2005 Nature Publishing Group

Cancer Research, Abbott

Laboratories, Department
R460, Building AP10-LL,
100 Abbott Park Road,
Abbott Park, Illinois 60064,
USA.
e-mail:
stephen.fesik@abbott.com
doi:10.1038/nrc1736
Published online
20 October 2005
Programmed cell death (apoptosis) is a natural
process for removing unwanted cells such as those
with potentially harmful mutations, aberrant sub-
stratum attachment, or alterations in cell-cycle
control. Deregulation of apoptosis can disrupt the
delicate balance between cell proliferation and cell
death and can lead to diseases such as cancer
1,2
.
In many cancers, pro-apoptotic proteins have
inactivating mutations or the expression of anti-
apoptotic proteins is upregulated, leading to the
unchecked growth of the tumour and the inability
to respond to cellular stress, harmful mutations and
DNA damage. In fact, the evasion of programmed
cell death has been recognized as one of the six
essential alterations in cell physiology that dictate
malignant growth and is a hallmark of most, and
maybe all, types of cancer
3
. Moreover, cancers that
possess alterations in proteins involved in cell death
signalling are often resistant to chemotherapy and
are more difficult to treat using chemotherapeutic
agents that primarily work by inducing apoptosis.
Therefore, drugs designed to restore programmed
cell death might be effective against many cancers
4
.
Selective killing of tumour cells might be achievable
with such drugs because unlike normal cells, cancer
cells are under stress, destined to die, and highly
dependent on aberrations of the apoptosis signalling
pathways to stay alive.
Possible drug targets for modulating programmed
cell death have been discovered from the elegant work
of many scientists in elucidating the protein components
and regulators of the apoptosis signalling pathways.
These pathways can be divided into two components
those that involve the mitochondria (intrinsic pathway)
or those that signal through death receptors (extrinsic
pathway) (FIG. 1). In the death receptor pathway, ligands
such as tumour-necrosis factor (TNF), FAS ligand (also
known as CD95L), or TNF-related apoptosis-inducing
ligand (TRAIL, also known as APO2 ligand (APO2L) or
TNF ligand superfamily member 10 (TNFSF10)) inter-
act with their respective death receptors (TNF receptor
1 (TNFR1), FAS (also known as CD95) and death recep-
tor 4 (DR4, also known as TRAIL receptor 1 (TRAILR1))
or DR5 (also known as TRAILR2), respectively). These
interactions ultimately lead to the recruitment of the
FAS-associated death domain (FADD) and the activa-
tion of the protease caspase 8 REF. 5. Caspase 8 cleaves
and activates caspase 3 and other downstream caspases,
which results in a proteolytic cascade that gives rise to
the cell death phenotype characterized by DNA frag-
mentation, chromatin condensation, cell shrinkage and
membrane blebbing. The intrinsic pathway involves the
release of cytochrome c from the intermembrane space
of the mitochondria. Cytochrome c interacts with apop-
totic protease-activating factor 1 (APAF1) and, together
with dATP (2-deoxyadenosine 5-triphosphate), they
PROMOTING APOPTOSIS AS A
STRATEGY FOR CANCER DRUG
DISCOVERY
Stephen W. Fesik
Abstract | Apoptosis is deregulated in many cancers, making it difficult to kill tumours.
Drugs that restore the normal apoptotic pathways have the potential for effectively treating
cancers that depend on aberrations of the apoptotic pathway to stay alive. Apoptosis
targets that are currently being explored for cancer drug discovery include the tumour-
necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors, the BCL2 family
of anti-apoptotic proteins, inhibitor of apoptosis (IAP) proteins and MDM2.
876 | NOVEMBER 2005 | VOLUME 5 www.nature.com/reviews/cancer
R E V I E WS
2005 Nature Publishing Group

ANTIBODYDEPENDENT
CELLULAR CYTOTOXICITY
Immune cells interact with the
Fc region of antibodies that are
bound to a target cell through
Fc receptors on their surface to
mediate cell killing.
COMPLEMENTDEPENDENT
CYTOTOXICITY
Antibodies can kill target cells
by binding the various
components of complement.
When combined, the
components form pores in the
cell membrane, which leads to
phagocytosis or lysis.
form a multimeric complex that recruits and activates
caspase 9, leading to the activation of downstream
caspases and the death response
6
.
In addition to the proteins that are directly
involved in cell death signalling, proteins that regulate
programmed cell death could also represent poten-
tial cancer drug targets. For example, BCL2 family
members
7
that localize to the mitochondria can
either prevent or cause the release of cytochrome c
from the mitochondria to either inhibit or promote
apoptosis. Another important protein family that
regulates apoptosis is the inhibitor of apoptosis (IAP)
proteins, which bind to and inhibit caspases

and block
apoptotic signalling
8
. IAPs are regulated by SMAC
(second mitochondria-derived activator of caspase
9
,
also known as DIABLO, direct IAP-binding protein
with low pI
10
), which is released from the mitochon-
dria once the cell has received a death signal. SMAC
binds to the IAPs and antagonizes their anti-apoptotic
activity.
MDM2 and p53 are also important regulators of
programmed cell death
11
. On sensing DNA damage,
p53 induces cell-cycle arrest and apoptosis. p53 is
mutated and inactivated in half of all cancers ana-
lysed, demonstrating the importance of disrupting
the p53 pathway in cancer development. Inactivation
of p53 can also occur by p53 binding to MDM2,
which leads to the degradation of p53 by the pro-
teasome. Therefore, disruption of the interaction
between p53 and MDM2 should stabilize p53 and
enable its apoptotic function
11
.
In this review, targets and strategies for pro-
moting apoptosis as a means for anticancer drug
discovery will be discussed. Although most of this
work is at an early stage, this overall strategy seems
promising.
TRAIL
The death receptors of the TNF superfamily represent
potential targets for promoting apoptosis in cancer
12
.
As death receptor-mediated apoptosis is thought to
be independent of p53, cancers with inactivating p53
mutations might be susceptible to treatment using
this approach. Although attempts have been made
to exploit death receptors for cancer drug discovery
using recombinant, soluble TNF and agonistic anti-
FAS antibodies, toxic side effects were observed with
these agents that limited their therapeutic use
12
. A
potentially more promising approach involves target-
ing the TRAIL receptors. Similar to other members
of the TNF family, TRAIL is a type II transmembrane
protein that exists as a homotrimer
1315
. TRAIL
induces apoptosis by binding to the DR4 and DR5
receptors, which causes the intracellular death
domains of these receptors to trimerize. This leads to
the recruitment of FADD and activation of caspase 8,
caspase 3 and caspase 7 REF. 16. Caspase 8 activa-
tion further amplifies the death signal by activating
the intrinsic apoptosis pathway through cleaving the
BCL2 family member BID. Cleaved BID binds to
BAX and BAK, causing the release of cytochrome c
and SMAC from the mitochrondria. This results in
the activation of caspase 9 and, subsequently, other
downstream caspases
16
.
In an attempt to develop effective agents for the
treatment of cancer by triggering TRAIL receptors,
agonistic antibodies against DR4 REF. 17 and DR5
REFS 18,19 have been made. These antibodies induce
apoptosis in cancer cells but not in normal cells
18
, and
slow the growth of tumours in xenograft tumour models
with no apparent systemic toxicity
18,19
.

Antibodies have
the advantage of having a relatively long half-life and
can use additional mechanisms for cell killing through
ANTIBODYDEPENDENT CELLULAR CYTOTOXICITY (ADCC) and
COMPLEMENTDEPENDENT CYTOTOXICITY (CDC) mechanisms
mediated by the Fc portion of the antibodies
20
.
Another approach for pharmacologically trigger-
ing the TRAIL receptors involves the use of soluble
truncated versions of TRAIL that contain the extra-
cellular domain. In preclinical studies, recombinant
TRAIL induced apoptosis in various cancer cell lines,
including those with p53 mutations, without affecting
normal cells. In addition, chemotherapeutic drugs
21

and histone deacetylase inhibitors
22
were shown to
augment the apoptotic activity of TRAIL. TRAIL also
displayed anti-tumour activity in vivo in mouse models
of colon
23
, glioma
24
, lung
25
and prostate cancer
26
, and
multiple myeloma
27
, when given as a single agent. In
addition, TRAIL caused tumour regression in mouse
models when given in combination with irinotecan
23
,
paclitaxel
25
or radiation
28
. Although some recombinant
forms of TRAIL have been shown to be toxic to hepato-
cytes and other normal cells, these effects are thought
to be related to the particular recombinant forms of the
protein rather than TRAIL itself
29
. Safety evaluations in
primates with TRAIL that did not contain extraneous
amino-acid residues showed no toxicities related to
TRAIL exposure
30
.
Summary
In many cancers, the normal process for eliminating unwanted cells (apoptosis) is
deregulated.
The deregulation of apoptosis leads to the unchecked growth of tumours and the
development of resistance to chemotherapy.
Drugs that restore apoptosis might selectively kill cancer cells that have
triggered a death signal and have become dependent on the deregulation of
apoptosis pathways.
Agonistic antibodies against the tumour-necrosis factor (TNF)-related apoptosis-
inducing ligand (TRAIL) receptors and a soluble, truncated TRAIL ligand are in
phase I/II clinical trials for the treatment of cancer.
BCL2 antisense oligonucleotides are in phase III clinical trials and pre-
registration, and small-molecule BCL2-family inhibitors are in early phase I
clinical trials and in late preclinical discovery for the treatment of chronic
lymphocytic leukaemia and solid tumours.
IAP (inhibitor of apoptosis) protein inhibitors, MDM2 antagonists and other
apoptosis-inducing compounds are under preclinical examination for possible use
in cancer therapy.
For these compounds to succeed, it will be important to test them in well-designed
clinical trials to determine the cancer patients that are most likely to respond to
the particular agent, the optimum dose and schedule, and the best combination
with other drugs.
NATURE REVIEWS | CANCER VOLUME 5 | NOVEMBER 2005 | 877
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D
D
BAK
BAD
BID
BAX
BCL2
APAF1
Cytochrome c
Caspase 9 Caspase 8
C
a
s
p
a
s
e

8
Caspase 3
BCL-X
L
SMAC
IAP
Mitochondrial pathway FAS pathway
dATP
D
E
D
D
E
D
D
E
D
D
D
D
D
D
D
FAS
Programmed cell death
F
A
D
D
P
r
o

d
o
m
a
i
n
P
r
o
c
a
s
p
a
s
e

8
FASL
Resistance to TRAIL can occur by various differ-
ent mechanisms. TRAIL can bind tightly to two decoy
receptors called DCR1 (decoy receptor 1) and DCR2.
Unlike DR4 and DR5, these receptors do not contain
an intact death domain and act as non-functional
binding partners for TRAIL, which can attenuate its
apoptotic activity
31
. Another mechanism of resistance
to TRAIL involves the loss or decrease in transport to
the cell surface of DR4 and DR5 REF. 32 that, in some
cases, can be overcome by the overexpression of DR4
REF. 33. Varying degrees of TRAIL sensitivity or resist-
ance have also been observed that are dependent on
the levels of pro- and anti-apoptotic proteins present in
a cell. RNA interference (RNAi)-mediated knockdown
of the anti-apoptotic proteins BCL2, XIAP (X-linked
IAP), FLIP or survivin, increased apoptosis in melanoma
cells when given in combination with TRAIL
34
. In addi-
tion, SMAC peptides that inhibit IAPs were shown to
increase the anti-tumour activity of TRAIL in a glioma
xenograft model
35
. Additional evidence that supports the
importance of other pro-apoptotic molecules in influenc-
ing TRAIL sensitivity is the finding that the efficacy of
TRAIL-mediated tumour cell killing correlates with the
levels of expressed BAX
3638
.
These approaches for targeting TRAIL receptors are
currently being tested in the clinic TABLE 1. Phase I
and II clinical trials have been initiated for an agonistic
antibody targeting DR4, and phase I clinical trials are
ongoing for an antibody that targets DR5. In addition,
recombinant TRAIL is in a phase I clinical trial for the
treatment of solid tumours.
BCL2-family inhibitors
The proteins of the BCL2 family have an important
role in the regulation of programmed cell death and
also serve as potential targets for cancer therapy.
Members of this family can either promote or inhibit
programmed cell death. The pro-apoptotic BCL2 pro-
teins can be further divided into those that possess
sequence homology to the BH1 (BCL2-homology 1),
BH2 and BH3 regions, and the BH3-only proteins
that only have homology with the BH3 region
39,40
.
Multidomain pro-apoptotic proteins such as BAX
and BAK can directly induce apoptosis on receiving
a death signal by forming oligomers in mitochondrial
membranes, leading to the release of cytochrome c,
aggregation of APAF1 and the activation of caspases
41
.
The anti-apoptotic BCL2 family members inhibit
cytochrome c release by blocking the activation of
BAX and BAK. The pro-apoptotic BH3-only proteins,
on the other hand, function either by directly inducing
the oligomerization of BAK and BAX or by binding to
and inhibiting the anti-apoptotic BCL2 proteins
41
.
The three-dimensional structures of several
BCL2 proteins have been solved
42
. The structures
are all similar and consist of two central, predomi-
nantly hydrophobic -helices surrounded by six or
seven amphipathic -helices. A hydrophobic groove
is present on the surface of the anti-apoptotic pro-
teins BCL2, BCL-X
L
and MCL1 this groove forms
the binding site for the BH3 region of pro-apoptotic
BCL2 family members
43,44
(FIG. 2a).
The anti-apoptotic BCL2 proteins represent
attractive targets for the treatment of cancer
45
. A
t(14;18)(q32;q21) chromosomal translocation is
present in over 60% of human follicular lymphomas
46,47
,
which results in the overexpression of BCL2 in this can-
cer
48
. BCL2 is also overexpressed in many other cancers,
including small cell lung carcinomas (SCLC)
49,50
, chronic
lymphocytic leukaemias (CLL)
51
, multiple myeloma
52
,
melanoma
53
, prostate
54,55
, ovarian
56
, cervical
57
, bladder
58
,
gastric
59
, pancreatic
60
, breast
6163
and colorectal
6467
can-
cers. For most cancers, the overexpression of BCL2 or
BCL-X
L
correlates with poor survival and progression
of the disease
5460
. However, contrary to what might be
expected, in breast
6163
and colorectal
6467
tumours the
over expression of BCL2 has been associated with favour-
able outcomes and improved survival. Overexpression
of BCL2 and BCL-X
L
is associated with resistance to
various cytotoxic agents
6869
and radiotherapy
7071
,
making anti-apoptotic BCL2 family proteins obvious
anticancer drug targets. In fact, cancer cell resistance
to a panel of 122 chemotherapeutic agents strongly
correlated with the expression levels of BCL-X
L
72
.
One approach for targeting anti-apoptotic BCL2
proteins is by reducing their expression levels using
antisense oligonucleotides. Indeed, an antisense
oligo nucleotide against BCL2, oblimersen
73
, has
Figure 1 | Simplified representation of the two main signalling pathways of
programmed cell death. The intrinsic pathway (left) involves the mitochondria. On
receiving a death signal, cytochrome c is released from the intermembrane space of the
mitochondrion, which, together with dATP (2-deoxyadenosine 5-triphosphate) and
apoptotic protease-activating factor 1 (APAF1), activates caspase 9. Caspase 9 activates
downstream caspases such as caspase 3, leading to apoptosis. BCL2 and BCL-X
L
inhibit
programmed cell death by preventing the release of cytochrome c, whereas pro-apoptotic
BCL2 family members (such as BAK, BAX, BAD and BID) promote the release of
cytochrome c. The extrinsic cell death pathway (right) is mediated by death receptors. As
an example, FAS ligand (FASL) interacts with the FAS receptor, leading to the interaction of
the death domains (DD) of FAS with the death domain of the adaptor protein, FAS-
associated death domain (FADD). The death effector domain (DED) of FADD then binds to
the DED of procaspase 8, which triggers the formation of activated caspase 8, the
activation of caspase 3, and the apoptotic response. IAP, inhibitor of apoptosis; SMAC,
second mitochondria-derived activator of caspase.
878 | NOVEMBER 2005 | VOLUME 5 www.nature.com/reviews/cancer
R E V I E WS
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ANTENNAPEDIA
HOMEOPROTEIN
Cationic peptides derived from
this protein can be attached to
molecules to allow their
transport into cells.
, DISUBSTITUTED
UNNATURAL AMINOACIDS
Amino-acids with two
additional chemical groups
attached to the -position.
TERPHENYL SCAFFOLD
A synthetic framework
containing three phenyl rings
that can be used to stabilize
-helical proteins and disrupt
proteinprotein interactions.
been in phase III clinical trials for melanoma, CLL
and multiple myeloma, as well as in phase II clini-
cal trials for several other cancers
74,75
TABLE 1. So
far, the results in the clinic are not very promising.
Oblimersen failed to reach the primary survival end-
point in a randomized, open label phase III trial in
melanoma when given with dacarbazine compared
with dacarbazine alone
76
, and therefore failed to be
approved by the Food and Drug Administration in
the United States. Furthermore, no clinical benefit
was observed in a phase III clinical trial for refrac-
tory multiple myeloma in patients who were receiv-
ing oblimersen and dexamethesome versus those
receiving dexamethesome alone
75
. However, the
overall response rate was found to be improved in
patients with CLL who were receiving fludaribine
and cyclophosphamide with oblimersen compared
with those receiving chemotherapy alone.
In addition to the lack of a clear clinical benefit of
oblimersen, there is also the possibility of off-target
effects and immunostimulatory activity owing to the
presence of phosphorothioates and two CpG motifs
in the oligonucleotide
77
. Also, oblimersen only targets
BCL2, and it might be necessary to target more than
one BCL2 family member to be effective and limit
the development of resistance. Towards this end, a bi-
specific antisense molecule against BCL2 and BCL-X
L

was developed that induces apoptosis in tumour cells
78
.
Another approach to inhibit BCL2 proteins involves
mimicking the activity of the pro-apoptotic BH3-only
proteins. Synthetic BH3 peptides have been designed
to interact with the hydrophobic cleft of BCL2 and
BCL-X
L
. To increase their cellular permeability and
direct the peptide to the membrane, the ANTENNAPEDIA
HOMEOPROTEIN internalization domain
79
or a fatty acid
was added to the peptide
80
. These peptides have been
shown to enter cells and induce apoptosis. BID-based
BH3-peptide analogues were also synthesized that
contained , DISUBSTITUTED UNNATURAL AMINOACIDS to
stabilize the -helix and make them more protease-
resistant and cell-permeable. These BID-based BH3-
peptide mimetics induced apoptosis in cells, inhibited
the growth of human leukaemia cells in vivo and
prolonged the survival of mice with leukaemia
81
.
Ideally, small organic molecules with suitable drug-
like properties could be obtained that mimic the BH3
peptides and bind into the hydrophobic groove of the
anti-apoptotic BCL2 proteins. Hamilton and co-work-
ers designed small molecules containing a TERPHENYL
SCAFFOLD to mimic the -helical BH3 peptides and
demonstrated that these compounds bind (dissocia-
tion constant (K
D
) = 114 nM for the tightest binder) to
BCL-X
L
82
. Using structure-based design and various
high-throughput screening techniques, additional
small molecules such as antimycin A
83
, chelerythrine
84
,
YC-137 REF. 85, HA14-1 REF. 86, Gossypol
8789
and oth-
ers
9092
, were discovered that bind to BCL2 or BCL-X
L
in
the low micromolar range and induce apoptosis in cells.
Recently, a more potent BCL2-family inhibitor, ABT-
737 (FIG. 2c), was reported
93
that binds to BCL2, BCL-X
L

and BCLW with sub-nanomolar affinity. ABT-737 and
structurally similar analogues occupy the same binding
Table 1 | Status of apoptosis-inducing agents
Compound Company Status
Anti-TRAILR1 agonistic antibody Human Genome Sciences/
Cambridge Antibody Technology/
Takeda
Phase I and II trials initiated for solid
tumours
Anti-TRAILR2 agonistic antibody Human Genome Sciences/
Cambridge Antibody Technology
Phase I studies initiated in the United
Kingdom and the United States
TRAIL Genentech/Amgen Phase I studies initiated in solid tumours
Oblimersen/Genasense (antisense
oligonucleotide targeting BCL2)
Genta Inc. Failed to meet primary end-point in
phase III trial in malignant melanoma and
multiple myeloma, phase III clinical trial
and pre-registration in CLL
SPC-2996 (antisense oligonucleotide
targeting BCL2)
Santaris Pharma Phase I/II in CLL in Europe
AT 101(()-Gossypol) Ascenta Therapeutics Inc. Phase I in CLL
Small-molecule BCL2-family inhibitor Gemin X Biotech Phase I in CLL
ABT-737 (small-molecule BCL2-
family inhibitor)
Abbott Laboratories/Fizer (Idun) Preclinical
IPI-983L/IPI-194 (small-molecule
BCL2-family inhibitors)
Infinity Pharmaceuticals Preclinical
XIAP-BIR2 inhibitor Burnham Institute Preclinical
XIAP-BIR3 inhibitor UT Southwestern Preclinical
XIAP-BIR3 inhibitor Abbott Laboratories Preclinical
Nutlins (MDM2 inhibitors) Wyeth Preclinical
BIR, baculovirus IAP repeat; CLL, chronic lymphocytic leukaemia; TNF, tumour-necrosis factor; TRAIL, TNF-related apoptosis-
inducing ligand; XIAP, X-linked inhibitor of apoptosis protein.
NATURE REVIEWS | CANCER VOLUME 5 | NOVEMBER 2005 | 879
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a b c
H
N
S
N
H
3
C CH
3
S
H
N
O O
O
N
N
Cl
NO
2
2,000
1,500
1,000
500
0
0 35 45 55 65 75 85 95 105
Days post inoculation
A
v
e
r
a
g
e

t
u
m
o
u
r

v
o
l
u
m
e

(
m
m

s
.
e
.
m
)
Therapy
Vehicle
25mg
1
kg
1
day
1
ip
50mg
1
kg
1
day
1
ip
75mg
1
kg
1
day
1
ip
100mg
1
kg
1
day
1
ip
site as the BH3-only protein BAD (FIG. 2b). ABT-737
showed synergistic cytotoxicity with chemotherapeutic
drugs or radiation in various cell lines and demonstrated
single-agent mechanism-based killing in lymphoma and
leukaemia cell lines and primary patient-derived cells.
This BCL2-family inhibitor also displayed potent activ-
ity by itself against SCLC cell lines. In vivo, ABT-737
induces complete regression in mouse models of non-
Hodgkin lymphoma when given in combination with
various chemotherapeutic agents and, in a significant
percentage of mice, causes the complete regression of
SCLC when given as a single agent
93
(FIG. 3). These results
indicate that BCL2-family inhibitors might be useful
for the treatment of lymphoma, leukaemia and SCLC
as monotherapies, and for other cancers when given in
combination with chemotherapy or radiation TABLE 1.
However, it is still too early to judge whether the small-
molecule BCL2-family inhibitors will have clinical ben-
efit as they are only in the preclinical and early phase I
stages of development TABLE 1.
XIAP inhibitors
The IAPs are important regulators of apoptosis that
function by binding to and inhibiting caspases
8
. They
are characterized by having one or more protein mod-
ules of 70 amino-acids called baculovirus IAP repeat
(BIR) domains that contain a CX
2
CX
16
HX
6
C signature
sequence and adopt a three-dimensional structure
resembling a classical zinc finger
94
. The most com-
prehensively studied IAP, XIAP, contains three BIR
domains and a ring finger, and inhibits caspase 3,
caspase 7 and caspase 9. The BIR2 domain and the
linker between BIR1 and BIR2 of XIAP are respon-
sible for inhibiting caspase 3 and caspase 7 REF. 95.
Caspase inhibition is achieved by the binding of the
BIR1BIR2 linker into the caspase active site
94,96
. By
contrast, XIAP-mediated inhibition of caspase 9 occurs
by a different mechanism
97,98
. In this case, the BIR3
domain of XIAP binds to a caspase 9 monomer and
prevents the formation of the active caspase 9 dimer
98
.
SMAC, once released from the mitochondria, binds to
the XIAP-BIR3 domain
99,100
at the same site as caspase 9
(FIG. 4a), relieves the XIAP-mediated inhibition of
caspase 9 and thereby promotes apoptosis.
The inhibition of XIAP and other IAPs is a pos-
sible mechanism for inducing apoptosis and treat-
ing cancer
101
. The XIAP protein is overexpressed in
many cancers and the expression of XIAP correlates
with apoptotic resistance. Knockdown of XIAP by
antisense oligonucleotides or RNAi induces apop-
tosis in cancer cell lines and promotes the synergistic
killing of cancer cells by TRAIL and actinomycin D.
In addition to using antisense oligonucleotides for
targeting XIAP
102
, peptidic and non-peptidic inhibi-
tors of XIAP have been reported. Cell-permeable
SMAC peptides can enhance the apoptotic effects of
Figure 2 | Three-dimensional structures of BCL-X
L
and a two-dimensional structure of a BCL2-family inhibitor.
a | Space-filling model of BCL-X
L
illustrating the hydrophobic groove that binds to a 25-mer peptide (green helix) derived from
the pro-apoptotic protein BAD. b | Structure of BCL-X
L
bound to a BCL2-family inhibitor. The compound binds to the same
hydrophobic groove of BCL-X
L
that interacts with the pro-apoptotic BAK and BAD proteins. c | Structure of the potent BCL2-
family inhibitor ABT-737.
Figure 3 | Anti-tumour activity of ABT-737 observed
in vivo in a small cell lung carcinoma xenograft mouse
model. ABT-737 was given intraperitoneally (ip) for 21 days at
the doses indicated. ABT-737 caused the complete regression
of established tumours, and the tumours did not grow back in
a high percentage of the mice (77% at the highest dose).
880 | NOVEMBER 2005 | VOLUME 5 www.nature.com/reviews/cancer
R E V I E WS
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W323
Q319
E314
D309
T308
L307
K299
K297
W310
a b
c
H
2
N
O
N
S
Br
HETEROCYCLES
Ring compounds with both
carbon atoms and atoms of
other elements in the ring.
CHALCONE DERIVATIVES
1,3-diphenyl-2-propen-1-one
analogues that are derived from
ketones and are important
intermediates in the synthesis of
flavonoids. They are used to
treat several diseases, including
cancer.
chemotherapeutic agents in vitro and in vivo
103108
, and
SMAC peptidomimetics have been shown to potently
induce caspase activation and apoptosis in cancer cells
and inhibit the growth of tumours in xenograft mouse
models
108
. Non-peptidic, small-molecule inhibitors
of XIAP that induce apoptosis and bind to the BIR3
domain have also been described
109112
. In one strategy
for discovering such inhibitors, HETEROCYCLES were used
to replace the valine in the SMAC-based AVPI peptide
(FIG. 4c). As shown in FIG. 4b, the same electrostatic and
hydrogen-bonding interactions are preserved in the
SMAC non-peptideBIR3 complex as those observed
in the SMAC peptideBIR3 structure (FIG. 4a). The
hydrophobic interaction between the proline of the
SMAC peptide and W323 of the BIR3 domain is
maintained by the phenyl ring, and the binding of
the SMAC isoleucine side chain to K297 and K299 is
mimicked by the bromophenyl group. Small molecules
that disrupt the XIAPcaspase 3 interaction
113
have
also been reported. However, at present, it is unclear
how they disrupt this interaction.
Another member of the IAP family that is differ-
ent from XIAP is survivin
114
. Survivin is the small-
est IAP family member and contains only one BIR
domain. In addition to inhibiting apoptosis (probably
by a mechanism that is different to that employed by
XIAP), survivin has an important function in mito-
sis. Survivin is expressed during mitosis and binds
to various components of the mitotic apparatus.
Interestingly, other than its cell-cycle-dependent
expression, survivin is not present in most normal
adult tissues but is overexpressed in many different
tumours and is important for tumour cell viability
115
.
Approaches for inhibiting survivin include the use of
antisense oligonucleotides
116
, vaccination strategies
against survivin-bearing tumour cells, and inhibiting
the phosphorylation of survivin to create a dominant-
negative phenotype
117
.
MDM2 antagonists
MDM2 is a protein that binds to the tumour sup-
pressor p53 and negatively regulates its activity
and stability
118
. The MDM2-mediated loss of p53
impairs a cells ability to block cell-cycle progression
and induce apoptosis in response to DNA damage,
which can contribute to the cancerous phenotype.
The observation that MDM2 is amplified or over-
expressed in several human tumour types
118
provides
further support for the involvement of MDM2 in
cancer. Therefore, strategies aimed at blocking the
interaction between MDM2 and p53 to inhibit
the degradation of p53 could be important for can-
cer drug discovery
119
. The X-ray crystal structure of
the p53MDM2 complex showed how p53 interacts
with MDM2 and aided in the design of p53 mimetics
that bind to MDM2 REF. 120. As shown in FIG. 5a,
the N-terminal region of p53 adopts an -helix,
and the side chains of F19, W23 and L26 within
this -helix interact with a hydrophobic pocket of
MDM2 REF. 20.
In the first attempt to identify MDM2 inhibitors,
peptide-based compounds were discovered that
bind tightly to the MDM2 cleft
121
. However, these
compounds lacked the ability to penetrate cells and
did not possess drug-like properties. Although sev-
eral small molecules (such as CHALCONE DERIVATIVES
122
,
polycyclic compounds
123
, chlorofusin
124
, sulfon-
amides
125
and benzodiazepines
126
) were also identi-
fied that bind to MDM2 and displace p53 peptides,
these compounds only bind weakly to MDM2.
Figure 4 | Structures of SMAC mimetics. a, b | Nuclear magnetic resonance-derived model of a second mitochondria-derived
activator of caspase (SMAC) peptide and a non-peptide SMAC mimetic complexed to the BIR3 domain of X-linked ihibitor of
apoptosis protein (XIAP). Solid black line indicates an electrostatic interaction between the N-terminal Ala of the ligands and E314 of
the XIAP-BIR3 domain, and the dotted lines indicate intermolecular hydrogen bonds. c | Structure of a non-peptide SMAC mimetic.
NATURE REVIEWS | CANCER VOLUME 5 | NOVEMBER 2005 | 881
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a b c
L26
W23
F19
N
N
O
N N
OH
O
CH
3
O
Br
Br
However, a more potent (50% inhibitory concen-
tration (IC
50
) = 100 nM) small-molecule MDM2
inhibitor was recently discovered within a series
of cis-imidazoline analogues called the Nutlins
127
(FIG. 5c). The binding of the Nutlins to MDM2 mim-
ics that of p53 with one bromophenyl group binding
in the W23 pocket, the other bromophenyl in the
L26 pocket, and the ethyl ether side chain occupying
the F19 site (FIG. 5b). As predicted, in cancer cells the
Nutlins caused the accumulation and activation of
p53 with subsequent cell-cycle arrest in G1 and G2.
The dose-dependent anti-proliferative and cytotoxic
activity was dependent on the status of p53, with
tumour cells containing wild-type p53 displaying
an increased sensitivity. The Nutlins inhibited the
growth of tumours in xenograft mouse models with-
out causing significant toxicities
127
. These results
indicate that small molecule inhibitors of MDM2
could be useful for the treatment of cancer, espe-
cially for those cancers that possess wild-type p53
but overexpress MDM2 TABLE 1.
Other strategies
There are many other compounds that induce apoptosis.
However, this might not be their primary function, or
the mechanism by which they induce programmed cell
death has not yet been defined. For example, in addition
to its other functions, the phosphatidylinositol 3-kinase
(PI3K)AKT pathway can regulate both the intrinsic
and extrinsic apoptotic pathways
128
. Activated AKT
decreases apoptosis by phosphorylating and inactivat-
ing BAD
129131
and apoptosis signal-regulating kinase
(ASK1)
132
. AKT also regulates FAS and IAP expression
through phosphorylating the forkhead (FOXO) tran-
scription factor family
133135
and nuclear factor of B
(NF-B)
136
, and stabilizing the phosphoprotein enriched
in astrocytes 15 kDa protein (PEA15)
137
. Furthermore,
the PI3KAKT pathway is deregulated in many can-
cers
138139
, indicating that this signalling pathway might
contain possible anticancer targets. Although inhibi-
tors of the PI3K family (such as LY294002) have been
shown to inhibit the growth of both cancer cells in vitro
and tumours in animal models, these compounds lack
selectivity and have mostly served as pharmacological
tools to study the functions of this pathway
138
. AKT
inhibitors have also been reported
140
that inhibit tumour
growth when used as a monotherapy or in combina-
tion with paclitaxel. Unfortunately, compounds within
this series were found to have a narrow therapeutic
window and show significant metabolic toxicities that
are probably mechanism-based. Another target in the
PI3KAKT pathway is mammalian target of rapamy-
cin (mTOR). Rapamycin analogues that inhibit mTOR
slow the growth of tumours in animal models without
displaying significant toxicity. These compounds are
currently in clinical trials for the treatment of breast,
colon and lung cancers
141
.
The proteasome could also be considered as a target
for inducing apoptosis, and proteasome inhibitors are
being used to treat cancer
142
. They have been shown to
promote apoptosis in part by inducing endoplasmic
reticulum stress and reactive oxygen species in head
and neck squamous cell carcinoma cells
143
. The pro-
teasome degrades ubiquitylated proteins, including
inhibitor of B (NF-BI), which inhibits NF-B
and modulates several pathways. However, the pro-
teasome is not part of the core apoptotic pathway and
degrades many other proteins besides NF-BI. Other
compounds that indirectly induce apoptosis include
inhibitor of B kinase (IKK) inhibitors
144
, arsenic tri-
oxide
145
and many chemotherapeutic agents that are
widely used for cancer therapy.
Conclusions and future perspectives
Targeting apoptosis is a promising strategy for can-
cer drug discovery. Antibodies and recombinant
TRAIL that target the TRAIL receptors are in clini-
cal trials assessing the response of solid tumours,
and BCL2-family inhibitors, IAP antagonists and
MDM2 inhibitors are showing impressive preclinical
efficacy in animal models TABLE 1. A common hur-
dle that all of these small-molecule-based therapies
have had to overcome is the difficulty in targeting a
proteinprotein interaction. To aid in this process, the
three-dimensional structures of the natural ligands
complexed to their protein targets were determined
Figure 5 | MDM2 inhibitors. a | X-ray structure of the MDM2p53 complex that illustrates how the key amino-acid
residues of p53 (L26, W23, and F19) interact with a hydrophobic pocket of MDM2. b | X-ray structure of an MDM2
Nutlin 2 complex, which shows how the different groups of Nutlin 2 bind to the same binding sites as L26, W23 and F19 of
p53. c | Structure of Nutlin 2.
882 | NOVEMBER 2005 | VOLUME 5 www.nature.com/reviews/cancer
R E V I E WS
2005 Nature Publishing Group

and used for inhibitor design. Linked fragment-based
approaches and parallel synthesis were also used. The
knowledge gained from developing these approaches
might prove useful in the future for designing inhibi-
tors for other difficult targets.
For these pro-apoptotic molecules to succeed
they will need to be properly tested in the clinic. As
cancer is a heterogeneous disease, it will be crucial
to conduct the clinical trials in the cancers that are
most likely to respond. For example, the BCL2-family
inhibitors were shown to be very effective as single
agents in preclinical studies against lymphomas and
SCLC
89
, indicating that these compounds should be
tested in these cancers in early clinical trials. From
preclinical studies, it will also be important to identify
the genetic bases for the development of resistance by
comparing the DNA, RNA and proteins in resistant
and sensitive cells. Tissues can then be collected in
any subsequent clinical trials to prove these hypoth-
eses. The optimum dose and schedule for delivering
the compounds also needs to be determined and
this can be aided by measuring pharmacodynamic
biomarkers in the clinic. Finally, effective treatment
for some tumours might require that the agent be
given in combination with other compounds. Indeed,
potent synergistic anti-tumour activity was observed
by administering TRAIL and IAP inhibitors in an
animal model
34
, and the BCL2-family inhibitors
exhibited synergistic cytotoxicity in various cancer
cell lines when given in combination with various
chemotherapeutic drugs or radiation.
Therefore, providing that apoptosis-inducers are
tested in well-designed clinical trials to reveal their
full potential, compounds that promote apoptosis
should become an important addition to the arsenal
of target-specific drugs in the fight against cancer.
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Acknowledgements
The author wishes to thank A. Petros and E. Olejniczak for help in
preparing the figures.
Competing interests statement
The author declares competing financial interests. See web
version for details.

Online links
DATABASES
The following terms in this article are linked online to:
Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db=gene
APAF1 | AKT | BAD | BAK | BAX | BCL2 | BID | caspase 3 |
caspase 7 | caspase 8 | caspase 9 | DR4 | DR5 | MDM2 | p53 |
SMAC | survivin | TRAIL | XIAP
National Cancer Institute: http://www.cancer.gov
leukaemias | melanoma | multiple myeloma | small cell lung
carcinomas
Access to this interactive links box is free online.
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