J Chem Biol (2008) 1:27–36
DOI 10.1007/s12154-008-0003-5
REVIEW
Rapamycin and mTOR kinase inhibitors
Lisa M. Ballou & Richard Z. Lin
Received: 19 February 2008 / Accepted: 11 March 2008 / Published online: 15 May 2008
# Springer-Verlag 2008
Abstract Mammalian target of rapamycin (mTOR) is a
protein kinase that controls cell growth, proliferation, and
survival. mTOR signaling is often upregulated in cancer
and there is great interest in developing drugs that target
this enzyme. Rapamycin and its analogs bind to a domain
separate from the catalytic site to block a subset of mTOR
functions. These drugs are extremely selective for mTOR
and are already in clinical use for treating cancers, but they
could potentially activate an mTOR-dependent survival
pathway that could lead to treatment failure. By contrast,
small molecules that compete with ATP in the catalytic site
would inhibit all of the kinase-dependent functions of
mTOR without activating the survival pathway. Several
non-selective mTOR kinase inhibitors have been described
and here we review their chemical and cellular properties.
Further development of selective mTOR kinase inhibitors
holds the promise of yielding potent anticancer drugs with a
novel mechanism of action.
L. M. Ballou : R. Z. Lin
Department of Medicine, Stony Brook University,
Stony Brook, NY 11794, USA
R. Z. Lin
Department of Physiology and Biophysics
and the Institute of Molecular Cardiology,
Stony Brook University,
Stony Brook, NY 11794, USA
R. Z. Lin
Department of Veterans Affairs Medical Center,
Northport, NY 11768, USA
R. Z. Lin (*)
Department of Medicine, Division of Hematology/Oncology,
Stony Brook University,
Stony Brook, NY 11794-8151, USA
e-mail: richard.lin@sunysb.edu
Keywords mTOR . Rictor . Raptor . Rapamycin .
Phosphatidylinositol 3-kinase . Akt
Phosphatidylinositol 3-kinase-related kinases
Mammalian target of rapamycin (mTOR) is a serine/
threonine protein kinase whose catalytic domain most
closely resembles the one found in lipid kinases of the
phosphatidylinositol (PI) 3-kinase (PI3K) family. PI3Ks
phosphorylate inositol phospholipids on the 3′-hydroxyl of
the inositol ring and are divided into three classes based on
structural features and substrate specificity (reviewed in
Ref. [1]). Class I PI3Ks consist of four different catalytic
subunits (p110α, p110β, p110δ and p110γ) that form
heterodimers with a variety of regulatory subunits. These
enzymes preferentially phosphorylate PI 4,5-bisphosphate
(PI(4,5)P2) in vivo. Crystal structures of p110γ bound to
ATP and to various inhibitors have been instrumental in
determining structure-activity relationships for PI3K inhib-
itors (discussed below). The structure of p110α bound to a
fragment of one of its regulatory subunits was also solved
recently [2]. Class II PI3Ks (PI3K C2α, C2β and C2γ)
phosphorylate PI and PI(4)P in vitro and contain a C2
homology domain at the C terminus that is not found in
other PI3Ks. The class III PI3K Vps34 only phosphorylates
PI and may play a role in the regulation of mTOR by amino
acids [3]. Type III PI 4-kinases (PI4Ks) phosphorylate the
4′-hydroxyl of the inositol ring and are closely related to
PI3Ks [1].
mTOR belongs to the family of PI3K-related protein
kinases (PIKKs) that includes ataxia–telangiectasia mutated
(ATM), ATM- and Rad3-related (ATR), DNA-dependent
protein kinase (DNA-PK) and suppressor of morphogenesis
in genitalia-1 (SMG-1; reviewed in Refs. [1, 4]). These are
28
large proteins (~300–500 kDa) that contain a conserved
kinase catalytic domain together with other regions that can
include HEAT repeats and FAT and FATC domains. mTOR
also has an FRB (FKBP12/rapamycin-binding) domain that
binds the drug rapamycin in complex with its intracellular
receptor protein FKBP12. ATM, ATR, DNA-PK, and
SMG-1 are involved in DNA and mRNA surveillance and
repair pathways. By contrast, mTOR integrates growth
factor-activated signals with permissive signals in the
presence of sufficient nutrients and energy to promote cell
growth, proliferation, and survival. mTOR signaling is
upregulated in many cancers and some benign growth or
proliferative disorders. Therefore, drugs that target mTOR
activity are expected to be useful therapies for these
conditions.
mTOR signal transduction and cellular functions
The discovery of mTOR and the understanding of its
biological functions were greatly facilitated by the use of
rapamycin, which inhibits some of the functions of mTOR.
Until recently, rapamycin sensitivity was the major criterion
used to identify mTOR-controlled events. However, it is
now known that mTOR binds to different regulatory
subunits to produce complexes with distinct signaling
functions and rapamycin sensitivity [5, 6]. The mTORC1
complex (containing mTOR, Raptor and mLST8) phos-
phorylates ribosomal protein S6 kinase (S6K) at Thr389
and the translation repressor 4EBP1 and is rapamycin
sensitive. Biological processes regulated by mTORC1
include translation, ribosome biogenesis, autophagy, glu-
cose metabolism, and the cellular response to hypoxia [7–
9]. The mTORC2 complex (containing mTOR, Rictor,
mLST8, and mSin1) phosphorylates the protein kinase Akt
at Ser473 and is insensitive to rapamycin. In comparison to
mTORC1, the biological function of mTORC2 is less clear.
However, available evidence suggests that this mTOR
complex controls cell survival and organization of the actin
cytoskeleton.
The physiological importance of the mTOR complexes
is underscored by genetic ablation of their molecular
components in mouse models. Mouse embryos lacking
mTOR die at E5.5–6.5 days [10, 11]. Ablation of Raptor to
disrupt mTORC1 is equally lethal at approximately
E6.5 days [12]. Mouse knockouts of Rictor or mSIN1
leading to disruption of mTORC2 are also embryonic lethal
[13, 14]. Not surprisingly, embryos lacking mLST8 also do
not survive [12]. Although Akt Ser473 phosphorylation
was blocked in cells isolated from Rictor−/− and mSIN1−/−
embryos, phosphorylation of several Akt substrates was not
inhibited, with the exception of FOXO transcription factors
[12, 13]. Part of the prosurvival function of Akt is to
J Chem Biol (2008) 1:27–36
phosphorylate and suppress the activity of FOXO proteins.
These results suggest that mTORC2 is necessary for Akt-
FOXO survival signaling. Additional studies using cells
derived from these animals might shed more light on the
specific cellular processes regulated by the distinct mTOR
complexes. These studies will also be facilitated if small
molecules that can specifically inhibit mTORC2 activity
become available.
A number of different signaling pathways regulate
mTORC1 activity, and the best characterized positive
effector is the growth factor/PI3K/Akt pathway (Fig. 1).
Akt plays an unusual role in mTOR signaling because it
acts upstream of mTORC1 and downstream of mTORC2.
Akt controls mTORC1 in part through tuberous sclerosis
complex (TSC) 2, a protein that has GTPase-activating
protein (GAP) activity toward Rheb, a small GTP-binding
protein related to Ras [15]. TSC2 forms a tight complex
with TSC1. The GAP activity of the TSC1/TSC2 hetero-
dimer converts Rheb to an inactive GDP-bound state and
thus suppresses mTOR activity [16]. In the presence of
growth factors that activate Akt, Akt phosphorylates TSC2
at Ser939 and Thr1462 and inhibits its GAP activity [17].
This allows Rheb to maintain an active GTP-bound form
that activates mTORC1 and increases signaling to S6K and
4EBP1.
An energy-sensing pathway also regulates mTOR activ-
ity. Inhibition of mTOR in response to low intracellular
energy levels is mediated by AMP-activated kinase
(AMPK) and its activator, the protein kinase LKB1 [18].
Under conditions in which intracellular ATP is depleted and
the level of AMP is increased, AMP binds to AMPK and
allows LKB1 to phosphorylate Thr172 on the catalytic α
subunit to activate AMPK [19]. AMPK then phosphor-
ylates TSC2 on Ser1345, which primes TSC2 for subse-
quent phosphorylation of Ser1341 and Ser1337 by
glycogen synthase kinase 3 [20]. Together, these modifica-
tions are thought to enhance the GAP activity of TSC2,
inactivate Rheb, and turn off mTORC1 signaling (Fig. 1).
Hypoxia and low amino acid levels also negatively
regulate mTOR activity. Signaling pathways that mediate
the mTOR response to these stimuli are less well
characterized. The class III PI3K, hVps34, seems to play
an important role in transducing the amino acid signal to
mTOR [3, 21]. Evidence indicates that the hypoxia signal is
mediated by a pathway from REDD1 (regulated in
development and DNA damage responses) to TSC1/2 and
then to mTOR [22].
Recent studies indicate that feedback from the
mTORC1/S6K pathway inhibits growth factor signaling to
PI3K (Fig. 1). TSC1−/− or TSC2−/− cells have abnormally
low Akt activity due to hyperactivation of mTORC1/S6K
[23–25]. Conversely, S6K1−/− cells are hypersensitive to
insulin activation of PI3K signaling [26, 27]. Treatment of
J Chem Biol (2008) 1:27–36
Fig. 1 mTOR signaling. Growth factor activation of PI3K causes
increased production of PI 3,4,5-trisphosphate (PI(3,4,5)P3). Akt is
activated upon binding to PI(3,4,5)P3 and phosphorylation of Thr308
by phosphoinositide-dependent protein kinase 1 (PDK1) and Ser473
by mTORC2. The TSC1/TSC2 complex inhibits mTORC1 by
stimulating the GTPase activity of Rheb, converting it to the inactive
GDP-bound state. Akt phosphorylation of TSC2 inhibits the TSC1/
TSC2 complex and allows Rheb to remain in the active GTP-bound
form. Phosphorylation by AMPK activates TSC2 and opposes the Akt
signal. In energy-poor conditions, increased AMP levels allow LKB1
to phosphorylate and activate AMPK. Decreased signaling through
Vps34 in the presence of low amino acid levels to inhibit mTOR
might be independent of TSC1/TSC2 and Rheb. mTOR is inhibited
some cancer cells with rapamycin upregulates Akt, which
can enhance survival under conditions that usually induce
apoptosis [23, 28–30]. As a result, there is concern that
reactivation of Akt in tumors following rapamycin treat-
ment could lead to resistance to other chemotherapeutic
agents. It has been proposed that small molecule inhibitors
that target the kinase domain of mTOR will display broader
antitumor activity than rapamycin because they should not
reactivate Akt [7, 31, 32]. Theoretically, a combination of
rapamycin plus an inhibitor of Akt or PI3K would have the
same effect.
Rapamycin and rapalogs
Rapamycin (sirolimus) is a macrocyclic antibiotic produced
by the bacterium Streptomyces hygroscopicus found in the
soil of Easter Island. Rapamycin was discovered as a potent
antifungal agent, but it also exhibited what was at first
29
during hypoxia due to activation of TSC1/TSC2 by REDD1. Rheb-
GTP binds to and activates mTORC1. mTORC1 phosphorylates the
translation repressor 4EBP1 and S6K at Thr389, leading to its
activation. Inhibitors that target the mTOR kinase domain block both
mTORC1 and mTORC2, whereas rapamycin only inhibits mTORC1.
Upregulation of S6K in cells with hyperactive mTORC1 signaling has
a feedback inhibitory effect on Akt. Therefore, treatment with
rapamycin leading to inhibition of S6K can upregulate Akt and
enhance cell survival. In contrast, mTOR kinase inhibitors block both
S6K and Akt activation and should not enhance cell survival. It is not
clear how TSC1/TSC2 and Rheb regulate the activity of mTORC2
[24]
considered to be an undesirable immunosuppressive effect,
which subsequently led to its development as a clinically
useful drug. The immunosuppressant FK506 (tacrolimus)
and rapamycin have similar chemical structures and bind to
the same intracellular receptor, FKBP12. Even though
FK506 and rapamycin bind to the same protein, they have
different mechanisms of action in cells. FK506 inhibits
T cell proliferation by blocking the Ca2+/calcineurin-
dependent transcriptional activation of genes required for
growth, whereas rapamycin interferes with growth-
promoting cytokine signaling [33]. Interleukin-2 (IL-2)
induced S6K activation in a T cell line was extremely
sensitive to rapamycin inhibition (IC50 = 0.05 nM) [34]. By
contrast, mTORC1 kinase activity in vitro was much less
sensitive to FKBP12/rapamycin [6]. The reason for this
apparent difference in mTORC1 sensitivity to FKBP12/
rapamycin inhibition in vivo and in vitro is not understood.
In addition to rapamycin, three rapamycin analogs
(rapalogs) are now in use in humans (Fig. 2). CCI779
30
Fig. 2 Chemical structure of rapamycin, rapalogs and mTOR kinase inhibitors. Rapalogs have the indicated O-substitutions at the C-40 position
of rapamycin (underline)
(temsirolimus) is a dihydroxymethyl propionic acid ester
prodrug of rapamycin. This modification makes the
compound water soluble and thus it can be administered
intravenously. Upon injection, CCI779 is rapidly converted
to rapamycin, which is probably responsible for most, if not
all, of its pharmacological effects. RAD001 (everolimus)
has a O-(2-hydroxyethyl) chain substitution at position C-
40 and AP23573 (deforolimus) has a phosphine oxide
substitution at the same position of the lactone ring of
rapamycin. Rapalogs have the same mechanism of action as
rapamycin. They bind to FKBP12 and interfere with the
FRB domain of mTOR.
Unlike kinase inhibitors that target the catalytic ATP-
binding site, these compounds are highly specific for
mTORC1. However, the exact mechanism of how the
interaction with the FRB domain leads to inhibition of
mTORC1 remains unclear. FKBP12/rapamycin inhibits
mTOR autophosphorylation and phosphorylation of
4EBP1 in vitro, suggesting that changes in the FRB domain
might exert an allosteric influence on the catalytic domain
[35]. It was initially thought that rapamycin interacts with
the FRB domain only after binding to FKBP12. The X-ray
crystal structure of the FKBP12-rapamycin-FRB complex
shows that rapamycin occupies two different hydrophobic
binding pockets in FKBP12 and the FRB domain simulta-
neously and brings the two proteins close together, but
there are relatively few interactions between the proteins
[36]. Subsequently, rapamycin was shown to bind to the
FRB domain without FKBP12, although with a lower
J Chem Biol (2008) 1:27–36
affinity [37]. Using this information and a solution NMR
structure of the FRB domain, Leone et al. virtually screened
a chemical library and discovered additional small mole-
cules that bind to the FRB domain in the absence of
FKBP12 [38]. However, these molecules were generally
ineffective at inhibiting mTOR kinase activity [38].
Another group used high-throughput screening to identify
4-[6-{[(1S,2R)-2-(benzyloxy)cyclopentyl]acetyl}-4-(2-
thienyl)pyridin-2-yl]-4-oxobutanoic acid (HTS-1) that
binds to the FRB domain in the low micromolar range
[39]. Unfortunately, they did not show if the compound
inhibits mTOR kinase activity in vitro. Interestingly, these
investigators also found that phosphatidic acid, which
activates mTOR [40], interacts with the FRB domain in
the same binding pocket as rapamycin. It remains unclear
why binding of phosphatidic acid activates mTOR whereas
rapamycin binding has the opposite effect. Interestingly,
mTOR with a mutation in the FRB domain that renders it
rapamycin resistant exhibits decreased catalytic activity
[41], supporting the hypothesis that this domain modulates
the catalytic domain. Future efforts may identify novel
compounds that bind to this area and potently inhibit
mTOR.
Rapamycin has also been proposed to inhibit mTOR by
destabilizing the mTOR-Raptor complex [42]. Interestingly,
mTOR has been shown to be inhibited by S-trans,trans-
farnesyl thiosalicylic acid (FTS), a compound that resem-
bles farnesylcysteine found in Ras family members and
other proteins. FTS dislodges farnesylated Ras proteins
J Chem Biol (2008) 1:27–36
from cell membranes by competing for their membrane
binding sites, thus facilitating their degradation [43]. FTS
inhibits mTORC1 activity in intact cells and cell extracts by
promoting the dissociation of Raptor [44]. It should be
noted that although rapamycin does not exert acute effects
on mTORC2, long-term treatment of some cell types with
the drug has been shown to prevent mTORC2 assembly,
thus inhibiting Akt [45]. This mechanism was proposed to
account for hyperlipidemia that is commonly seen in
patients treated with the drug and may also account for
hyperglycemia, which is another common side-effect.
A recent study provided evidence that the intracellular
protein FKBP38 is an endogenous inhibitor of mTORC1 [46].
FKBP38 bound to the FRB domain of mTOR and inhibited
its activity in vitro, similar to FKPB12/rapamycin. Further-
more, the FKBP12/rapamycin complex competed with
FKBP38 for binding to mTOR in vitro, and cells treated
with rapamycin had reduced association between FKBP38
and mTOR [46]. Upon growth factor stimulation, Rheb–
GTP bound strongly to FKBP38 and displaced it from
mTOR, thus activating the enzyme. These data raise the
possibility that the FKBP12/rapamycin complex might
inhibit mTOR because it cannot be displaced by GTP–Rheb.
Clinical uses of rapamycin and rapalogs
Rapamycin is an oral drug and its bioavailability is low.
RAD001 is also an oral compound and an often-cited
rationale for its development is that it has improved
bioavailability. However, in a clinical pharmacokinetic
study, RAD001 was found to also have a relatively low
bioavailability (~15%) [47]. Rapamycin and RAD001 are
both approved for use as immunosuppressants for organ
transplant patients. Due to their pharmacokinetic properties,
drug level monitoring of these drugs is necessary and they
require daily dosing. Rapamycin inhibits IL-2-induced T
cell proliferation [34] that is due at least in part to a specific
down-regulation of ribosomal protein mRNA translation
[48]. It is believed that the immunosuppressive effect of
RAD001 is caused by a similar mechanism of action. Post-
transplant malignancy is a serious complication of solid
organ transplantation. In contrast with calcineurin inhibitors
such as FK506, the use of mTOR inhibitors as immuno-
suppressants has the added benefit of being antitumori-
genic. Indeed, rapamycin is now the established treatment
for post-transplant Kaposi’s sarcoma in patients who were
on other immunosuppressive regimens [49].
CCI779 and AP23573 can be given intravenously and
are being used as anticancer drugs. CCI779 was recently
approved by the U.S. Food and Drug Administration (FDA)
for treatment of renal cell carcinoma. AP23573 is in clinical
trials for treating a variety of cancers, including sarcomas.
31
RAD001 and rapamycin are also being tested in clinical
studies for treatment of both hematologic and solid
malignancies. Aside from a possible direct antiproliferative
effect on tumor cells, experiments in a mouse model of
metastatic cancer suggest that rapamycin limits the growth
of solid tumors by blocking angiogenesis [50]. Intravenous
CCI779 and AP23573 are administered on intermittent
weekly schedules. Given in this fashion, these drugs do not
appear to cause immunosuppression. This is surprising
because rapalogs have long half-lives (RAD001, 24–35 h;
CCI779, 13–25 h; AP23573, 45–74 h). In a rat model, a
single dose of RAD001 suppressed mTOR signaling
(measured by S6K activity and 4EBP1 Thr70 phosphory-
lation) in peripheral blood mononuclear cells for longer
than 72 h [51]. A possible explanation for this discrepancy
is that the immunosuppressive effect of rapamycin is
mediated by other effectors that are not inhibited by the
drug concentrations achieved during these treatments. S6K
activity in blood cells or tissue might not be a good
indicator of pharmacodynamic effectiveness because this
enzyme is exquisitely sensitive to rapamycin inhibition.
Rapamycin-eluting coronary artery stents were approved
for use by the U.S. FDA in 2003. Rapamycin inhibits
vascular smooth muscle cell migration and proliferation and
attenuates reocclusion of coronary arteries following an-
gioplasty. In a randomized, double-blind trial with 1058
patients, patients treated with rapamycin-eluting stents had
a restenosis rate of 8.6% vs. 21% in patients treated with a
standard non-drug-eluting stent [52]. It is conceivable that
the antiproliferative effect of mTOR inhibitors might be
utilized to treat other non-malignant proliferative disorders
such as polycystic kidney disease [53].
mTOR kinase inhibitors
A small molecule designed to compete with ATP in the
catalytic site of mTOR would be expected to inhibit all of
the kinase-dependent functions of mTORC1 and mTORC2,
unlike rapalogs that only target mTORC1. Most if not all of
the non-rapalog mTOR inhibitors described to date in the
scientific literature were developed to inhibit other
enzymes, especially class I PI3Ks. Because PI3K regulates
mTOR activity (see Fig. 1), inhibitors that target both
enzymes are generally not useful as research tools to study
mTOR regulation or function. However, drugs that are dual
PI3K/mTOR inhibitors might have a therapeutic advantage
over single-target inhibitors in certain disease settings.
Wortmannin (Fig. 2) is a toxic steroidal furan that is
produced by various fungi including Penicillium wortmanni
[54]. The first kinase shown to be targeted by wortmannin
was smooth muscle myosin light chain kinase (IC50 =
0.17 μM). Inhibition was irreversible and the enzyme was
32
partially protected by incubation in the presence of ATP
[55]. Later studies exploring wortmannin inhibition of
agonist-induced responses in neutrophils and basophils led
to the discovery that the compound is a potent inhibitor of
class I PI3Ks (IC50 ~1–3 nM) [56, 57]. Biochemical
analyses and the X-ray crystallographic structure of
wortmannin bound to p110γ showed that the mechanism
of inhibition is two-fold. First, wortmannin binds with high
affinity to the ATP-binding site to block substrate binding.
Second, the ε-amino group of a lysine in the active site
forms a covalent bond with carbon-20 of the wortmannin
furan ring to irreversibly inhibit the enzyme [58, 59]. The
active site lysine (Lys802 in p110α and Lys833 in p110γ)
is critical for catalysis and is highly conserved in lipid and
protein kinases. Wortmannin also targets class II PI3K C2β
(IC50 =1.6 nM), the human class III PI3K hVps34p (IC50 =
2.5 nM), type III PI4Ks (IC50 =50–300 nM) and polo-like
protein kinases that regulate mitosis (IC50 =24–48 nM) [60–
64]. Not surprisingly, considering the similarity between the
PI3K and PIKK catalytic domains, wortmannin also
irreversibly inhibits PIKK family members. DNA-PK is
the most sensitive (IC50 =16 nM), followed by SMG-1
(IC50 ∼ 60 nM), ATM (IC50 ~100–150 nM), mTOR (IC50 ~
200 nM) and ATR (IC50 =1.8 μM) [65–68]. Wortmannin
forms a covalent bond with mTOR, presumably at Lys2187
in the ATP-binding site [65]. Based on its potency and
selectivity, wortmannin at 100 nM has been recommended
for use in cells to assess the role of PI3K [69], although
effects on mTOR at this concentration should be consid-
ered. The use of wortmannin as a therapeutic agent is
limited by its toxicity and instability in biological solutions.
The wortmannin derivative PX-866 is more stable and less
toxic than the parent compound and exhibits antitumor
activity in mice, but it was reported to be inactive against
mTOR at concentrations up to 10 μM [70].
Theophylline and caffeine are naturally occurring meth-
ylxanthines that have pleiotropic effects on the nervous,
respiratory, cardiovascular, and renal systems. Caffeine is
the world’s most widely consumed stimulant and reaches
plasma concentrations of ~50 μM in moderate coffee
drinkers. Plasma levels above 200 μM are toxic in humans.
Caffeine is used medically to treat apnea of prematurity and
is a component of various headache and pain remedies [71].
Theophylline causes bronchodilator and antiinflammatory
responses and has long been used clinically for the
treatment of asthma and other respiratory diseases. The
therapeutic serum levels range from 55–111 μM, while
concentrations >111 μM are considered to be toxic [72].
Many biochemical actions of methylxanthines have been
identified, including antagonism of adenosine receptors,
inhibition of cyclic nucleotide phosphodiesterases, and
increased Ca2+ release from the sarcoplasmic reticulum.
These compounds also inhibit mTOR and related kinases,
J Chem Biol (2008) 1:27–36
most likely by acting as low affinity ATP analogs.
Theophylline (5 mM) strongly inhibits mTOR kinase
activity in vitro and blocked insulin activation of Akt in
3T3-L1 adipocytes [73]. However, since theophylline and
caffeine also target class I PI3Ks with IC50 values ranging
from 75 μM to 1 mM [74], effects on Akt cannot be
attributed solely to mTOR inhibition. Caffeine has been
used extensively in cell-based experiments to investigate
cell cycle checkpoints in G1/S and G2/M that are induced
by DNA damage. These studies led to the discovery that
caffeine inhibits ATM (IC50 ~0.2 mM ), ATR (IC50 ~
1.1 mM), SMG-1 (IC50 =0.3 mM), and mTOR (IC50 ~0.2–
0.4 mM ). There is disagreement about whether DNA-PK is
also inhibited by caffeine [41, 68, 75–77]. Interestingly,
TORC1 signaling is an important target for caffeine in
yeast, and several caffeine-resistant mutants of the TOR1
protein have been identified [78]. These proteins contain
two mutations: one in the kinase domain that probably
impairs binding to both caffeine and ATP, resulting in
greatly reduced kinase activity, and a second mutation in
the FRB that increases the kinase activity. The double
mutants exhibit strong caffeine resistance and kinase
activity that is high enough to support TORC1 function in
vivo. By contrast, mutations in the FRB that cause
rapamycin resistance decrease TOR1 kinase activity, again
highlighting the idea that this domain regulates TOR1
activity in multiple ways. Caffeine and theophylline also
block the activity of class II PI3K C2α (IC50 ~0.4 mM) [74].
The field of PI3K signaling was revolutionized by the
introduction of LY294002 (2-(4-morpholinyl)-8-phenyl-
4H-1-benzopyran-4-one) (Fig. 2), a synthetic PI3K inhib-
itor whose structure is based on the naturally occurring
flavonoid quercetin [79]. LY294002 behaves as a compet-
itive inhibitor for the ATP-binding site and is much less
potent against class I PI3Ks than wortmannin (IC50 ~0.3–
4 μM) [80]. However, its superior chemical stability in
solution has led to its widespread use as a PI3K inhibitor in
cell-based experiments, where it is usually used at concen-
trations of 10–50 μM. Despite its frequent description as a
“specific” PI3K inhibitor, LY294004 blocks the activity of
a number of different protein kinases unrelated to PI3K
[81]. In addition, use of immobilized LY294002 showed
that the drug binds tightly to numerous non-kinase proteins
with diverse functions, with as yet unknown biological
consequences [80]. Class II PI3Ks (IC50 =6.9–19 μM) and
PI4Ks are relatively resistant to LY294002 as compared
with the class I enzymes [60, 61]. By contrast, the PIKK
family members SMG-1, DNA-PK, and mTOR are targeted
at low micromolar concentrations that also inhibit class I
PI3Ks [41, 65, 82, 83]. The X-ray crystallographic structure
of p110γ bound to LY294002 shows that the morpholino
oxygen forms a hydrogen bond with the backbone amide of
Val882 in the pocket that can be occupied by adenine in
J Chem Biol (2008) 1:27–36
ATP [58]. Substitution of this oxygen with nitrogen yields a
compound (LY303511) that is essentially inactive against
PI3K [79]. Treatment of cells with LY303511 inhibited
agonist-induced S6K Thr389 phosphorylation and auto-
phosphorylation of mTOR at Ser2481, suggesting that
LY303511 might be an mTOR inhibitor. However, effects
on mTOR activity were not directly tested using in vitro
assays, so whether or not LY303511 is a bona fide mTOR
inhibitor remains an open question [84].
Since the advent of LY294002, there has been an
explosion of interest in the synthesis of small molecule
PI3K inhibitors that show isoform selectivity and improved
potency and specificity [1]. Unfortunately, so far there have
been no reports of an analogous effort to systematically
produce mTOR inhibitors based on a structural or func-
tional understanding of its catalytic site. However, some of
the new compounds developed as PI3K inhibitors have also
been evaluated for activity against mTOR. Knight et al.
[85] determined the specificity profile of PI3K inhibitors
from nine different chemical classes by measuring IC50
values in vitro against 55 purified kinases, including 15
PI3K family members. Six compounds were reported to
inhibit mTORC1 activity with IC50s≤10 μM. Interestingly,
two of the compounds were less potent against mTORC2
than mTORC1, suggesting that the catalytic site of the
common mTOR subunit might be subtly altered by its
association with the distinct binding partners in the two
complexes. The most potent mTOR inhibitor in this group
was PI-103 (Fig. 2; IC50 =0.02 μM and 0.083 μM against
mTORC1 and mTORC2, respectively). A model of the
three-dimensional structure of PI-103 bound to the active site
of p110γ showed that the inhibitor forms a hydrogen bond
with Val882 in the “adenine pocket” and stretches deep into
an “affinity pocket” not accessed by ATP that contains the
side chain of Ile879 [85]. The residues in mTOR at
equivalent positions are Val2240 and Ile2237, respectively,
suggesting that binding of PI-103 to the catalytic site of
mTOR involves similar interactions in this region.
Although PI-103 represents the first potent synthetic
inhibitor of mTOR, it is essentially equipotent against class
I PI3Ks (IC50 = 0.008–0.15 μM). Although this dual
specificity limits the use of PI-103 as a probe to study
mTOR function, PI-103 has proved to be useful as an
experimental compound in cancer research. PI-103 was
found to be the most active compound in a group of ten
isoform-selective PI3K inhibitors that were evaluated for
the ability to block the proliferation of glioma cells in vitro
[86]. The cytostatic property of the compound was
attributed to its ability to inhibit both p110α and mTOR.
PI-103 treatment of mice also reduced the size of
established tumor xenografts at doses that produced no
observable toxicity [86]. These results suggest that dual-
specificity PI3K/mTOR inhibitors, or use of a PI3K
33
inhibitor in combination with rapamycin, might be a viable
therapeutic option for the treatment of certain cancers.
Programs to develop potent and specific inhibitors of
DNA-PK have also yielded some compounds that inhibit
mTOR [83, 87]. In general, these compounds inhibit DNA-
PK (IC50s in the sub-millimolar range) and p110α and
mTOR (low millimolar IC50s), but not ATM or ATR. One
exception to this rule is 2-(morpholin-1-yl)pyrimido[2,1-α]
isoquinolin-4-one (compound 13 in Ref. [83]), which does
not inhibit PI3K. We used this compound (named 401 in
Ref. [88]) to examine the cellular effect of mTOR inhibition
without complicating side effects on PI3K. The chemical
structure of 401 is shown Fig. 2. First we performed in vitro
kinase assays to confirm that 401 is selective for mTOR
over p110α and p110β. The specificity profile was further
widened by assaying 40 different protein kinases in the
presence of 5 μM 401. As expected for an mTOR inhibitor,
treatment of cells with 401 blocked growth factor-induced
phosphorylation of S6K Thr389 (an mTORC1 site) and Akt
Ser473 (an mTORC2 site). By contrast, phosphorylation of
Erk was not affected. These effects on cell signaling were
not due to inhibition of DNA-PK, as incubation of cells that
lack DNA-PK with 401 also decreased S6K and Akt
phosphorylation. Finally, we examined the effect of 401 on
the growth and survival of mouse embryo fibroblasts
(MEFs) deficient for TSC1. These cells have abnormally
high mTORC1/S6K signaling and abnormally low Akt
activity due to feedback inhibition from the hyperactivated
mTORC1/S6K pathway. Long term treatment of these cells
with rapamycin turns off the negative pathway and
upregulates Akt, which can provide a survival signal [23].
By contrast, we found that Akt phosphorylation remained
low in TSC1−/− MEFs cultured in the presence of 401.
Furthermore, 401-treated cells exhibited strong growth
inhibition and increased apoptosis as compared with MEFs
treated with rapamycin [88]. Treatment of TSC1−/− MEFs
with an Akt inhibitor also increased apoptosis, suggesting
that the cytotoxic effect of 401 might be due to suppression
of mTORC2/Akt signaling. These results suggest that
inhibition of mTOR kinase activity by a small molecule
inhibitor such as 401 might be more effective than
rapamycin at killing cancer cells that exhibit hyperactivated
mTOR signaling.
Conclusions and outlook
Upregulation of the PI3K/mTOR/Akt pathway is a common
feature of many proliferative disorders, including cancer.
Up to now, rapamycin and rapalogs are the most well
studied mTOR inhibitors and they are now clinically used
as cancer treatments. However, the possibility that inhibi-
tion of mTORC1 with these drugs might lead to Akt
34
upregulation and outgrowth of more aggressive lesions is a
concern. Use of rapamycin plus an inhibitor of Akt or PI3K
is one way to circumvent this problem, and another is to use
a dual-specificity agent such as PI-103 that targets both
PI3K and mTOR. Another strategy is to develop drugs that
selectively target the mTOR kinase domain, which should
inhibit both mTORC1 and mTORC2. A treatment combin-
ing an mTORC2 inhibitor plus rapalogs would have a
similar effect. One caveat regarding these strategies is that
each drug class is expected to cause a distinct spectrum of
toxicities. For example, mTORC2 inhibition might be less
toxic than PI3K or Akt inhibitors because it would affect
mainly FOXO signaling and not other downstream effectors
of Akt.
Considerable progress has been made in the last several
years toward elucidating the structure and regulation of the
two mTOR complexes, and it is now possible to assay each
one in vitro. Determination of the three-dimensional struc-
ture of the mTOR kinase catalytic domain would be a major
breakthrough that would aid in the design and analysis of
new inhibitors. Development of drugs specific for the mTOR
kinase domain or that disrupt the mTORC2 complex should
narrow the gaps in our knowledge about the importance of
mTORC1 and mTORC2 in health and disease.
Acknowledgments The authors’ research was supported by Carol
M. Baldwin Breast Cancer Research Awards, and grants from the
Department of Veterans Affairs and the National Institutes of Health
DK62722. We greatly appreciate the expert graphical assistance of Jun
Yong Choi.
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