Modifications of the GSK3β substrate sequence to produce substrate-mimetic inhibitors of Akt as potential anti-cancer therapeutics

Article abstract of DOI:10.1016/j.bmcl.2007.01.004

Amplification, overexpression, and elevated activation of Akt have been detected in many human malignancies making it an important target for cancer therapy. The Akt substrate-binding site offers a large number of potential interactions to an appropriatel

Full text of DOI:10.1016/j.bmcl.2007.01.004

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2068–2073
Modifications of the GSK3b substrate sequence to produce
substrate-mimetic inhibitors of Akt as potential
anti-cancer therapeutics
Katherine J. Kayser,a Matthew P. Glenn,a Said M. Sebti,b,c Jin Q. Chengb,c,d
and Andrew D. Hamiltona,*
aDepartment of Chemistry, Yale University, PO Box 208107, New Haven, CT 06520, USA
bDrug Discovery Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA
cDepartment of Interdisciplinary Oncology, Tampa, FL 33612, USA
dDepartment of Pathology, University of South Florida College of Medicine, Tampa, FL 33612, USA
Received 3 November 2006; revised 20 December 2006; accepted 5 January 2007
Available online 12 January 2007
Abstract—Amplification, overexpression, and elevated activation of Akt have been detected in many human malignancies making it
an important target for cancer therapy. The Akt substrate-binding site offers a large number of potential interactions to an appro-
priately designed small molecule and can form the basis for the development of selective inhibitors. Here, we report the progression
of GSK3b substrate-mimetic inhibitors towards the development of a potent, small molecule substrate-mimetic inhibitor of Akt.
Ó 2007 Elsevier Ltd. All rights reserved.
Akt, or protein kinase B, is a serine/threonine protein ki-
nase that has been found to be amplified, overexpressed,
and/or activated in many human malignancies.1–6 It ex-
ists as three separate isoforms: Akt1/PKBa, Akt2/
PKBb, and Akt3/PKBc, that are structurally very simi-
lar.7 Each isoform consists of an N-terminal pleckstrin
homology (PH) domain, a central catalytic domain,
and a C-terminal regulatory tail.7
ovarian, breast, prostate, and pancreatic cancer.1–6 Its
role in malignancies is accomplished by the simulta-
neous promotion of cell survival, growth, and migra-
tion, and the down-regulation of apoptosis.11–13
Overall, Akt activation down-regulates the expression
of pro-apoptotic proteins and up-regulates the expres-
sion of anti-apoptotic proteins.11,12,14 Inhibition of Akt
function has been shown to stimulate apoptosis in a
number of cancer cell lines.15 These observations estab-
lish Akt as a valuable cancer target and suggest that the
development of potent and selective inhibitors of Akt
could lead to novel anti-cancer therapeutics.
The events that lead to the activation of Akt have been
elucidated.8–10 Activation requires agonist-induced stim-
ulation of phosphoinositide 3-kinase (PI3K) to generate
phosphatidylinositol-3,4,5-triphosphate (PIP3) that
associates with the PH domain of Akt, and localizes
the protein to the intracellular side of the plasma mem-
brane. Once anchored to the plasma membrane, residues
Thr308 and Ser473 are phosphorylated, inducing maxi-
mal catalytic activity. Once activated, the protein is re-
leased to target cytosolic and nuclear substrates.
Much effort has been applied to the development of Akt
inhibitors that target the ATP-binding domain of the
protein, but these have the potential to affect other ki-
nases or the many ATP utilizing enzymes.16–19 Recently,
there has also been an emergence of allosteric inhibitors
that has been met with some success.20,21 However, as a
general point of strategy, targeting inhibitors to the pep-
tide substrate-binding site has the potential for greater
selectivity since it has evolved to respond to a highly spe-
cific sequence of amino acids.22,23
Elevated activation of specific isoforms of Akt have
been detected in many human malignancies including
Keywords: Protein kinase B; Akt; Cancer; Peptidomimetics; Substrate-
mimetic.
In addition, peptide or peptidomimetic substrates conju-
gated to ATP mimetics have provided potent bisubstrate
*
Corresponding author. E-mail: andrew.hamilton@yale.edu
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.01.004

K. J. Kayser et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2068–2073
2069
inhibitors.24,25 The substrate-binding site offers a large
number of potential interactions to a small molecule de-
rived through mimicry of the key peptide sequence,
which provides a good opportunity for development of
an Akt selective inhibitor. The X-ray crystal structure
of an activated Akt ternary complex with a 10-residue
sequence of a cellular substrate glycogen synthase kinase
3 (GSK3b) and an ATP analogue identifies the GSK3b-
peptide (GRPRTTSFAE) to be bound in an essentially
extended conformation, with discrete sections of
b-strand on either side of the modifiable serine residue
(Fig. 1).26 A closely related substrate, GRPRTSSF,
was found to have a Km of 8 lM.27 Extensive hydrogen
bonding interactions are observed between acidic pock-
ets of the protein and N-terminal basic residues of
GSK3b, while C-terminal interactions are essentially
hydrophobic. The consensus substrate for processing
has been shown to be Arg-Xaa-Arg-Yaa-Zaa-S/T-Hyd,
where Xaa may be any amino acid, Yaa and Zaa any
small amino acid other than glycine, and Hyd represents
a large hydrophobic amino acid.27
and tested for in vitro inhibition of Akt (Table 1). Inhib-
itors were tested utilizing a fluorescence polarization
assay system.28 Initial investigations of our substrate-
mimetic inhibitors included evaluation of the contribu-
tion of a number of amino acids, starting with the
phosphorylatable serine residue of the GSK3b peptide.
Numerous serine substitutions were made by scanning
the position with several other L-amino acids to provide
weakly binding peptidic inhibitors (1–7). Replacement
of the reactive serine with a valine residue (a non-nucle-
ophilic surrogate) in peptide 4 provided a starting point
in our design.
Inclusion of a small hydrophobic group, benzyl (Bn), at
the C-terminus to complement the unoccupied hydro-
phobic pocket afforded inhibitor 8 with a two-fold in-
crease in potency (IC50 of 239lM). C-terminal
coupling was conducted with benzylamine (BnNH2),
HATU, and DIPEA in DMF to limit the racemization
of the terminal Phe residue. Both diastereomers of 10
were tested and found to have comparable activity
(IC50 of 28 and 40 lM), therefore further diastereomers
of later compounds were not separated, but tested as
mixtures.
In this paper, we report the synthesis and activity of sub-
strate-mimetic inhibitors of Akt based on the truncated
GSK3b-peptide substrate sequence GRPRTTSF.
Further refinement included the replacement of the
internal -TT- residues, which make few interactions with
the protein surface. Replacement of this dipeptide with -
AA- resulted in a 2-fold decrease in activity, however,
substitution with a conformationally restricted scaffold,
p-aminobenzoic acid (Abz), afforded inhibitor 10 with a
10-fold increase in activity (IC50 of 28lM). Docking
studies suggest that the Abz spacer can reproduce the
hydrophobic interactions of the native discrete dipeptide
b-strand, while reducing the entropy cost of the extend-
ed binding conformation of the unbound inhibitor.
A simple series of peptidomimetic inhibitors was gener-
ated directly from the minimal substrate sequence by
systematic replacement of the non-critical amino acids
A small screen of hydrophobic groups appended to the
C-terminus (11–17) demonstrated similar activity to
C-terminal benzyl derivative 10. These C-terminal mod-
ifications and the reported X-ray structure suggest the
hydrophobic pocket is extensive (11, 13–14). Further
structural refinement at this position should lead to sig-
nificant improvement in activity. As expected these
hydrophilic peptidic inhibitors showed no cellular
activity.
Modification of the N-terminal GRPR sequence repre-
sents a challenge in the stepwise development of non-
peptidic inhibitors, as previous alanine scanning had
demonstrated a strict requirement for conservation of
both arginines.27,30 The presence of these polar residues
would also likely hinder cell penetration and useful in
vivo activity. Having introduced hydrophobic contacts
(Abz, Hyd) to inhibitors 10–17, with a concomitant
improvement in Akt affinity, we reexamined the depen-
dence of N-terminal hydrophilic contacts using an ala-
nine scan (18–20, Table 1). From this we concluded
that only one arginine residue was necessary to maintain
activity, with 18 and 19 possessing similar potency to 10.
This series also suggests that the arginine residue adja-
cent to the hydrophobic spacer contributes more signif-
icantly to binding.
Figure 1. (a) Synopsis of GSK3b–Akt interactions by residue. (b)
Crystal structure of the enzyme-bound conformation of GSK3b
(green) and an ATP analogue (yellow) against the solvent-accessible
surface area of Akt (Gradient color scale—Red, hydrophobic; blue,
hydrophilic).26

2070
K. J. Kayser et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2068–2073
Table 1. Initial peptidic inhibitors and their contributions to binding
a
Compound
Structure
IC50 (lM)
1
2
3
4
5
6
7
Ac-
Ac-
Ac-
Ac-
Ac-
Ac-
Ac-
G
G
G
G
G
G
G
R
R
R
R
R
R
R
P
P
P
P
P
P
P
R
R
R
R
R
R
R
T
T
T
T
T
T
T
T
T
T
T
T
T
T
W
F
F
F
F
F
F
F
F
–OH
–OH
–OH
–OH
–OH
–OH
–OH
>500
>500
>500
445
H
V
A
C
N
409
241
229
H
N
8
Ac-
Ac-
Ac-
Ac-
Ac-
Ac-
Ac-
Ac-
Ac-
Ac-
Ac-
Ac-
Ac-
G
G
G
G
G
G
G
G
G
G
G
G
G
R
R
R
R
R
R
R
R
R
R
A
R
A
P
R
R
R
R
R
R
R
R
R
R
R
A
A
R
R
R
R
T
T
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
239
438
28
H
N
9
P
A
A
H
N
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
P
Abz
Abz
Abz
Abz
Abz
Abz
Abz
Abz
Abz
Abz
Abz
Abz
Abz
Abz
Abz
P
36
N
H
H
N
P
52
P
62
N
H
N
P
94
N
H
H
N
P
96
H
N
H
N
O
P
322
>500
41
O
H
N
P
N
H
N
P
H
N
P
79
H
N
P
>500
51
Ac-
Ac-
Ac-
Ac-
N
H
89
N
H
H
N
104
119
H
N

K. J. Kayser et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2068–2073
2071
Table 1 (continued)
Compound
25
a
Structure
IC50 (lM)
H
N
Ac-
R
Abz
V
V
F
F
163
NH2
O
H
N
26
Abz
193
HN
N
H
a Reported values are an average of three independent-binding curves utilizing an IMAPÒ Akt Assay Kit (Molecular Devices). Reactions were
conducted in wells with 20.0 lL of 10 mM Tris–HCl (pH 7.2), 10 mM MgCl2, 0.1% BSA, 0.05% NaN3, 1 mM DTT, 100 nM 5FAM-
GRPRTSSFAEG-COOH, 5lM ATP, 0.067 U/mL of Akt1, and inhibitor. Reaction mixtures were incubated for 1 h at room temperature and then
quenched with 60lL of the IMAP-binding solution. The reactions were equilibrated for 1 h at room temperature then data points were collected
and analyzed.
Modifications to the N-terminal hydrophilic residues
were concentrated on increasing the rigidity and hydro-
phobicity of the inhibitors. Rigidifying modifications
were focused on decreasing the length and rotational
freedom of the essential guanidinium functionality to
project it directly into an acidic pocket of Akt (Scheme 1).
Different length linkers were explored, with 0, 1, 2,
and 3 atoms separated from the aromatic spacer to af-
ford inhibitors that showed comparable or better affinity
than 24, which contains the entire Arg residue. Inhibi-
tors with 0–2-atom linkers, 32a–c, respectively, were
synthesized by the guanidinylation of commercially
available aminobenzoic acids followed by solid-phase
coupling, cleavage, and C-terminal coupling. Inhibitor
Figure 2. Flexible ligand docking GOLD29 of 24 (green) against the
31 possesses a three-atom linker and was synthesized
solvent-accessible surface area of Akt (Gradient color scale—Red,
by the reductive amination of methyl 4-aminobenzoate
hydrophobic; blue, hydrophilic).
with N-Boc-2-aminoacetaldehyde, with subsequent
saponification and deprotection to afford 29. Guanid-
inylation of 29, followed by solid phase coupling, cleav-
age, and C-terminal coupling, provided 31. Inhibitor 32a
provides the best affinity in this series with an IC50 of
77 lM, suggesting that a one-atom linker is sufficient
to reach the hydrophilic pocket.
Evaluation of the role of the arginine residues led us
to truncate the inhibitors, thereby eliminating the
N-terminal -GRP- tripeptide sequence. This produced
a set of inhibitors with only three amino acids and
with the optimal hydrophobic substituents coupled
to the C-terminus of AcR-Abz-V-F-OH (21–25).
These truncated inhibitors are significantly more
hydrophobic than peptidomimetics 1–20, but retain
almost identical inhibition potency. Further trunca-
tion of the N-terminal acylated amine 26 resulted in
an almost two-fold loss of activity when compared
to 24, highlighting the importance of hydrophobic
interactions and the interaction of the carbonyl or
amide proton with an adjacent residue (Fig. 2).
Docking studies suggest that this acylated amine
could occupy a hydrophobic pocket of Akt previously
occupied by one of the Thr residues of the GSK3b
peptide or it could be hydrogen bonding to residues
within the active site of Akt: E315, E279, D275, or
K277. The Boc-protected modified arginine residue
lacking the a-amine 27 was synthesized by guanidiny-
lation of 5-aminovaleric acid with N,N0-bis(tert-butox-
ycarbonyl)-1H-pyrazole-1-carboxyamidine (BisBocPCH)
and Et3N in CH3OH.
Additional N-terminal modifications focused on scaf-
folds with a functionalizable handle to access a hydro-
phobic pocket previously occupied by one of the Thr
residues of the GSK3b peptide. This handle was also
used to incorporate a hydrogen-bonding functionality
to investigate the hypothesis that the N-terminal acyl
group of the Arg residue is hydrogen bonding to an ac-
tive site residue. A simple series of inhibitors was synthe-
sized to probe both interactions (36a–c, Scheme 2). Two
derivatives were synthesized by coupling 27 and methyl
4-aminobenzoate to afford 33 which was then alkylated
on the amide nitrogen with the corresponding bromide
to provide 35a–b. Reductive amination of methyl 4-ami-
nobenzoate with 3-phenyl-propionaldehyde and subse-
quent coupling to 27 via an in situ acid chloride
formation afforded 35c.31 Saponification of 35a–c fol-
lowed by solid phase coupling, cleavage, and C-terminal
coupling afforded inhibitors 36a–c.
Incorporation of an amide functionality and a large
hydrophobic group gave a significant increase in activi-
ty, with the benzyl derivative 36a showing an IC50 of
14 lM, which is significantly higher than that of the
unacylated analogue 26. Docking studies of 36a suggest
that the benzyl substituent projects into a large pocket
Further refinement of inhibitor 24 toward non-peptidic,
small molecule substrate-mimetics was focused on three
main areas of modification: the N-terminal hydrophilic
residues, the interior hydrophobic spacer, and the C-ter-
minal hydrophobic contacts.

2072
K. J. Kayser et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2068–2073
˚
Scheme 1. Reagents: (a) i—N-Boc-2-aminoacetaldehyde, AcOH, CH3OH, 4A mol. sieves, ii—NaCNBH3; (b) LiOH, THF/H2O; (c) TFA; (d)
BisBocPCH, Et3N, CH3OH; (e) NH2-Val-Phe-Wang, HBTU, DIPEA, DMF; (f) TFA, 5% thioanisole, 1% TIPS, 1% H2O; (g) BnNH2, HATU,
DIPEA, DMF (*Synthesized from commercially available amino benzoic acids following the sequences d–g.).
˚
Scheme 2. Reagents: (a) Compound 27, DIC, CH2Cl2, cat. DMAP; (b) R–Br, Base, DMF; (c) i—3-phenyl-propionaldehyde, AcOH, CH3OH, 4A
mol. sieves, ii—NaCNBH3; (d) Compound 27, Ph3PCl2, CHCl3; (e) LiOH, THF/H2O; (f) NH2-Val-Phe-Wang, HBTU, DIPEA, DMF; (g) TFA, 5%
thioanisole, 1% TIPS, 1% H2O; (h) BnNH2, HATU, DIPEA, DMF.
within the active site of Akt, previously occupied by res-
idues of the GSK3b peptide (Fig. 3). The t-butyl deriv-
ative 36b was slightly less potent with an IC50 of
58 lM. Inhibitor 36c was synthesized with similar
hydrophobic character as 36a, but lacking the hydrogen
bonding potential. Its affinity is comparable to inhibi-
tors 36a and 36b, suggesting that hydrophobic contacts
in the Thr pocket are the key interactions promoting in-
creased affinity.
studies of substrate-mimetic inhibitors demonstrate that
limited structural modification of the initial peptidic
substrate provides inhibitors with decreasing peptidic
character and increasing lipophilicity, which with future
modifications should lead to in vivo activity. Modifica-
tions of these inhibitors toward increasing potency and
selectivity for specific Akt isoforms will provide a useful
set of molecular probes to assist in the validation of Akt
as a potential target for anti-cancer drug design.
Many malignancies display elevated levels of specific
isoforms of Akt identifying it as an attractive target
for cancer therapy. Divergence in Akt isoform-specific
function presumably occurs at the level of substrate
selection on the basis of sequence diversity. Preliminary
Acknowledgments
K.J.K. thanks Sanofi-Aventis for their funding of the
American Chemical Society division of Medicinal

K. J. Kayser et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2068–2073
2073
12. Ozes, O. N.; Mayo, U. D.; Gustin, J. A.; Pfeffer, S. R.;
Pfeffer, L. M.; Donner, D. B. Nature (London, UK.) 1999,
401, 82.
13. Datta, S. R.; Brunet, A.; Greenberg, M. E. Genes Dev.
1999, 13, 2905.
14. Pugazhenthi, S.; Nesterova, A.; Sable, C.; Heidenreich, K.
A.; Boxer, L. M.; Heasley, L. E.; Reusch, J. E. B. J. Biol.
Chem. 2000, 275, 10761.
15. Yang, L.; Dan, H. C.; Sun, M.; Liu, Q.; Sun, X.-m.;
Feldman, R. I.; Hamilton, A. D.; Polokoff, M.; Nicosia, S.
V.; Herlyn, M.; Sebti, S. M.; Cheng, J. Q. Cancer Res.
2004, 64, 4394.
16. Li, Q.; Li, T.; Zhu, G.-D.; Gong, J.; Claibone, A.; Dalton,
C.; Luo, Y.; Johnson, E. F.; Shi, Y.; Liu, X. Bioorg. Med.
Chem. Lett. 2006, 16, 1679.
17. Reuveni, H.; Livnah, N.; Geiger, T.; Kleid, S.; Ohne, O.;
Cohen, I.; Benhar, M.; Gellerman, G.; Levitzki, A.
Biochemistry 2002, 41, 10304.
18. Breitenlechner, C. B.; Wegge, T.; Berillon, L.; Graul, K.;
Marzenell, K.; Friebe, W.-G.; Thomas, U.; Schumacher,
R.; Huber, R.; Engh, R. A.; Masjost, B. J. Med. Chem.
2004, 47, 1375.
19. Woods, K. W.; Fischer, J. P.; Claiborne, A.; Li, T.;
Thomas, S. A.; Zhu, G. D.; Diebold, R. B.; Liu, X. S.; Shi,
Y.; Klinghofer, V.; Han, E. K.; Guan, R.; Magnone, S. R.;
Johnson, E. F.; Bouska, J. J.; Olson, A. M.; de Jong, R.;
Oltersdorf, T.; Luo, Y.; Rosenberg, S. H.; Giranda, V. L.;
Li, Q. Bioorg. Med. Chem. 2006, 14, 6832.
Figure 3. Flexible ligand docking GOLD29 of 36a (green) against the
solvent-accessible surface area of Akt (Gradient color scale—Red,
hydrophobic; blue, hydrophilic).
Chemistry predoctoral fellowship. We also thank the
National Institutes of Health (1R01 CA107078-01) for
financial support of this work.
Supplementary data
20. Lindsley, C. W.; Zhao, Z.; Leister, W. H.; Robinson, R.
G.; Barnett, S. F.; Defeo-Jones, D.; Jones, R. E.;
Hartman, G. D.; Huff, J. R.; Huber, H. E.; Duggan, M.
E. Bioorg. Med. Chem. Lett. 2005, 15, 761.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2007.01.004.
21. Zhao, Z.; Leister, W. H.; Robinson, R. G.; Barnett, S. F.;
Defeo-Jones, D.; Jones, R. E.; Hartman, G. D.; Huff, J.
R.; Huber, H. E.; Duggan, M. E.; Lindsley, C. W. Bioorg.
Med. Chem. Lett. 2005, 15, 905.
References and notes
1. Cheng, J. Q.; Godwin, A. K.; Bellacosa, A.; Taguchi, T.;
Franke, T. F.; Hamilton, T. C.; Tsichlis, P. N.; Testa, J. R.
Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 9267.
2. Cheng, J. Q.; Ruggeri, B.; Klein, W. M.; Sonoda, G.;
Altomare, D. A.; Watson, D. K.; Testa, J. R. Proc. Natl.
Acad. Sci. U.S.A. 1996, 93, 3636.
3. Staal, S. P. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 5034.
4. Bellacosa, A.; Defeo, D.; Godwin, A. K.; Bell, D. W.;
Cheng, J. Q.; Altomare, D. A.; Wan, M. H.; Dubeau, L.;
Scambia, G.; Masciullo, V.; Ferrandina, G.; Panici, P. B.;
Mancuso, S.; Neri, G.; Testa, J. R. Int. J. Cancer 1995, 64,
280.
5. Jones, P. F.; Jakubowicz, T.; Hemmings, B. A. Cell
Regulation 1991, 2, 1001.
6. Nakatani, K.; Thompson, D. A.; Barthel, A.; Sakaue, H.;
Liu, W.; Weigel, R. J.; Roth, R. A. J. Biol. Chem. 1999,
274, 21528.
22. Livnah, N.; Levitzki, A.; Senderovitz, H.; Yechezkel, T.;
Salitra, Y.; Litman, P.; Ohne, O.; (Develogen Israel Ltd.,
Israel). WO 2004/110337.
23. Luo, Y.; Smith, R. A.; Guan, R.; Liu, X. S.; Klinghofer,
V.; Shen, J. W.; Hutchins, C.; Richardson, P.; Holzman,
T.; Rosenberg, S. H.; Giranda, V. L. Biochemistry 2004,
43, 1254.
24. Livnah, N.; Yechezkel, T.; Salitra, Y.; Perlmutter, B.;
Ohne, O.; Cohen, I.; Litman, P.; Senderowitz, H.; (Peptor
Ltd., Israel). WO 2003/010281.
25. Parang, K.; Cole, P. A. Pharmacol. Ther. 2002, 93, 145.
26. Yang, J.; Cron, P.; Good, V. M.; Thompson, V.; Hem-
mings, B. A.; Barford, D. Nat. Struct. Biol. 2002, 9, 940.
27. Alessi, D. R.; Caudwell, F. B.; Andjelkovic, M.; Hem-
mings, B. A.; Cohen, P. FEBS Lett. 1996, 399, 333.
28. Molecular Devices, IMAPÒ technology <http://
www.moleculardevices.com/pages/reagents/imap.html>.
29. Jones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor,
R. J. Mol. Biol. 1997, 267, 727.
7. Brodbeck, D.; Cron, P.; Hemmings, B. A. J. Biol. Chem.
1999, 274, 9133.
8. Downward, J. Curr. Opin. Cell Biol. 1998, 10, 262.
9. Chan, T. O.; Rittenhouse, S. E.; Tsichlis, P. N. Annu. Rev.
Biochem. 1999, 68, 965.
30. Alessi, D. R.; Andjelkovic, M.; Caudwell, B.; Cron, P.;
Morrice, N.; Cohen, P.; Hemmings, B. A. EMBO J. 1996,
15, 6541.
10. Kandel, E. S.; Hay, N. Exp. Cell Res. 2000, 254, 196.
11. Downward, J. Nat. Cell Biol. 1999, 1, E33.
31. Azumaya, I.; Okamoto, T.; Imabeppu, F.; Takayanagi, H.
Tetrahedron 2003, 59, 2325.