A novel inhibitor of the PI3K/Akt pathway based on the structure of inositol 1,3,4,5,6-pentakisphosphate.

Abstract

Owing to its role in cancer, the phosphoinositide 3-kinase (PI3K)/Akt pathway is an attractive target for therapeutic intervention. We previously reported that the inhibition of Akt by inositol 1,3,4,5,6-pentakisphosphate (InsP(5)) results in anti-tumour properties. To further develop this compound we modified its structure to obtain more potent inhibitors of the PI3K/Akt pathway.
Cell proliferation/survival was determined by cell counting, sulphorhodamine or acridine orange/ethidium bromide assay; Akt activation was determined by western blot analysis. In vivo effect of compounds was tested on PC3 xenografts, whereas in vitro activity on kinases was determined by SelectScreen Kinase Profiling Service.
The derivative 2-O-benzyl-myo-inositol 1,3,4,5,6-pentakisphosphate (2-O-Bn-InsP(5)) is active towards cancer types resistant to InsP(5) in vitro and in vivo. 2-O-Bn-InsP(5) possesses higher pro-apoptotic activity than InsP(5) in sensitive cells and enhances the effect of anti-cancer compounds. 2-O-Bn-InsP(5) specifically inhibits 3-phosphoinositide-dependent protein kinase 1 (PDK1) in vitro (IC(50) in the low nanomolar range) and the PDK1-dependent phosphorylation of Akt in cell lines and excised tumours. It is interesting to note that 2-O-Bn-InsP(5) also inhibits the mammalian target of rapamycin (mTOR) in vitro.
InsP(5) and 2-O-Bn-InsP(5) may represent lead compounds to develop novel inhibitors of the PI3K/Akt pathway (including potential dual PDK1/mTOR inhibitors) and novel potential anti-cancer drugs.

Full text

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A novel inhibitor of the PI3K/Akt pathway based on the structure
of inositol 1,3,4,5,6-pentakisphosphate
M Falasca*,1, D Chiozzotto1, HY Godage2, M Mazzoletti3, AM Riley2, S Previdi3, BVL Potter2,4, M Broggini3,4and
T Maffucci1
1Queen Mary University of London, Barts and The London School of Medicine and Dentistry, Blizard Institute of Cell and Molecular Science, Centre for
Diabetes, Inositide Signalling Group, 4 Newark Street, London E1 2AT, UK;2Wolfson Laboratory of Medicinal Chemistry, Department of Pharmacy and
Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY, UK;3Laboratory of Molecular Pharmacology, Department of Oncology, Istituto di
Ricerche Farmacologiche Mario Negri, Via La Masa 19, Milan 20156, Italy
BACKGROUND: Owing to its role in cancer, the phosphoinositide 3-kinase (PI3K)/Akt pathway is an attractive target for therapeutic
intervention. We previously reported that the inhibition of Akt by inositol 1,3,4,5,6-pentakisphosphate (InsP5) results in anti-tumour
properties. To further develop this compound we modified its structure to obtain more potent inhibitors of the PI3K/Akt pathway.
METHODS: Cell proliferation/survival was determined by cell counting, sulphorhodamine or acridine orange/ethidium bromide assay;
Akt activation was determined by western blot analysis. In vivo effect of compounds was tested on PC3 xenografts, whereas in vitro
activity on kinases was determined by SelectScreen Kinase Profiling Service.
RESULTS:The derivative 2-O-benzyl-myo-inositol 1,3,4,5,6-pentakisphosphate (2-O-Bn-InsP5) is active towards cancer types resistant to
InsP5in vitro and in vivo. 2-O-Bn-InsP5possesses higher pro-apoptotic activity than InsP5in sensitive cells and enhances the effect of
anti-cancer compounds. 2-O-Bn-InsP5specifically inhibits 3-phosphoinositide-dependent protein kinase 1 (PDK1) in vitro (IC50in the
low nanomolar range) and the PDK1-dependent phosphorylation of Akt in cell lines and excised tumours. It is interesting to note that
2-O-Bn-InsP5also inhibits the mammalian target of rapamycin (mTOR) in vitro.
CONCLUSIONS:InsP5and 2-O-Bn-InsP5may represent lead compounds to develop novel inhibitors of the PI3K/Akt pathway (including
potential dual PDK1/mTOR inhibitors) and novel potential anti-cancer drugs.
British Journal of Cancer (2010) 102, 104–114. doi:10.1038/sj.bjc.6605408
& 2010 Cancer Research UK
www.bjcancer.com
Keywords: phosphoinositide 3-kinase; inositol polyphosphates; protein kinase B-Akt; 3-phosphoinositide-dependent protein
kinase 1; apoptosis
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Phosphoinositide 3-kinase (PI3K) isoforms catalyse the phosphory-
lation of the 3-hydroxyl group within the inositol ring of
phosphoinositides generating lipid products, which in turn
mediate the activation of several proteins (Maffucci and Falasca,
2001; Vanhaesebroeck et al, 2001). The best characterised PI3K
effector is the Serine/Threonine kinase protein kinase B (PKB)/
Akt, which regulates a plethora of intracellular processes,
including cell survival, growth, proliferation, migration and
regulation of cell size (Vivanco and Sawyers, 2002; Manning and
Cantley, 2007). Upon PI3K activation, interaction between Akt
pleckstrin homology (PH) domain and the PI3K product
phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) recruits
Akt to the plasma membrane, where it is activated through
phosphorylation at its residues Thr308 and Ser473. Phosphory-
lation of Thr308 is mediated by 3-phosphoinositide-dependent
protein kinase 1 (PDK1), which itself possesses a PH domain able
to bind PtdIns(3,4,5)P3(Komander et al, 2004). Mutations in PDK1
PH domain that abolish PtdIns(3,4,5)P3binding strongly inhibit
Akt activation in homozygous knock-in embryonic stem cell and
knock-in mice (McManus et al, 2004; Bayascas et al, 2008). On the
other hand, binding of Akt PH domain to PtdIns(3,4,5)P3is critical
to induce a conformational change that allows PDK1-dependent
phosphorylation (Calleja et al, 2007). Among other targets, Akt
activates the multi-protein complex mTORC1 containing the
enzyme mammalian target of rapamycin (mTOR), which regulates
several intracellular functions including cell growth, cell cycle
progression and autophagy (Wullschleger et al, 2006). It is
interesting to note that a second mTOR-containing complex
(mTORC2) is involved in the phosphorylation of Akt at its
residue Ser473 as well as its activation (Sarbassov et al, 2005).
The mechanism of mTORC2-dependent Akt phosphorylation at
Ser473 is still not completely understood, but it does not seem to
involve phosphoinositides, as in the case of PDK1, since mTOR
does not appear to possess phosphoinositide-binding domains.
Deregulation of PI3K-dependent signalling pathways is linked to
the development of cancer (Maehama and Dixon, 1999; Shayesteh
et al, 1999; Vivanco and Sawyers, 2002; Bader et al, 2005; Shaw and
Cantley, 2006; Vogt et al, 2007) and to increased resistance to
treatment with chemotherapeutic agents (Clark et al, 2002; Liang
et al, 2003; She et al, 2003). Accumulation of PtdIns(3,4,5)P3either
Received 10 August 2009; revised 5 October 2009; accepted 8 October
2009
*Correspondence: Dr M Falasca; E-mail: m.falasca@qmul.ac.uk
4These authors contributed equally to this work.
British Journal of Cancer (2010) 102, 104–114
& 2010 Cancer Research UKAll rights reserved 0007– 0920/10$32.00
www.bjcancer.com
Translational Therapeutics

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due to gain of function of PI3K activity (Vivanco and Sawyers,
2002; Vogt et al, 2007) or loss of the enzyme phosphatase and
tensin homolog deleted on chromosome 10 (PTEN), which
specifically dephosphorylates PtdIns(3,4,5)P3
Dixon, 1999), has been detected in almost 50% of all tumour
types (Carracedo and Pandolfi, 2008). Reducing the levels of PDK1
in PTENþ/?mice strongly protects them from developing a wide
range of tumours (Bayascas et al, 2005). Furthermore, it has
recently been reported that PDK1 is overexpressed in several
human breast cancers and that increased copy number of the gene
encoding for PDK1 is associated with upstream pathway lesions
and patient survival (Maurer et al, 2009), highlighting the
importance of PDK1 in cancer development. Elevated Akt activity
has been found in several forms of cancer (Sun et al, 2001; Bacus
et al, 2002; Altomare et al, 2004) and evidence suggest that
mTORC1 is one of the key effectors in PI3K/Akt-mediated
tumourigenesis (Guertin and Sabatini, 2007). The crucial role of
mTORC2 in tumorigenesis driven by Pten loss has also been
reported (Guertin et al, 2009). The PI3K/Akt pathway is, therefore,
at present being considered to be an attractive target for
therapeutic intervention, and several compounds targeting the
different components of the pathway have been developed or are in
development (Vivanco and Sawyers, 2002; Luo et al, 2003;
Hennessy et al, 2005; Guertin and Sabatini, 2007; Liu et al,
2009), with some of them currently in clinical trials for cancer
treatment (Liu et al, 2009). Toxicity, low therapeutic index,
insolubility and aqueous instability have prevented the use of
generic PI3K inhibitors wortmannin and LY294002 as anti-cancer
agents despite their anti-tumour activity (Hu et al, 2000; Ng et al,
2001). More specific approaches are needed to selectively block
only the deregulated rather than all the PI3Ks-dependent path-
ways. Small molecule inhibitors of PDK1 have recently been
developed, most of which target the ATP-binding site of PDK1, but
they often possess poor physicochemical properties and inade-
quate selectivity profiles (Peifer and Alessi, 2008). Similarly several
Akt inhibitors have been designed including compounds targeting
its ATP-binding domain or allosteric inhibitors and pseudosub-
strates (Luo et al, 2005; Crowell et al, 2007; Lindsley et al, 2008).
However, some of these agents show toxic side effects either
because of non-specific effects or blockade of all Akt isoforms,
thus resulting in the alteration of normal glucose homeostasis.
The in vitro and in vivo effects on Akt of chemopreventive com-
pounds, such as the rotenoid deguelin have also been reported (Lee
et al, 2005). Finally, several mTOR inhibitors are at present
available, whose effects have been investigated in many solid
tumours (Guertin and Sabatini, 2007; Fasolo and Sessa, 2008).
Despite several efforts, there is still a need to develop novel, more
potent inhibitors of the PI3K/Akt pathway to overcome problems
of lack of specificity and chemoresistance.
A few years ago we were the first to propose an alternative
mechanism to block Akt activation based on the inhibition of its PH
domain-mediated translocation to the plasma membrane (Berrie and
Falasca, 2000). The critical role of the PH domain in Akt-driven
tumourigenesis has recently been highlighted by the detection of a
somatic mutation in Akt1 PH domain resulting in Akt1 activation in
breast, colorectal and ovarian cancers (Carpten et al, 2007). It is
interesting to note that this mutant is able to induce leukaemia in
mice (Carpten et al, 2007). Our strategy was based on the hypothesis
that specific exogenous inositol polyphosphates can compete with
PtdIns(3,4,5)P3by binding to Akt PH domain, and thus prevent
recruitment to the plasma membrane and activation of Akt (Berrie
and Falasca, 2000). Indeed we reported that inositol 1,3,4,5,6-
pentakisphosphate (InsP5) specifically blocks Akt activation and
possesses pro-apoptotic (Razzini et al, 2000; Piccolo et al, 2004),
anti-angiogenic and anti-tumour activity in vivo (Maffucci et al,
2005). In addition, some of us recently demonstrated the targeting of
the Akt PH domain with an unusual inositol polyphosphate mimic
(Mills et al, 2007). Other phosphatidylinositol-based Akt inhibitors
(Maehama and
also act by inhibiting Akt targeting to the plasma membrane,
including ether lipid analogues and PH domain-targeting inhibitors
(Kozikowski et al, 2003; Gills et al, 2006; Crowell et al, 2007) such as
perifosine, the most developed Akt inhibitor currently available
(Kondapaka et al, 2003).
In order to explore early structure-activity relationships for
InsP5and possibly obtain more potent and specific inhibitors of
the PI3K/Akt pathway, we synthesised novel compounds based on
the InsP5structure. Here we show that the derivative 2-O-benzyl-
myo-inositol 1,3,4,5,6-pentakisphosphate (2-O-Bn-InsP5) exhibits
more efficient and potent activity than InsP5not only in inhibiting
Akt phosphorylation but also in inducing apoptosis in different
human cancer cell lines. It is interesting to note that 2-O-Bn-InsP5
promotes apoptosis in cell lines normally resistant to treatment
with InsP5 and markedly inhibits the in vivo growth of
InsP5-resistant xenografts. Kinase profiling analysis reveals that
2-O-Bn-InsP5strongly inhibits PDK1 activity in vitro with an IC50
in the low nanomolar range. This is mirrored by the inhibition of
Akt phosphorylation at its residue Thr308 in 2-O-Bn-InsP5-treated
cells and in tumours from 2-O-Bn-InsP5-treated mice. Further-
more, the effect of 2-O-Bn-InsP5 is highly specific, as this
compound only inhibits PDK1 and to a lesser extent mTOR in a
panel of almost 60 kinases. These data represent the first attempt
to exploit InsP5as a potential lead compound for the development
of potent small molecule inhibitors of the PI3K/Akt pathway.
MATERIALS AND METHODS
Materials
Inositol 1,3,4,5,6-pentakisphosphate was synthesised as previously
reported (Godage et al, 2006). 2-O-Bn-InsP5was synthesised in a
similar manner from 2-O-benzyl-myo-inositol. Each compound
was purified to homogeneity by ion-exchange chromatography on
Q-Sepharose Fast Flow resin (GE Healthcare Life Sciences,
Little Chalfont, Buckinghamshire, UK) and used as the triethy-
lammonium salt, which was fully characterized by
spectroscopy and accurately quantified by total phosphate assay.
For the in vivo experiments, InsP5and 2-O-Bn-InsP5were each
converted into the hexasodium salt by treatment with Dowex
50WX2-100ion-exchangeresin
Dorset, UK), followed by addition of sodium hydroxide (6
equivalents) and lyophilisation. Sulphorhodamine (SRB), curcu-
min, paclitaxel and 4-hydroxy-tamoxifen were purchased from
Sigma-Aldrich; anti-phospho Ser473 Akt, anti-phospho Thr308
Akt and anti-Akt from (Cell Signaling Technologies, Danvers, MA,
USA) or Santa Cruz Biotechnology (Santa Cruz, CA, USA).
31P and
1H
(Sigma-Aldrich,Gillingham,
Cell lines
SKOV-3 and PC3 were cultured in RPMI 1640; all other cell lines
were cultured in DMEM. Media were supplemented with 10% FBS,
penicillin/streptomycin and glutamine.
Cell survival and apoptosis assays
Cells seeded in a 24-well plate were treated with the indicated
compounds in serum free DMEM or DMEM containing 0.5% FBS
(PC3). After 72h, the number of surviving cells was assessed by
manual cell counting or by using the cell counter CDA-500
(Sysmex, Milton Keynes, UK). Alternatively, after 48h, the number
of apoptotic cells was assessed by acridine orange/ethidium
bromide assay as described (Piccolo et al, 2004; Maffucci et al,
2005). SRB test was carried out in SKOV-3 and PC3 seeded in 96-
well plate (3800 cells per well or 5800 cells per well, respectively)
after 72h of treatment as described (Cappella et al, 2001).
A novel PDK1 inhibitor
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In vivo studies
Male nude athymic CD-1 nu/nu mice (8-weeks old) were obtained
from Harlan (San Pietro al Natisone, Italy) and maintained under
specific pathogen-free conditions with food and water provided
ad libitum. The general health status of the animals was monitored
daily. Procedure involving animals and their care were conducted
in conformity with the institutional guidelines that are in
compliance with national and international laws and policies.
Toxicity assay
Male nude CD-1 mice were treated with a single dose of
750mgkg?1InsP5 or 2-O-Bn-InsP5/mouse administered intra-
peritoneally (i.p.). Each group consisted of 2–3 mice. Body weight,
deaths and any other sign of toxicity and changes in behaviour
(such as motility, eating and drinking habits) were recorded.
Anti-tumour activity assay
Exponentially growing PC3 cells were harvested, washed twice and
resuspended in PBS at a concentration of 2.5?107cellsml?1. A
suspension of 5?106PC3 cells was injected subcutaneously (s.c.)
into the left flank of the recipient mice. When tumours reached a
size of B70mm3(approximately 15 days after tumour cell
implant), mice were divided into seven groups (n¼7). InsP5and
2-O-Bn-InsP5were administered by daily i.p. injections at different
doses of 12.5–25–50mgkg?1day-1for 14 consecutive days.
Control mice were treated with water in an equal volume.
The diameters of s.c. growing tumours were measured with a
caliper twice a week and the experiment was ended at day 28 after
the implantation.
Data analysis and in vivo tumour parameters
The volume of s.c. growing tumours was calculated by the formula:
Tumour weight (mg)¼(length?width2)/2. Differences in s.c
tumour growth between the treatment groups were evaluated with
a one-way ANOVA followed by Fisher’s test using the StatView
statistical package (SAS Institute, Cary, NC, USA). The percentage
of tumour growth was calculated as T/C%¼(RTV-treated animals/
RTV-control animals)?100, where RTV was the mean relative
tumour volume calculated as RTV¼Vt/V0. Vtwas the tumour
volume on the day of measurement and V0was the tumour volume
at the beginning of the treatment. The percentage of tumour
weight inhibition (TWI%) was calculated using the formula:
TWI%¼100–T/C%. The log cell kill (LCK) was calculated using
the formula: LCK¼T?C/3.32?Td, where T?C is the tumour
growth delay calculated as the difference in median time (in days)
required for the tumours in the treatment (T) and control group
(C) to reach a predetermined size (i.e., 1000mg). Td is the tumour
volume doubling time in days, determined in the exponential
growth phase of the control group from a best-fit straight line.
Median doubling time was 3 days in control animals.
Western blot
Mice with s.c. growing tumours were treated with a single dose
of InsP5and 2-O-Bn-InsP5(50mgkg?1) or vehicle. Animals were
killed 24h after treatment and tumour samples were collected and
snap frozen. Frozen specimens of tumour tissue were homogenised
with a Polytron homogeniser in a lysis buffer (ratio 1:1 w/v)
containing 50mM Tris-HCl (pH 7.4), 5mM EDTA, 0.1% Nonidet
NP-40, 250mM NaCl, 50mM NaF and proteases and phosphatase
inhibitors. After centrifugation at 13000r.p.m. for 10min at 41C,
80mg of protein was separated on SDS–PAGE and transferred to a
polyvinylidene difluoride membrane (Millipore, MA, USA).
Membranes were probed with the indicated antibodies.
Protein kinase profiling
Effect of the indicated compounds on the activity of various
kinases was assessed by SelectScreen Kinase Profiling Service
(Invitrogen-Life Technologies, Paisley, UK). Assays were per-
formed using 1mM of the tested compounds and ATP concentra-
tion as indicated in the corresponding tables. In the case of InsP5a
screen using 10mM of the compound was also carried out, as
indicated in the corresponding table.
RESULTS
Synthesis of novel potential inhibitors of the PI3K/Akt
pathway and in vitro screening
We have recently reported that InsP5is a novel inhibitor of the
PI3K/Akt pathway, which possesses pro-apoptotic, anti-angiogenic
and anti-tumour activity (Razzini et al, 2000; Piccolo et al, 2004;
Maffucci et al, 2005). To explore the design of novel inhibitors of
the PI3K/Akt pathway, potentially more active than InsP5, we
decided to modify the structures of either Ins(1,3,4,5)P4or InsP5.
Different strategies were used to modify the parent molecules and
several compounds were synthesised and tested. For Ins(1,3,4,5)P4-
related compounds, modifications were made at C-6 because X-ray
structures of the Akt (Thomas et al, 2002) and PDK1 (Komander
et al, 2004) PH domains in complex with Ins(1,3,4,5)P4indicated
that the 6-hydroxyl group of Ins(1,3,4,5)P4is not directly involved
in binding. Furthermore, the X-ray structure of Akt PH domain
showed that a tyrosine residue near the 6-OH of bound
Ins(1,3,4,5)P4might interact with an aromatic group. In the case
of InsP5analogs, modifications were on either the 2-O-atom or the
5-phosphate, thus maintaining the symmetry of the parent
molecule. The derivatives were first tested for their ability to
inhibit Akt activation in cell lines characterised by constitutive
activation of the PI3K/Akt pathway and with a reported sensitivity
to InsP5, namely ovarian cancer cells SKOV-3 and breast cancer
cells SKBR3 (Piccolo et al, 2004; Maffucci et al, 2005). In this
original screening we observed that the InsP5 derivative 2-O-
benzyl-myo-inositol 1,3,4,5,6-pentakisphosphate
named 2-O-Bn-InsP5) showed the highest efficiency in inhibiting
Akt activation in all cell lines tested (data on the other derivatives
will be published elsewhere). More specifically, we found that 2-O-
Bn-InsP5inhibited Akt phosphorylation at its residue Ser473 more
efficiently than InsP5in SKOV-3, being already active after 8h of
treatment and at a concentration of 20mM (Figure 1B). Inhibition
of Akt phosphorylation at residue Thr308 was also detected
(Figure 1B). More interestingly, we found that 2-O-Bn-InsP5was
able to block Akt phosphorylation in cell lines resistant to InsP5,
such as prostate cancer cells PC3 (Figure 1C) and pancreatic
cancer cells ASPC1 (results not shown). Taken together these data
indicate that structural modification at the C-2 of InsP5 can
enhance its inhibitory properties towards Akt activation.
(Figure1A,
Analysis of the biological activity of 2-O-Bn-InsP5
We next compared the effects of 2-O-Bn-InsP5 and InsP5 on
proliferation/survival of cancer cells in vitro. Treatment with 2-O-
Bn-InsP5 strongly reduced the number of surviving SKBR3
(Figure 2A) and SKOV-3 (Figure 2B) assessed by cell counting.
In particular, 2-O-Bn-InsP5was more active than InsP5in both the
cell lines. Acridine orange/ethidium bromide assay confirmed that
the percentage of apoptotic cells was higher in 2-O-Bn-InsP5-
treated compared with InsP5-treated SKBR3 (Figure 2C) and
SKOV-3 (Figure 2D). Based on the data on Akt phosphorylation we
then decided to analyse the effect of 2-O-Bn-InsP5on the survival
of cell lines normally very resistant to InsP5treatment. 2-O-Bn-
InsP5was more potent than InsP5at a concentration of 50mM in
pancreatic cancer cells BxPc-3 (Figure 3A), whereas it was more
A novel PDK1 inhibitor
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active than InsP5at almost all concentrations tested in pancreatic
cancer cells ASPC1 (Figure 3B). A stronger activity of 2-O-Bn-InsP5
compared with InsP5 was also observed in breast cancer cells
MDA-MB-468 (Figure 3C) and in PC3 (Figure 3D), consistent with
data on Akt phosphorylation. Higher activity of 2-O-Bn-InsP5in
PC3 cells was also observed in SRB assays (Figure 3E). It is
important to note that although InsP5had no effect in PC3 at
concentrations up to 50mM, concentrations of 200–300mM were
eventually able to mimic the effect of 2-O-Bn-InsP5in PC3 cells
(Figure 3F), thus suggesting that 2-O-Bn-InsP5is acting on the
same intracellular pathway as InsP5. Taken together these data
demonstrate that addition of a benzyl group to the axial 2-O atom
of InsP5potentiates the pro-apoptotic properties of the compound
not only in cells sensitive to InsP5but also in cells normally very
resistant to treatment with the parent inositol compound.
In vivo anti-tumour activity of 2-O-Bn-InsP5on
InsP5-resistant xenografts
We then decided to test the therapeutic efficacy of 2-O-Bn-InsP5in
human tumour xenografts characterised by the activation of PI3K/
Akt pathway and higher sensitivity to 2-O-Bn-InsP5compared with
InsP5. We specifically implanted PC3 cells in nude mice and 15
days after the implantation we treated groups of mice with
different concentrations (12.5, 25 and 50mgkg?1) of InsP5 or
2-O-Bn-InsP5for 14 consecutive days (from day 15 to day 28).
Tumour growth was followed for further 12 days after the end of
the treatment (upto day 40). Data revealed that 2-O-Bn-InsP5at
doses of 12.5 and 25mgkg?1clearly decreased the growth of
tumours compared with untreated mice, although the differences
were statistically significant only on the last day of measurement
(Figure 4A and C). A strong reduction in tumour growth was
obtained in the group treated with 50mgkg?12-O-Bn-InsP5, with a
statistically significant difference vs controls detectable from day
22 after tumour cells implant onwards (Figure 4A and C). Data on
in vivo anti-tumour activity parameters relative to 2-O-Bn-InsP5
are shown in Figure 4C, bottom table. More than 50% inhibition of
tumour weight was achieved in the 50mgkg?1-treated group, with
a tumour growth delay (T?C) of almost 9 days between this group
and the untreated (control) group. In agreement with our in vitro
data, we observed that InsP5had no effect on concentrations up to
50mgkg?1(Figure 4B). At the end of the experiment, western blot
analysis revealed that 24h-treatment with 2-O-Bn-InsP5markedly
reduced Akt phosphorylation at its residue Ser473 in all 2-O-Bn-
InsP5-treated mice (Figure 4D). Furthermore, a clear inhibition of
Akt phosphorylation at its residue Thr308 was detected in five out
of seven 2-O-Bn-InsP5-treated mice (Figure 4D). It is noteworthy
that no evidence of toxicity was observed in different groups of
mice at all the tested doses of either 2-O-Bn-InsP5or InsP5and the
body weight of the treated animals was not different from the
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Figure 1
(A) Structure of inositol 1,3,4,5,6-pentakisphosphate (InsP5) and 2-O-benzyl-myo-inositol 1,3,4,5,6-pentakisphosphate (2-O-Bn-InsP5). (B, C) SKOV-3 were
treated for 8h or 24h with the indicated concentrations of InsP5or 2-O-Bn-InsP5in serum free medium, (B) while prostate cancer PC3 cells were treated
for 24h with the indicated concentrations of InsP5or 2-O-Bn-InsP5in medium containing 0.5% FBS (C). Akt activation was assessed by monitoring
phosphorylation at its residues Ser473 and Thr308. Membranes were then stripped and re-probed with the indicated antibodies.
In vitro activity of inositol 1,3,4,5,6-pentakisphosphate (InsP5) and 2-O-benzyl-myo-inositol 1,3,4,5,6-pentakisphosphate (2-O-Bn-InsP5).
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untreated mice throughout the entire experiment (Figure 4E).
Moreover, a single treatment with a very high dose of 2-O-Bn-InsP5
or InsP5 (750mgkg?1) did not cause any major toxic effect
(Figure 4F). Taken together these data demonstrate that 2-O-Bn-
InsP5is able to inhibit growth of InsP5-resistant tumours through a
more efficient blockade of Akt phosphorylation in vivo.
In vitro kinase profiling of InsP5and 2-O-Bn-InsP5
To determine the mechanism responsible for the higher activity of
2-O-Bn-InsP5, we decided to carry out a protein kinase activity
screen for InsP5and 2-O-Bn-InsP5(SelectScreen Kinase Profiling
Service, Invitrogen-Life Technologies). Among almost 60 protein
kinases screened, 2-O-Bn-InsP5 (1mM) showed a very high
inhibitory activity towards PDK1 (79% inhibition) and a lower
activity towards mTOR (Table 1A, Supplementary Table 1). 2-O-
Bn-InsP5did not inhibit (percentage of inhibition o40%) any of
all the other tested kinases, including AGC kinases, such as GSK3,
RSK, S6K and members of the PKC family, AMPK and several
members of the MAPK family (Supplementary Table 1). Further-
more 2-O-Bn-InsP5did not directly inhibit any of the class I PI3K
isoforms tested or any Akt isoforms (Table 1A, Supplementary
Table 1). InsP5 showed a reduced inhibitory effect on PDK1
compared with 2-O-Bn-InsP5 (Table 1B and C, Supplementary
Table 2 and 3). As 2-O-Bn-InsP5, when tested on a panel of over 50
kinases and at a concentration of 10mM, InsP5did not significantly
inhibit any of the tested kinases (Supplementary Table 3) including
Akt isoforms (Table 1B). In contrast to 2-O-Bn-InsP5, InsP5
did not inhibit mTOR, even when tested at a concentration of
10mM (Supplementary Table 3). Comparing the effect of 1 mM of
different natural inositol polyphosphates on PDK1, InsP5 pos-
sessed the highest inhibitory activity towards PDK1 (71%
inhibition) with only Ins(1,3,4,5)P4 also showing some effect
(56% inhibition). None of the other polyphosphates had any
significant effect (Table 1C). These data indicate that 2-O-Bn-InsP5
and InsP5 inhibit PDK1 very specifically, with 2-O-Bn-InsP5
possessing the highest inhibitory activity towards PDK1. Indeed
results from SelectScreen Kinase Profiling Service (Invitrogen-Life
Technologies) 10-point titration revealed that the IC50of InsP5
towards PDK1 was 613nM whereas the corresponding IC50of 2-O-
Bn-InsP5was a striking 26.5nM (Table 1A and B). These data
clearly indicate that 2-O-Bn-InsP5is a novel, potent and highly
selective PDK1 inhibitor. This is consistent with the detected
inhibition of Thr308 phosphorylation in 2-O-Bn-InsP5-treated
SKOV-3 and PC3 cells (Figure 1B and C) and 2-O-Bn-InsP5-treated
mice (Figure 4D). Furthermore, 2-O-Bn-InsP5, but not InsP5, is
able to inhibit mTOR selectively in vitro with an IC50of 1.3mM
(Table 1A).
In vitro effects of 2-O-Bn-InsP5in combination with
anti-cancer compounds
Parallel RNAi and compound screens have recently revealed that
PDK1 is a critical determinant of sensitivity to tamoxifen in breast
120
100
80
60
40
20
0
No. of cells (% control)
InsP5
2-O-Bn-InsP5
**
**
010 2050
Concentration (?M)
Concentration (?M)
01020 50
Concentration (?M) Concentration (?M)
**
**
InsP5
2-O-Bn-InsP5
InsP5
2-O-Bn-InsP5
**
30
25
20
15
10
5
0
01 2.55 102050
01 2.5510 20 50
InsP5
2-O-Bn-InsP5
120
100
80
60
40
20
0
No. of cells (% control)
% of apoptotic cells
30
25
20
15
10
5
0
% of apoptotic cells
Figure 2
sphate (InsP5). (A, B) SKBR3 (A) and SKOV-3 (B) were treated for 72h with the indicated concentrations of InsP5or 2-O-Bn-InsP5. The number of
surviving cells was assessed by cell counting. Data are mean±s.e. of n¼4 (A) and n¼2 (B) independent experiments. **¼Po0.05. (C, D) SKBR3 (C) and
SKOV-3 (D) were treated with the indicated concentrations of InsP5or 2-O-Bn-InsP5. The number of apoptotic cells was assessed by acridine orange/
ethidium bromide assay. Data are mean±s.e. of three independent experiments. **¼Po0.05.
2-O-benzyl-myo-inositol 1,3,4,5,6-pentakisphosphate (2-O-Bn-InsP5) possesses higher pro-apoptotic activity than inositol 1,3,4,5,6-pentakispho-
A novel PDK1 inhibitor
M Falasca et al
108
British Journal of Cancer (2010) 102(1), 104–114
& 2010 Cancer Research UK
Translational Therapeutics

Page 6

cancer cells MCF7 (Iorns et al, 2009). Based on this result we
decided to investigate whether inhibition of PDK1 by 2-O-Bn-InsP5
was able to sensitise MCF7 to the pro-apoptotic effect of
tamoxifen. Our data revealed that treatment with 4-OH tamoxifen
(the active metabolite of tamoxifen) for 72h reduced the number
of survivingcells, whereas 2-O-Bn-InsP5
(Figure 5A). It is interesting to note that the combination of
2-O-Bn-InsP5and 4-OH tamoxifen strongly enhanced the effect
of 4-OH tamoxifen or 2-O-Bn-InsP5alone (Figure 5A). We then
tested the effects of 2-O-Bn-InsP5 in combination with several
natural anti-cancer compounds. The concentrations of the different
compounds used in these experiments were minimally effective
had littleeffect
based on preliminary dose-response experiments (results not
shown). A combination of 2-O-Bn-InsP5and curcumin, a compo-
nent of turmeric (Curcuma longa), strongly reduced the number of
surviving PC3 cells, resulting in a more than additive effect
(Figure 5B) and was able to enhance the effect of curcumin in
ASPC1 (Figure 5C) and in MDA-MB-468 (Figure 5D). A combina-
tion of 2-O-Bn-InsP5and paclitaxel clearly reduced the number of
surviving MDA-MB-468 (Figure 5D), SKOV-3 (Figure 5E) and PC3
(Figure 5F) cells compared with the corresponding single treat-
ments. An additive effect was detected when combining 2-O-Bn-
InsP5with rapamycin in SKOV-3 (Figure 5E) and PC3 (Figure 5F)
cells. These data clearly indicate that the combination of 2-O-Bn-
05
Concentration (?M)
1020
50
051020 50
Concentration (?M)
0 1020 50
Concentration (?M)
05 10
20
50
Concentration (?M)
05
Concentration (?M)
102050
0
50
Concentration (?M)
100200150
300
*
**
**
**
*
120
100
80
60
40
20
0
No. of cells (% control)
120
100
80
60
40
20
0
No. of cells (% control)
120
100
80
60
40
20
0
No. of cells (% control)
120
100
80
60
40
20
0
No. of cells (% control)
120
100
80
60
40
20
0
No. of cells (% control)
**
*
120
100
80
60
40
20
0
No. of cells (% control)
InsP5
2-O-Bn-InsP5
InsP5
2-O-Bn-InsP5
InsP5
2-O-Bn-InsP5
InsP5
2-O-Bn-InsP5
InsP5
2-O-Bn-InsP5
Figure 3
pentakisphosphate (InsP5). (A–D) BxPc-3 (A), ASPC1 (B), MDA-MB-468 (C) and PC3 (D) were treated for 72h with the indicated concentrations of InsP5
or 2-O-Bn-InsP5. The number of surviving cells was assessed by cell counting. Data are mean±s.e. of n¼3 (A), n¼6 (B), n¼3 (C) and n¼4 (D)
independent experiments carried out in duplicate. *¼Po0.01; **¼Po0.05. (E, F) PC3 were treated with the indicated concentrations of InsP5and 2-O-
Bn-InsP5(E) or increasing concentrations of InsP5(F). After 72h the number of surviving cells was assessed by SRB assay. Data are mean±s.e. of n¼2
independent experiments.
2-O-benzyl-myo-inositol 1,3,4,5,6-pentakisphosphate (2-O-Bn-InsP5) possesses pro-apoptotic activity in cell lines resistant to inositol 1,3,4,5,6-
A novel PDK1 inhibitor
M Falasca et al
109
British Journal of Cancer (2010) 102(1), 104–114
& 2010 Cancer Research UK
Translational Therapeutics

Page 7

InsP5 and natural anti-cancer compounds results in additive or
more than additive effects, and therefore suggest that 2-O-Bn-InsP5
can potentially be used in combination with natural compounds to
increase their anti-cancer activity.
DISCUSSION
The in vivo anti-tumour activity of InsP5together with the lack of
toxicity observed using this compound (Maffucci et al, 2005),
40
35
30
25
20
15
10
5
0
Body weight (g)
40
35
30
25
20
15
10
5
0
Body weight (g)
Controls
2-O-Bn-InsP5 12.5 mg kg–1
2-O-Bn-InsP5 25 mg kg–1
2-O-Bn-InsP5 50 mg kg–1
Controls
InsP5 12.5 mg kg–1
InsP5 25 mg kg–1
InsP5 50 mg kg–1
1520
Days after cell implant
253035 4045
Days after treatment
0
510 15 2025
15 20
Days after cell implant
25303540
45
121234567
pThr308
Akt
Akt
Controls2-O-Bn-InsP5-treated
Controls
P-value 2-O-Bn-InsP5
vs controls
12.5
mg kg–1
mg kg–1
Days
15
19
22
26
29
36
40
NS
0.0325
2-O-Bn-InsP5
Dosage (mg kg–1)
Route
i.p.
i.p.
i.p.qdx15
qdx15
qdx15
ScheduleTWI%
19.5
22
52 8.7
3.3
3
T-C
(days)
LCK
0.14
0.15
0.40
12.5
25
50
0.0139 00135
0.0131
0.012
0.0035
0.0091
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
2550
mg kg–1
14 18 2226 3034 3842
Days after cell implant
Controls
InsP5 12.5 mg kg–1
InsP5 25 mg kg–1
InsP5 50 mg kg–1
1400
1200
1000
800
600
400
200
0
121234567
pSer473
Akt
Akt
2-O-Bn-InsP5-treated
2
1.6
1.2
0.8
0.4
Controls
2-O-Bn-InsP5
InsP5
0
Relative body weight
Tumour weight (mg)
1400
1200
1000
800
600
400
200
0
Tumour weight (mg)
Controls
2-O-Bn-InsP5 12.5 mg kg–1
2-O-Bn-InsP5 25 mg kg–1
2-O-Bn-InsP5 50 mg kg–1
141822 263034 3842
Days after cell implant
A novel PDK1 inhibitor
M Falasca et al
110
British Journal of Cancer (2010) 102(1), 104–114
& 2010 Cancer Research UK
Translational Therapeutics

Page 8

suggested that InsP5might represent a lead compound to design
novel inhibitors of the PI3K/Akt pathway to be eventually brought
into clinical testing. InsP5possesses very few sites for chemical
modification, the axial 2-hydroxyl group being the most realistic
possibility. Here we describe one InsP5derivative, 2-O-Bn-InsP5,
which possesses enhanced pro-apoptotic and anti-tumour activity
compared with the parent molecule. In this respect 2-O-Bn-InsP5
represents a first step towards the development of novel efficient
anti-cancer drugs targeting the PI3K/Akt pathway and based on
the InsP5structure. Kinase profiling assays revealed that 2-O-Bn-
InsP5 potently and specifically inhibits PDK1 in vitro and the
PDK1-dependent phosphorylation of Thr308 Akt in cell lines and
in vivo. These results are particularly important considering that,
to our knowledge, no specific and selective PDK1 inhibitors are at
present available and they make 2-O-Bn-InsP5an interesting new
molecule to use as a model for designing novel specific PDK1
inhibitors. In addition 2-O-Bn-InsP5is able to inhibit mTOR at
least in vitro (albeit to a lesser extent than PDK1). It is noteworthy
that PDK1 and mTOR were the only enzymes to be inhibited by
2-O-Bn-InsP5in a screen of almost 60 different kinases, indicating
that 2-O-Bn-InsP5may represent an interesting lead compound to
design novel and potent dual PDK1 and mTOR inhibitors.
2-O-benzyl-myo-inositol 1,3,4,5,6-pentakisphosphate was selected
in a screen of several different compounds that we synthesised and
first tested for their ability to inhibit Akt phosphorylation in cells
sensitive to InsP5. In this original screen, 2-O-Bn-InsP5was not
only more efficient than the parent molecule in inhibiting Akt
phosphorylation and inducing apoptosis in InsP5-sensitive cell
lines but it was also able to induce apoptosis in InsP5-resistant cell
lines including pancreatic cancer cells. The pro-apoptotic activity
of 2-O-Bn-InsP5detected in pancreatic cancer cells represents an
extremely important result taking into account the high resistance
of this cancer to chemotherapeutic treatment and the urgent need
for novel therapeutics in clinical treatment. Furthermore, 2-O-Bn-
InsP5was able to inhibit the in vivo growth of InsP5-resistant
prostate cancer xenografts. It is noteworthy that, although 2-O-Bn-
InsP5acted in the micromolar range in in vitro studies, it was able
to inhibit tumour growth in vivo at 12.5, 25 and 50mgkg?1, doses
commonly used to test the in vivo effect of potential anti-tumour
compounds. In particular, 2-O-Bn-InsP5induced a tumour weight
inhibition of 52% in prostate cancer xenografts when dosed at
50mgkg?1once daily for 14 days.
We then decided to investigate in more detail the mechanisms of
action of InsP5 and 2-O-Bn-InsP5, and to explain the higher
activity of 2-O-Bn-InsP5compared with the parent molecule. Our
previous and current data demonstrated that the in vitro and in
vivo properties of InsP5and 2-O-Bn-InsP5were because of reduced
Akt phosphorylation and activation (Piccolo et al, 2004; Maffucci
et al, 2005). Results from the kinase profiling assays here show that
InsP5and 2-O-Bn-InsP5do not inhibit Akt kinase activity itself in
vitro, whereas they are both able to directly inhibit PDK1 kinase
activity (albeit with different potency), thus indicating that the
detected Akt inhibition is due to blockade of the activity of its
upstream regulatory kinase. This is consistent with the observed
enhanced activity of 2-O-Bn-InsP5compared with InsP5, likely due
to its higher inhibitory activity towards PDK1. Furthermore, this
difference could explain the strong effect of 2-O-Bn-InsP5in InsP5-
resistant cell lines such as the PTEN mutant cells MDA-MB-468
and PC3, and would be consistent with the proposed key role of
PDK1 in tumourigenesis driven by Pten loss (Bayascas et al, 2005).
It should be noted that the resistance to InsP5treatment in these
cells is consistent with the delayed onset of inhibition in cells with
mutant PTEN observed using the ether lipid analogues (Castillo
et al, 2004). The observation that higher concentrations of InsP5
are eventually able to mimic the pro-apoptotic effect of 2-O-Bn-
InsP5 in these cells further supports the conclusion that the
different activity is because of different potency of the two
compounds towards PDK1. In this respect, it is interesting to
notice that the addition of the benzyl group to InsP5confers such a
higher inhibitory activity towards PDK1 on the derivative 2-O-Bn-
InsP5. It would be interesting to investigate whether the resulting
small increase in the hydrophobicity of the molecule enhances its
activity possibly by improving its binding to PDK1. Moreover, it is
noteworthy that such a modification confers to 2-O-Bn-InsP5a
selective inhibitory activity towards mTOR in vitro, providing
crucial information to develop novel specific dual PI3K/mTOR
inhibitors. Indeed one intriguing possibility is that 2-O-Bn-InsP5is
more active than InsP5because of its unique capability to inhibit
simultaneously and very specifically PDK1 and mTOR. In
particular the dual activity of 2-O-Bn-InsP5can explain its effect
Table 1
Life Technologies)
Results from SelectScreen kinase profiling service (Invitrogen–
Compound
[ATP]
tested (lM)
Kinase
testedIC50(nM)
(A)
2-O-Bn-InsP5
2-O-Bn-InsP5
2-O-Bn-InsP5
2-O-Bn-InsP5
100
75
200
10
PDK1
Akt1 (PKBa)
Akt2 (PKBb)
FRAP (mTOR)
26.5
4100000
77000
1300
(B)
InsP5
InsP5
InsP5
100
75
200
PDK1
Akt1 (PKBa)
Akt2 (PKBb)
613
4100000
38300
Compound
[ATP]
tested (lM)
Kinase
tested
% Inhibition-
mean
(C)
Ins(1,4,5)P3
Ins(1,3,4,5)P4
Ins(1,4,5,6)P4
Ins(3,4,5,6)P4
InsP5
Ins(1,2,3,4,5,6)P6
100
100
100
100
100
100
PDK1
PDK1
PDK1
PDK1
PDK1
PDK1
17
56
33
35
71
18
Abbreviations:
benzyl-myo-inositol 1,3,4,5,6-pentakisphosphate. 10-point titration for 2-O-Bn-InsP5.
10-point titration for InsP5. Single point for the indicated inositol polyphosphates. A
concentration of 1mM of the compounds was used in the assays.
InsP5¼inositol1,3,4,5,6-pentakisphosphate; 2-O-Bn-InsP5¼2-O-
Figure 4
(InsP5)-resistant xenografts and it is not associated with toxicity in vivo. Male athymic CD-1 nu/nu mice were inoculated subcutaneously (s.c.) with PC3 and
treated with the indicated concentrations of either InsP5or 2-O-Bn-InsP5from day 15 after cell implantation. The inositol compounds (12.5–25–
50mgkg?1day?1) and vehicle (water) were given daily by intraperitoneal (i.p.) injections for 14 consecutive days (days 15–28). Tumour size was assessed
twice weekly. (A, B) Tumour growth in 2-O-Bn-InsP5-treated mice (A) and InsP5-treated mice (B) compared with control group measured for the duration
of the experiment. Results are expressed as mean±s.e. (C) Top: Table showing all P-values for the indicated doses of 2-O-Bn-InsP5compared to controls at
the indicated days of treatment (NS¼not significant). Bottom: In vivo anti-tumour activity parameters. The percentage of tumour weight inhibition (TWI%),
the tumour growth delay (T?C) and the log cell kill (LCK) were calculated as described in the MATERIALS AND METHODS section. The highest inhibition
of tumour volume is reported. (D) Mice with s.c. growing tumours were treated with a single dose (50mgkg?1) of InsP5, 2-O-Bn-InsP5or water (control).
Tumours were excised 24h after treatment. Phosphorylation of Akt at its residues Ser473 and Thr308 was assessed by using specific antibodies. Membranes
were then stripped and re-probed with an anti-Akt antibody. (E) Body weights of mice treated with 2-O-Bn-InsP5or InsP5for 14 consecutive days. (F) Body
weights of mice treated with a single dose of 750mgkg?1InsP5or 2-O-Bn-InsP5.
2-O-benzyl-myo-inositol 1,3,4,5,6-pentakisphosphate (2-O-Bn-InsP5) possesses anti-tumour activity on inositol 1,3,4,5,6-pentakisphosphate
A novel PDK1 inhibitor
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111
British Journal of Cancer (2010) 102(1), 104–114
& 2010 Cancer Research UK
Translational Therapeutics

Page 9

in prostate cancer cells PC3, consistent with the recently reported
key role of mTORC2 in the development of loss of Pten-driven
prostate cancer (Guertin et al, 2009). Furthermore, a potential
2-O-Bn-InsP5-mediated inhibition of mTORC2 would also increase
the inhibitory activity towards Akt, by preventing its Ser473
phosphorylation, indicating that 2-O-Bn-InsP5represents a useful
compound to be tested in cancer types specifically dependent
on mTOR activation. Studies have recently revealed the existence
of a negative-feedback loop by which mTORC1 inhibition leads
to upregulation of Akt (Manning, 2004; O’Reilly et al, 2006)
and/or ERK/MAPK pathway (Carracedo et al, 2008), activating
proliferative and anti-apoptotic signals in certain cancer types.
120
100
80
60
40
20
0
No. of cells (% control)
120
100
80
60
40
20
0
No. of cells (% control)
Ctr
Rapamycin
Rapamycin+
2-O-Bn-InsP5
2-O-Bn-InsP5
Paclitaxel
Paclitaxel+
2-O-Bn-InsP5
Paclitaxel
Curcumin
Paclitaxel+
2-O-Bn-InsP5
Curcumin
Curcumin+
2-O-Bn-InsP5
Curcumin+
2-O-Bn-InsP5
2-O-Bn-InsP5
2-O-Bn-InsP5
Ctr
Ctr
2-O-Bn-InsP5
Curcumin
Curcumin+
2-O-Bn-InsP5
Ctr
Ctr EtOH
4-OH Tamoxifen
2-O-Bn-InsP5
4-OH Tamoxifen
+ 2-O-Bn-InsP5
Ctr
2-O-Bn-InsP5
Ctr
Rapamycin
Rapamycin+
2-O-Bn-InsP5
2-O-Bn-InsP5
Paclitaxel
Paclitaxel+
2-O-Bn-InsP5
2-O-Bn-InsP5
SKOV-3
PC3
ASPC1
MCF-7
PC3
MDA-MB-468
120
100
80
60
40
20
0
No. of cells (% control)
120
100
80
60
40
20
0
No. of cells (% control)
120
100
80
60
40
20
0
No. of cells (% control)
120
100
80
60
40
20
0
No. of cells (% control)
AB
CD
EF
Figure 5
more than additive effects. (A) MCF7 were treated with 20nM 4-OH Tamoxifen, 50mM 2-O-Bn-InsP5alone or in combination. Data are mean±s.e. of n¼4
independent experiments carried out in duplicate. 4-OH Tamoxifenþ2-O-Bn-InsP5: Po0.01 vs 4-OH Tamoxifen; Po0.01 vs 2-O-Bn-InsP5. (B) PC3 were
treated with 5mM 2-O-Bn-InsP5, 10mM curcumin alone or in combination. Data are mean±s.e. of n¼5 independent experiments carried out in duplicate.
Curcuminþ2-O-Bn-InsP5: Po0.01 vs 2-O-Bn-InsP5; Po0.05 vs curcumin. (C) ASPC1 were treated with 5mM 2-O-Bn-InsP5, 10mM curcumin alone or in
combination. Data are mean±s.e. of n¼6 independent experiments carried out in duplicate. Curcuminþ2-O-Bn-InsP5: Po0.05 vs 2-O-Bn-InsP5; Po0.05 vs
curcumin. (D) MDA-MB-468 were treated with 10mM 2-O-Bn-InsP5, 10mM curcumin alone, 1nM paclitaxel or the indicated combination. Data are
mean±s.e. of n¼2 independent experiments carried out in duplicate. In all cases (A–D), cells were treated for 72h and the number of surviving cells was
assessed by cell counting. (E) SKOV-3 were treated with 20mM 2-O-Bn-InsP5, 30nM paclitaxel, 20nM rapamycin or with the indicated combinations. (F) PC3
were treated with 10mM 2-O-Bn-InsP5, 1nM rapamycin alone or in combination and with 20mM 2-O-Bn-InsP5, 40nM paclitaxel alone or in combination. In all
cases (E, F), cells were treated for 72h and the number of surviving cells was assessed by SRB assay and data are mean±s.e. of two experiments carried out
in quadruplicate.
Combination of 2-O-benzyl-myo-inositol 1,3,4,5,6-pentakisphosphate (2-O-Bn-InsP5) with anti-cancer compounds in vitro results in additive or
A novel PDK1 inhibitor
M Falasca et al
112
British Journal of Cancer (2010) 102(1), 104–114
& 2010 Cancer Research UK
Translational Therapeutics

Page 10

The possibility of blocking PDK1 and mTOR simultaneously in
these tumours is likely to be very effective therefore, because of its
dual inhibitory activity towards both enzymes, it would be
interesting to investigate the effect of 2-O-Bn-InsP5 in these
cellular contexts.
Our future strategies to design novel compounds will take into
consideration the possibility that, besides its direct inhibitory
activity towards PDK1 and possibly mTOR, binding of 2-O-Bn-
InsP5to non-catalytic domains may affect the activity of kinases or
their mechanism of activation in vivo. Indeed in our previous work
we proposed that InsP5can inhibit Akt activation by binding to
Akt PH domain and preventing Akt recruitment to the plasma
membrane (Berrie and Falasca, 2000). It was also proposed that
inositol phosphates can bind PDK1 PH domain and retain this
kinase in the cytosol, preventing Akt phosphorylation at Thr308
(Komander et al, 2004). These data suggest that the detected
inhibitory effect of both 2-O-Bn-InsP5and InsP5on Akt activation
in vivo can result from a combination of a direct effect on PDK1
kinase activity and effect on Akt/PDK1 recruitment to the plasma
membrane. Similarly, the possibility that in vivo the inositol
polyphosphates can bind and increase the activity of phosphatases
which regulate Akt, such as PH domain leucine-rich repeat protein
phosphatases 1 and 2 (Gao et al, 2005; Brognard et al, 2007) will be
taken into consideration in our future strategies. In this respect it
would be interesting to develop binding experiments of cellular
lysates to immobilized 2-O-Bn-InsP5 and InsP5 to determine
whether the compounds only bind PDK1, as indicated by the
kinase profiling assays, or the in vivo mechanisms of action is
more complex. These experiments would also give more informa-
tion of whether the compounds may indirectly act on other kinases
without directly affecting their catalytic activity.
It must be noted that, like InsP5, 2-O-Bn-InsP5is a water soluble
compound and it is well tolerated in vivo even at concentrations 15
times higher the active dose. In addition, combination of 2-O-Bn-
InsP5with other anti-cancer compounds including natural com-
pounds results in additive or more than additive effects, indicating
that such a compound (or derivatives) may prove particularly useful
in combinatorial therapies. In particular 2-O-Bn-InsP5increases the
effect of tamoxifen in breast cancer cells MCF7, consistent with the
reported role of PDK1 inhibition in tamoxifen sensitisation (Iorns
et al, 2009). It is worth mentioning that our in vitro assays revealed
that InsP5itself is able to inhibit PDK1 (although less than 2-O-Bn-
InsP5). This raises the interesting possibility that the endogenous
intracellular InsP5may act as an endogenous PDK1 inhibitor and
regulator of the PI3K/Akt pathway. This hypothesis is currently
being investigated in our laboratory.
In conclusion, here we have described the in vitro and in vivo
properties of 2-O-Bn-InsP5, a derivative of InsP5, which possesses
similar solubility and lack of toxicity in vivo but enhanced pro-
apoptotic and anti-tumour activity compared with the parent
molecule. In particular 2-O-Bn-InsP5possesses specific inhibitory
activity towards PDK1. Data also indicate that 2-O-Bn-InsP5can
inhibit mTOR, at least in vitro. It is interesting to note that InsP5
does not possess such an inhibitory activity towards mTOR, thus
suggesting that comparison of the two molecules can give useful
information towards developing specific dual PDK1/mTOR
inhibitors. Taken together these data indicate that InsP5 and
2-O-Bn-InsP5 may represent promising models for further
development of novel anti-cancer drugs.
ACKNOWLEDGEMENTS
This work was supported by the European Commission FP6
program Apotherapy (EC contract number 037344; http://apotherapy.
med.uoc.gr, to M.F. and M.B.), American Institute for Cancer
Research and Pancreatic Cancer Research Fund (to M.F.), Well-
come Trust (Programme Grant No. 082837 to A.M.R. and
B.V.L.P.), Italian Association for Cancer Research (to M.B.) and
Fondazione Carichieti. D.C. was supported by British Heart
Foundation (grant PG/04/033/16906 to M.F.). M.M. is recipient of
a fellowship from the Italian Foundation for Cancer Research
(FIRC).
Supplementary Information accompanies the paper on British
Journal of Cancer website (http://www.nature.com/bjc)
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