Novel PI analogues selectively block activation of the pro-survival serine/threonine kinase Akt

Article abstract of DOI:10.1021/ja0285159

The synthesis from l-quebrachitol of a series of 3-deoxygenated ether lipid-type phosphatidylinositol (PI) analogues is reported, that selectively block activation of Akt and downstream substrates without affecting activation of the upstream kinase, PDK-1

Full text of DOI:10.1021/ja0285159

Published on Web 01/08/2003
Novel PI Analogues Selectively Block Activation of the Pro-survival Serine/
Threonine Kinase Akt
Alan P. Kozikowski,*,† Haiying Sun,† John Brognard,‡ and Phillip A. Dennis*,‡
Drug DiscoVery Program, Department of Neurology, Georgetown UniVersity, 3900 ReserVoir Road, NW,
Washington, DC 20007, and Cancer Therapeutics Branch, Center for Cancer Research, National Cancer Institute,
Bethesda, Maryland 20889
Received September 11, 2002 ; E-mail: kozikowa@georgetown.edu
Phosphorylation regulates many cellular processes, and abnormal
regulation of kinases or phosphatases that control phosphorylation
contributes to human diseases such as cancer. Considerable attention
is therefore being directed toward the design of selective kinase
inhibitors.1 The serine/threonine kinase Akt (or PKB) is the
prototypic kinase that promotes cellular proliferation, growth, and
survival in vitro and tumor formation in vivo. Through inactivation
of the regulatory phosphatase PTEN, activation of ras or growth
factor receptors, or amplification of the upstream kinase, phos-
phatidylinositol 3-kinase, active Akt has been detected in many
types of human cancers (Figure 1).2 Despite the greater understand-
ing of the role of Akt in cancer biology, however, the study of Akt
has been limited by the lack of specific inhibitors directed against
Akt. Development of Akt inhibitors would not only facilitate in
vitro studies but would also identify lead compounds for potential
drug development. Herein we disclose a novel class of modified
phosphatidylinositol (PI) analogues that selectively block activation
of Akt and downstream substrates without affecting activation of
Figure 1. The Akt pathway. Akt can be activated in a PI3K-dependent
manner by stimulation of G protein-coupled receptors (GPCR), growth factor
receptors such as IGF-IR or EGFR or ras. PI3K generates the phospholipids,
the upstream kinase, PDK-1, or other kinases downstream of ras
such as MAPK in cancer cells that have high levels of constitutively
active Akt. Our studies suggest that these analogues are effective
lead compounds for the development of drugs designed to inhibit
Akt activity and ultimately treat neoplastic diseases.
phosphatidylinositol 3,4-bisphosphate, and phosphatidylinositol 3,4,5-
trisphosphate. PIP2 and PIP3 bind to PDK-1 and Akt and induce translocation
to the plasma membrane where phosphorylation of Akt occurs. Levels of
PIP2 and PIP3 are regulated by the tumor suppressor phosphatase, PTEN.
Activation of Akt subsequently increases phosphorylation of downstream
substrates such as the kinase c-Raf, the transcription factors AFX, 4EBP-
1, or p70S6K, which control the initiation phase of protein translation.
Previously, we had reported on the ability of the phosphatidyl-
inositol analogue DPIEL (3-deoxyphosphatidylinositol ether lipid)
to inhibit platelet derived growth factor-stimulated activation of
Akt and growth factor-stimulated cell growth. Using this as our
lead structure, we now sought compounds of improved metabolic
stability and of anticancer potential through manipulation of the
2-OH group. As the degradation of inositol phospholipids occurs
through the enzymatic action of PI-specific phospholipase C (PI-
PLC) with formation of a 1,2-cyclic phosphate, we chose to explore
the effect of both alkylation as well as deletion of the 2-OH group
to block formation of the cyclic phosphate.3
Scheme 1 a
Compound 1 was prepared from quebrachitol according to our
previous method4 and transformed into intermediates 3-6 as shown
in Scheme 1. These compounds were carried on to the ether lipids
13-16 as shown in Scheme 2. From 3 and 6, the carbonate
analogues 20 and 21 were also prepared (Scheme 3).
a Reagents and conditions: (a) Bu2SnO, toluene, reflux; p-MeOC6H4CH2Cl,
CsF, DMF, rt, 90%; (b) (i) NaH, MeI, or isobutyl bromide or cyclohexyl-
methyl bromide, 0 °C, DMF; (ii) CAN, CH3CN-H2O 4:1 (v/v), 0 °C to rt;
(c) (i) NaH, CS2, then MeI, 0 °C, DMF; (ii) Bu3SnH, AIBN, toluene, reflux;
(iii) CAN, CH3CN-H2O 4:1 (v/v), 0 °C to rt, 81% over three steps.
To test the biological activity of these PI analogues, we chose
two lung cancer cell lines that we had previously shown to have
high endogenous levels of Akt activity, H157 cells and H1703 cells.5
H157 cells have increased Akt activity as a result of mutant K-ras
and mutant PTEN status. H1703 cells have wild-type PTEN, and
the cause of their increased Akt activity is unknown. Compounds
13, 16, 7, or 20 were added at 10 µM to both cell lines, and
phosphorylation of S473, which is necessary and indicative of
activation of Akt, was assessed by immunoblotting with phospho-
specific antibodies directed against S473 (Figure 2). Phosphorylation
of Akt was decreased by 13 and 16, but not by 7, which lacks the
inositol headgroup, or by 20, which contains a carbonate linker.
Similar decreases in Akt phosphorylation were observed when 14
or 15 were added to these cells (data not shown). Interestingly,
DPIEL was ineffective in these assays, indicating that, while DPIEL
previously inhibited PDGF-induced Akt phosphorylation, it could
not inhibit endogenous Akt phosphorylation or activity in these cells.
Levels of native Akt were not affected by these compounds. To
† Georgetown University.
‡ National Cancer Institute.
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J. AM. CHEM. SOC. 2003, 125, 1144-1145
10.1021/ja0285159 CCC: $25.00 © 2003 American Chemical Society

C O M M U N I C A T I O N S
Scheme 2 a
added to H157 cells, only inhibition of Akt and downstream
components was observed. Phosphorylation of PDK-1 was not
affected. Akt phosphorylation was decreased by 13 or 16, but not
7. Levels of total Akt were unaffected. Of the downstream substrates
tested, phosphorylation of 4EBP-1 was attenuated most by 13 or
16. Phosphorylation of AFX or p70S6K was attenuated less. c-Raf
phosphorylation was also inhibited by 13 or 16. Interestingly, this
correlated with increased phosphorylation of ERK1/2, which is
consistent with prior observations that phosphorylation of c-Raf
by Akt can inhibit the MEK/ERK pathway.10,11 Phosphorylation
of p38, a MAPK kinase activated by cellular stress, was also
increased. Similar inhibition of Akt and downstream substrates was
observed when 14 or 15 was tested (data not shown). Together,
our data indicate that 2-modified, 3-deoxy PI analogues are capable
of decreasing activation of Akt. PI analogues with phosphate linkers
were more effective than those with carbonate linkers (compare
13 or 16 with 20). Most importantly, 13 and 16 inhibited activation
of Akt and select downstream substrates without decreasing
phosphorylation of PDK-1, or other kinases downstream of ras such
as MAPK. On the basis of the increased phosphorylation of p38, a
kinase whose activity is increased under conditions of cellular stress,
we have begun experiments evaluating these compounds for toxicity
in these cell lines. Compounds 13 and 16 are potent inducers of
apoptosis and selectively kill a variety of cancer cell lines that
contain high levels of active Akt and depend on Akt for survival
(data not shown). Thus, this report is the first to identify PI
analogues as effective Akt inhibitors that decrease Akt activity and
cause apoptosis in cancer cells. These compounds have potential
for use as therapeutic agents in cancer therapy. Future studies will
characterize the mechanism of inhibition of Akt and suitability of
these analogues for in vivo use.12
a Reagents and conditions: (a) BnOP[N(i-Pr)2]2, diisopropylammonium
tetrazolide, CH2Cl2, rt, 98%; (b) (i) 8, 1H-tetrazole, rt, CH2Cl2; (ii) m-CPBA,
0 °C to rt, CH2Cl2; (c) H2, 20% Pd(OH)2-C, t-BuOH, rt, 1 atm.
Scheme 3 a
Acknowledgment. We are indebted to the National Institutes
of Health (CA61015-07) and to the intramural program of the NCI
for their support of these investigations.
a Reagents and conditions: (a) (imd)2CO, toluene, reflux; (b) 17, DBU,
toluene, reflux; (c) H2, 20% Pd(OH)2-C, t-BuOH, rt, 1 atm.
Supporting Information Available: Figure 3 and experimental
procedures and characterization data for new compounds (PDF). This
material is available free of charge via the Internet at http://pubs.acs.org.
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Figure 2. Effects of novel PI analogues on Akt phosphorylation. H157
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test whether the PI analogues affected other components in the Akt
pathway, we assessed the activation state of multiple components
in the Akt pathway using phospho-specific antibodies in immuno-
blotting experiments (Figure 3). We chose to test the activation
state of PDK-1, a kinase upstream of Akt that activates Akt,6 and
four proteins whose phosphorylation is increased in response to
Akt activation, 4EBP-1,7 p70S6K,8 AFX,9 and c-Raf.10 We also
tested two members of the MAPK family, ERK, which is activated
downstream of ras, and p38. When compound 13, 16, or 7 was
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