sensitive to inhibition, and this was
observed with only a subset of the AQZs. No appreciable inhibition was
observed with caspase-7, which is the isoform with highest structural
homology to caspase-3. This selectivity is not seen with the isatin
sulfonamides, which are at most <3-fold selective versus caspase-7.
Therefore, the AQZs represent useful pharmacological tools to help
delineate the relative importance of caspase-3 versus caspase-7 in the
apoptotic response. These results also indicate that specificity for
the AQZs does not fit within the subgroup families as defined by the P4
determinants, i.e., structural features other than S4 are important in
defining the binding of AQZs to caspases.
The caspase-3 cellular assay described in this report has advantages over other cell-based assays described in the literature. The protocol is a simple mix-and-measure assay that can be used with both adherent and nonadherent cells. It can be used as an endpoint assay and is compatible with high density plates for use in random high throughput screening. Transfection of a reporter gene or other construct is not required, although it is compatible should one choose to evaluate the influence of other gene products on caspase-3 activity. Importantly, it is a direct measure of intracellular DEVDase activity rather than a downstream readout that could be modulated by other biochemical pathways.
Although the AQZs are relatively potent inhibitors of isolated caspase-3 with several members having Ki values in the 100 to 500 nM range, typically one needs 5- to 10-fold or higher concentrations to inhibit caspase-3 in intact cells (and downstream measures of caspase-3 activity). A 10-fold loss in potency from enzyme to whole cell assay is not unusual when pursuing inhibitors of intracellular targets, e.g., protein kinases. The difference in inhibitor potencies for the isolated enzyme versus a cell response may be due to one of several possibilities including poor cell penetration, metabolism by the cell, protein binding, and/or high enzyme catalytic activity, which requires the need to inhibit >90% of the active enzyme before seeing efficacy in a cell response. The efficacy of these compounds in the cellular assays is not due to an induction of oncosis and thereby redirecting cell death via a nonapoptotic pathway. The AQZs show no cytotoxic activity at 10 µM as measured by the Alamar Blue assay, trypan blue exclusion, or lactate dehydrogenase release (data not shown).
The NGF withdrawal model has been widely used to explore the
biochemical pathways involved in the induction and regulation of
apoptosis in a neuronal-like environment. The results from these
studies indicate that caspase-3 is activated as part of the apoptotic
response and plays an important role in the execution phase of
apoptosis (Haviv et al., 1997; Kim et al., 1999), although studies with
peptide inhibitors have suggested that a separate caspase-2 pathway is
also involved in the death process (Stefanis et al., 1998). These
results required the use of very high concentrations of peptide
inhibitors (relative to that required to inhibit the isolated enzyme)
to achieve adequate cell exposure. It is possible that under such
conditions the selectivity profile of these inhibitors is lost, and/or
the compounds act on other biochemical processes. Indeed, the caspase-3
peptidic inhibitor z-DEVD-fmk has proapoptotic activity in PC12 cells
under conditions where the isatin sulfonamides are protective (Nuttall
et al., 2001). The results in Fig. 7 indicate that caspase-3-selective
AQZs can prevent DNA fragmentation in PC12 cells and do so at
concentrations where they do not inhibit caspase-2. These results
support the role of caspase-3 as a key caspase in the apoptotic pathway
activated upon withdrawal of NGF.
The AQZs represent new tools to further explore the role of caspase-3 in various cellular models of apoptosis. This should facilitate a better understanding of in vivo settings where this enzyme plays a critical function in the execution of the apoptotic death response. Such studies will help elucidate both the therapeutic value and potential mechanism-based side effect liabilities of caspase-3 inhibition.
| |
Footnotes |
|---|
Accepted for publication September 9, 2002.
Received for publication June 4, 2002.
DOI: 10.1124/jpet.102.039651
Address correspondence to: Dr. Clay Scott, LW208, AstraZeneca Pharmaceuticals, 1800 Concord Pike, Wilmington, DE 19810. E-mail: clay.scott{at}astrazeneca.com
| |
Abbreviations |
|---|
NGF, nerve growth factor; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; AQZs, anilinoquinazolines; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; FCS, fetal calf serum; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay.
| |
References |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
protection by lithium.
J Biol Chem
275:
7583-7590This article has been cited by other articles:
|
|
 |
|
  J.-Q. Du, J. Wu, H.-J. Zhang, Y.-H. Zhang, B.-Y. Qiu, F. Wu, Y.-H. Chen, J.-Y. Li, F.-J. Nan, J.-P. Ding, et al. Isoquinoline-1,3,4-trione Derivatives Inactivate Caspase-3 by Generation of Reactive Oxygen Species J. Biol. Chem., October 31, 2008; 283(44): 30205 - 30215. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Okun, S. Malarchuk, E. Dubrovskaya, A. Khvat, S. Tkachenko, V. Kysil, D. Kravchenko, and A. Ivachtchenko Screening for Caspase-3 Inhibitors: Effect of a Reducing Agent on Identified Hit Chemotypes J Biomol Screen, September 1, 2006; 11(6): 694 - 703. [Abstract] [PDF]
|
|
|
|
|
|
I. Okun, S. Malarchuk, E. Dubrovskaya, A. Khvat, S. Tkachenko, V. Kysil, A. Ilyin, D. Kravchenko, E. R. Prossnitz, L. Sklar, et al. Screening for Caspase-3 Inhibitors: A New Class of Potent Small-Molecule Inhibitors of Caspase-3 J Biomol Screen, April 1, 2006; 11(3): 277 - 285. [Abstract] [PDF]
|
|
|
|
 |
|
 U. Fischer and K. Schulze-Osthoff New Approaches and Therapeutics Targeting Apoptosis in Disease Pharmacol. Rev., June 1, 2005; 57(2): 187 - 215. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
) or 1µM
staurosporine (
). The fluorescent product was quantified in
individual wells at various times using a microplate reader as
described under Materials and Methods. SH-SY5Y cells
were also treated with buffer (
) or 1 µM staurosporine (
) for
260 min, and the spent medium was collected. The medium was then
incubated with 50 µM (Z-DEVD)2-R110 to see if
appreciable DEVDase activity had been released from the cells. B-C,
SH-SY5Y cells were incubated for 4 h with either 1 µM
staurosporine and various concentrations of (Z-DEVD)2-R110
(B) or with 50 µM (Z-DEVD)2-R110 and different
concentrations of staurosporine (C) and the fluorescence was measured.
D, inhibition of the staurosporine response by various caspase-3
inhibitors. SH-SY5Y cells were incubated with 50 µM
(Z-DEVD)2-R110, 1 µM staurosporine, and various
concentrations of either AQZ-3 (
). After 4 h the cells
were lysed, and the amount of DNA fragmentation measured by ELISA as
described under Materials and Methods.
