LY294002-geldanamycin heterodimers as selective inhibitors of the PI3K and PI3K-related family

Article abstract of DOI:10.1016/S0960-894X(01)00099-3

Several LY294002-GM heterodimers were synthesized with the intent of modulating their activity in the presence of hsp90 and thereby creating selective inhibitors of PI3K and PI3K-related family.

Full text of DOI:10.1016/S0960-894X(01)00099-3

Bioorganic & Medicinal Chemistry Letters 11 (2001) 909–913
LY294002-geldanamycin Heterodimers as Selective Inhibitors of
the PI3K and PI3K-related Family
y
Gabriela Chiosis,* Neal Rosen and Laura Sepp-Lorenzino
Department of Molecular Oncogenesis, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA
Received 29 September 2000; accepted 6February 2001
Abstract—Several LY294002-GM heterodimers were synthesized with the intent of modulating their activity in the presence of
hsp90 and thereby creating selective inhibitors of PI3K and PI3K-related family. # 2001 Elsevier Science Ltd. All rights reserved.
The specificity and affinity of a ligand–protein interac-
tion can be modulated by borrowing additional surface
contacts from another protein. By linking a weak and/
or unselective ligand to the ligand of a physiologically
abundant protein one could envision increasing the effi-
cacy of the interaction if the created contact among the
protein partners is favorable. In this fashion, selective
inhibition of proteins that have similar pockets could be
achieved.
types form tight heterodimers with a p85 regulatory
unit, the assembly being activated by binding to auto-
phosphorylated growth factor receptors or their sub-
strates. Other PI3Ks (e.g., p110g) seem to propagate
signals from seven transmembrane helix receptors as
they are activated by G-protein bg-subunits. The PI3K-
related proteins, ataxia-telangiectasia mutated (ATM),
ataxia-telangiectasia related (ATR) and DNA-depen-
dent protein kinase (DNA-PK) play important roles in
checkpoints that operate to permit cell survival follow-
ing many forms of DNA damage. Emerging experi-
mental evidence suggests that although these proteins
posses similar substrate specificity, they exhibit over-
lapping but distinct functions in vivo. ATM, ATR, and
DNA-PK have been involved in regulating p53, and
additionally, ATM has been found to play an important
role in the control of the non-receptor tyrosine kinase
Abl. Their counterparts in yeast have been identified as
rad3p, Mec1p, Tel1p, and Esr1p. Another PI3K-related
protein that possesses the kinase domain is FRAP
1
Phosphoinositol-3 kinase (PI3K) and a newly emerging
subfamily called PI3K-related kinases share a similar
2
carboxy-terminus catalytic domain. Given the sequence
homology in the kinase domain of these proteins it is
expected that they will all be inactivated by the known
3
PI3K inhibitors wortmannin and LY294002. The PI3K
kinases consist of enzymes composed of various catalytic
subunits of the p110a, p110b, and p110g and the yeast
homologue Vps34p types. The p110a and p110b sub-
(mTOR). FRAP is homologous to the yeast Tor1p/2p
believed to control aspects of several diverse cellular
functions, protein stability, translocation and transla-
tion as well as glucose metabolism. For example, FRAP
controls pathways that are activated by transmembrane
s6
receptors, such as regulation of p70 kinase.
Selective inhibitors of the members of this family
would constitute important tools for investigating
the regulatory functions mediated by each protein.
Additionally, due to the radiosensitive phenotypes
exhibited by cells defective in ATM, ATR, or DNA-PK,
inhibitors of these kinases might enhance the cytotoxic
effects of ionizing radiation or DNA damaging cancer
chemotherapeutic agents.
Figure 1. The protein assembly takes place only if favorable interac-
tions among the two partners are allowed by the nature of the het-
erodimer inhibitor.
*Corresponding author. Fax: +1-212-717-3627; e-mail: chiosisg@
mskmail.mskcc.org
y
Present address: Merck & Co., Inc. MRL WP16-327, West Point, PA
19486, USA.
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00099-3

910
G. Chiosis et al. / Bioorg. Med. Chem. Lett. 11 (2001) 909–913
In this paper we describe a new strategy for the synth-
esis of selective PI3K inhibitors. An LY294002-like
molecule was chosen as the kinase domain inhibitor and
Due to the relatively similar inhibitory effects of the 7-
and 8-substituted derivatives in the LY series, we chose
LY292223 as the kinase domain-binding skeleton. OH
or Br derivatized 2-hydroxy-3-phenylacetophenones
were chosen to be the starting points for the facile
reconstruction of such a skeleton (Scheme 1). These
functionalities permit the attachment of several linkers
through a Mitsunobu or a modified Sagatoshira reac-
tion, respectively. Construction of the LY-skeleton was
performed, with the appropriate modifications, based
4
the hsp90 chaperone as the interaction modulator pro-
tein (Fig. 1). The choice of this protein comes from its
abundance in the cytosol and the existence of a high
5
affinity binder, geldanamycin (GM). Additionally,
hsp90 is expressed at 2- to 10-fold higher levels in tumor
cells compared to their normal counterparts. One would
expect that given the abundance of hsp90 and the high
affinity of GM for the protein, heterodimers of
the LY294002-GM type would predominantly bind to
hsp90 and be presented to the PI3K family only if
the interactions in this tertiary complex are favorable
6
on the method published by the Eli Lilly group. Con-
densation of the benzyl protected hydroxy-acet-
ophenones (1) with carbon disulphide in the presence of
potassium t-butoxide in benzene gave the correspond-
ing thionocoumarins. These were converted to thio-
ethers by alkylation with iodoethane and potassium
carbonate.
(
Fig. 1).
A very important factor in determining the nature of the
interaction would be the length, nature, and mode of
attachment of the linker. A preferential complex for-
mation would result in selective binding and, therefore,
compounds with more restricted cellular effects than the
parent LYs.
If the linkers were to be attached through the Mitsu-
nobu reaction, the benzyl group was first deprotected
using boiling trifluoroacetic acid. Alkylation was carried
out on the resulting phenols (2) with several N-Boc
protected amino alcohols in a mixture of toluene/di-
chloromethane. After the attachment of the linker, the
2-sulphinyl moiety was replaced by morpholinyl
through a nucleophilic displacement by simple refluxing
in morpholine under acetic acid catalysis. Having the
LY-skeleton and the linker assembled, GM was intro-
duced through the ability of this molecule to undergo
smooth Michael reactions with primary amines.
ꢀ
Scheme 1. Synthesis of the LY-GM heterodimers. (a) BnBr, K
ꢀ
2
CO , DMF, 6 0 C, 16h; (b) 10 equiv t-BuOK, 3 equiv CS
3
2
, benzene, 16h; (c) EtI,
, 1.3 equiv DEAD, toluene/DCM (10:1),
K
2
CO , acetone, reflux 30 min; (d) TFA, PhSMe, 70 C, 16h; (e) 3 equiv HO-linker-NHBoc, 1.3 equiv PPh
3
8 h; (f) morpholine, cat. AcOH, 75 C, 2–3 h; (g) TFA/DCM (1:4), 1 h, rt; (h) GM, 3 equiv TEA, DMF, 15–24 h; (i) AlCl
3
ꢀ
ꢀ
1
3
, 120 C, 16h; (j) 1.7 equiv
N-Boc protected alkyl-e-ynylamine, Pd(PPh , PPh , CuI, morpholine.
3
)
4
3

G. Chiosis et al. / Bioorg. Med. Chem. Lett. 11 (2001) 909–913
911
If the attachment mode is the Sagatoshira reaction, the
whole LY-skeleton is assembled first to give 2-morpho-
linyl-8-bromochromone (3), followed by addition of the
N-Boc protected alkyl-e-ynylamine linker. After stan-
dard Boc deprotection, the resulting amine was coupled
to GM, giving the derivatives PI3K-1 and PI3K-2.7
members are very weak inhibitors of PI3K with IC s
50
over 300 mM, however, they inhibit DNA-PK at con-
centrations comparable to the parent Lilly compound.
The most selective candidate, LY6-GM is over two
orders of magnitude more active against DNA-PK than
PI3K. The LYnMe-GM series shows only 1.5- to 16-
fold selectivity, while, most surprisingly, the PI3K-n-
GM series has the exact potency in inhibiting both
proteins. GM (at 40 mM) and DMSO (8% v/v) had no
effect on the activity of DNA-PK.
To assess the effect of the mode of attachment and size
of the linker on the activity of these dimers, we tested
them for their ability to inhibit PI3K and DNA-PK,
and additionally, to compete with solid-phase immobilized
GM for hsp90 binding.
Hsp90 binding assays10
GM immobilized on solid phase was utilized to deter-
mine the ability of the synthesized heterodimers to bind
to the hsp90 a protein. Drugs were pre-incubated with
protein and then added to the GM-beads. After an
incubation time, the amount of protein left for binding
to the solid support was separated and quantified. The
ability of drugs to compete with the immobilized GM
for 50% of the hsp90 was measured (Table 1). It was
expected that the addition of a floppy linker on the GM
molecule would diminish somewhat its affinity for
hsp90. It is rather unexpected however, that some
small variations in the linker substantially affected
hsp90 binding. The only difference between LY5-GM
and LY4O-GM is the substitution in the linker of one
C atom with O, however, the latter is 6times less active.
Inhibition of PI3-kinase by heterodimers8
The PI3K enzyme was immunopurified from insulin
stimulated MCF-7 breast cancer cell lines using protein
A immobilized anti-p85 antibody. Its ability to catalyze
the phosphorylation of phosphatidylinositol by ATP in
the presence and absence of drugs was determined. The
hybrids LY3-GM, LY4-GM, LY5-GM, LY6-GM, and
LY4O-GM showed significantly lower activity than the
parent compound LY292223 (Table 1). Surprisingly,
LY4OMe-GM, LY6Me-GM, and LY7Me-GM that
differ from the above series just by the presence of a
methyl group at the 8 position of the LY-skeleton,
exhibited potencies comparable to the Lilly derivative,
while the derivatives PI3K-1-GM, PI3K-2-GM, and
LY6Me-GM were more potent. Free GM (at 500 mM)
and DMSO (2.5% v/v) had no effect on the kinase
activity of PI3K at the studied concentrations (data not
shown).
Modulation of LY5-GM binding to DNA-PK by hsp90
LY5-GM is the tightest hsp90 a binder and a modest
DNA-PK inhibitor. It was, therefore, an attractive
candidate to study the influence of hsp90 on kinase
activity. Pre-incubation of the heterodimer with hsp90 a
increased its inhibition of DNA-PK by 50% (Table 1).
No effect on enzyme activity was observed in the
samples that hsp90 was pre-incubated with DMSO (not
shown).
Inhibition of DNA-PK by heterodimers9
A peptidic sequence derived from p53 was used as the
substrate to determine the kinase activity of DNA-PK
in the presence of the heterodimers. There is a consistent
correlation between linker attachment site and potency
of inhibition of DNA-PK and PI3-K. All the LYn-GM
The study points out how unpredictable and important
a small modification in the nature, length, and mode of
attachment of the linker can be. The presence of a
methyl group at the 8 position of LY292223 seems to
have a crucial role in determining the selectivity of the
inhibitors. The methylated derivatives are good PI3K
and DNA-PK inhibitors and have modest affinity for
Hsp90. Meanwhile, compounds without the 8-methyl
are good DNA-PK inactivators and maintain good
hsp90 binding, however, PI3K is 3 log more resistant to
their action. DNA-PK may be important for suppres-
sion of apoptosis. Therefore, compounds that selectively
affect DNA-PK and not PI3K may be of therapeutic
use in cancer and other disorders.
Table 1. Inhibition of PI3K activity and competition for hsp90
binding by the synthesized heterodimers
a
a
Compounds IC50, mM for
inhibition of
PI3K activity
IC50, mM for
inhibition of
EC50, mM drug
necessary to
DNA-PK activity compete for
0% of Hsp90
a binding
5
ꢁ
hsp90 a +hsp90 a
17AAG
WTM
LY292223
LY3-GM
LY4-GM
>500
ND
>40
0.02
1
ꢂ50
ꢂ50
26
10
2.5
4
16
>50
46
14 (ꢃ2)
4.3 (ꢃ0.6)
>500
200 (ꢃ12) 13.9 (ꢃ0.8)
28 (ꢃ1)
LY5-GM
LY6-GM
310 (ꢃ23)
10 (ꢃ0.4) 5 (ꢃ0.1)
>500
85 (ꢃ15)
6.1 (ꢃ0.2)
33 (ꢃ1.8)
It is noteworthy that PI3K-2-GM has increased PI3-
kinase and DNA-PK inhibitory effect compared to the
parent Lilly derivative and binding for Hsp90 analogous
to 17AAG.
LY4O-GM
LY4OMe-GM
LY6Me-GM
LY7Me-GM
PI3K-1-GM
PI3K-2-GM
70 (ꢃ13) 12.2 (ꢃ1.8)
5 (ꢃ1.5) 3.7 (ꢃ1)
28 (ꢃ3)
1.75 (ꢃ0.15)
28
ꢂ50
13.5 (ꢃ2.5) 13.4 (ꢃ1.3)
3 (ꢃ1) 3 (ꢃ0.2)
7
Our study suggests that it is possible to modulate the
activity of dual ligands, at least in in vitro systems.
Additionally, it implies that the strategy outlined here
aValues are means of two experiments, standard deviation is given in
parentheses. 17AAG is a GM derivative with similar potency.

912
G. Chiosis et al. / Bioorg. Med. Chem. Lett. 11 (2001) 909–913
can be used to generate a family of inhibitors that dif-
ferentially inhibit members of the PI3K-family. Screen-
ings in mammalian cell culture systems dependent on
various PI3K-related members or in functional yeast
hybrid systems will assess the in vivo activity of the
heterodimers.
J=11.5 Hz), 6.88 (d, 1H, J=8.7 Hz), 6.54 (m, 2H), 5.87 (m,
2H), 5.42 (s, 1H), 5.19 (s, 1H), 4.73 (bs, 2H), 4.27 (m, 3H),
3
3
.93 (m, 2H), 3.86 (m, 6H), 3.71 (m, 2H), 3.49 (m, 6H), 3.34 (s,
H), 3.26 (s, 3H), 2.68 (m, 2H), 2.32 (m, 1H), 2.26 (s, 3H), 1.65
(
m/z 877.6(M+H).
m, 3H), 0.99 (d, 3H, J=6.9 Hz), 0.95 (d, 3H, J=6.6 Hz). MS
1
LY6Me-GM: H NMR (400 MHz, CDCl
.96(d, 1H, J=8.7 Hz), 7.25 (s, 1H), 6.94 (d, 1H, J=12 Hz),
3
) d 9.12 (s, 1H),
7
6
5
1
.88 (d, 1H, J=8.7 Hz), 6.57 (t, 1H, J=11.1 Hz), 6.26 (bt, 1H),
.88 (m, 2H), 5.42 (s, 1H), 5.17 (s, 1H), 4.72 (bs, 2H), 4.28 (d,
H, J=10 Hz), 4.07 (m, 2H), 3.83 (m, 4H), 3.55 (m, 7H), 3.32
Acknowledgements
The work was funded in part by the Leukemia Research
Foundation (GC), the Belfer Foundation and a SPORE
grant.
(s, 3H), 3.25 (s, 3H), 2.69 (m, 2H), 2.39 (m, 1H), 2.24 (s, 3H),
2.01 (s, 3H), 1.84 (m, 2H), 1.79 (s, 3H), 1.74 (m, 4H), 1.57 (m,
4
8
H), 0.99 (d, 3H, J=6.9 Hz), 0.95 (d, 3H, J=6.6 Hz). MS m/z
89.6(M+H).
1
LY7Me-GM: H NMR (400 MHz, CDCl ) d 9.16(s, 1H),
3
7
6
.95 (d, 1H, J=8.5 Hz), 7.26(s, 1H), 6. 94 (d, 1H, J=12 Hz),
.86 (d, 1H, J=8.5 Hz), 6.57 (t, 1H, J=11.5 Hz), 6.24 (bt, 1H),
References and Notes
5.87 (m, 2H), 5.43 (s, 1H), 5.19 (s, 1H), 4.85 (bs, 2H), 4.30 (d,
H, J=9.9 Hz), 4.15 (m, 2H), 3.83 (m, 4H), 3.55 (m, 2H), 3.46
1
1
. Briesewitz, R.; Ray, G. T.; Wandless, T. J.; Crabtree, G. R.
Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 1953.
. Kuruvilla, F. G.; Schreiber, S. L. Chem. Biol. 1999, 6,
R129.
. Sarkaria, J. N.; Tibbets, R. S.; Busby, E. C.; Kennedy,
A. P.; Hill, D. E.; Abraham, R. T. Cancer Res. 1998, 58, 4375.
(m, 5H), 3.35 (s, 3H), 3.26(s, 3H), 2. 62 (m, 2H), 2.35 (m, 1H),
2.25 (s, 3H), 2.00 (s, 3H), 1.82 (m, 2H), 1.78 (s, 3H), 1.72 (m,
3H), 1.44 (m, 6H), 0.99 (d, 3H, J=6.9 Hz), 0.94 (d, 3H,
2
J=6.6 Hz). MS m/z 902.6(M+H).
1
3
PI3K-1-GM: H NMR (400 MHz, CDCl
3
) d 9.12 (s, 1H),
8.08 (d, 1H, J=7.9 Hz), 7.62 (d, 1H, J=7.5 Hz), 7.27 (m, 2H),
6.93 (d, 1H, J=11.3 Hz), 6.58 (t, 1H, J=11.5 Hz), 6.37 (bt,
1H), 5.87 (m, 2H), 5.50 (s, 1H), 5.19 (s, 1H), 4.79 (bs, 2H),
4.29 (d, 1H, J=9.8 Hz), 4.15 (bs, 1H), 3.85 (m, 4H), 3.72 (m,
2H), 3.54 (m, 5H), 3.33 (m, 1H), 3.27 (s, 3H), 3.26(s, 3H), 2.66
(m, 4H), 2.41 (m, 1H), 2.08 (s, 3H), 2.00 (m, 1H), 1.78 (m, 3H),
1.67 (m, 5H), 1.01 (d, 3H, J=6.9 Hz), 0.92 (d, 3H, J=6.6 Hz).
4
5
. Buchner, J. Trends Biochem. Sci. 1999, 24, 136.
. Whitesell, L.; Mimnaugh, E. G.; De Costa, B.; Myers, C. E.;
Neckers, L. M. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 8324.
. Vlahos, C. J.; Matter, W. F.; Hui, K. Y.; Brown, R. F. J.
Biol. Chem. 1994, 269, 5241.
6
1
7
3
. LY6-GM: H NMR (400 MHz, CDCl ) d 9.18 (s, 1H), 8.03
(d, 1H, J=8.8 Hz), 7.27 (s, 1H), 6.95 (d, 1H, J=11.2 Hz), 6.88
(dd, 1H, J=8.8 Hz, J=2.2 Hz), 6.73 (d, 1H, J=2.2 Hz), 6.58
(t, 1H, J=11.4 Hz), 6.28 (bt, 1H), 5.84 (m, 2H), 5.42 (s, 1H),
MS m/z 841.7 (M+H).
1
PI3K-2-GM: H NMR (400 MHz, CDCl
3
) d 9.09 (s, 1H),
8.14 (d, 1H, J=7.9 Hz), 7.63 (d, 1H, J=7.5 Hz), 7.33 (s, 1H),
7.31 (dd, 1H, J=7.9 Hz, J=7.5 Hz), 6.95 (d, 1H, J=11.5 Hz),
6.58 (t, 1H, J=11.7 Hz), 6.37 (bt, 1H), 5.88 (m, 2H), 5.50 (s,
1H), 5.21 (s, 1H), 4.74 (bs, 2H), 4.58 (m, 2H), 4.32 (d, 1H,
J=9.5 Hz), 3.91 (m, 1H), 3.82 (m, 4H), 3.55 (m, 6H), 3.36 (s,
3H), 3.28 (s, 3H), 2.72 (m, 2H), 2.41 (m, 1H), 2.04 (s, 3H), 1.80
(s, 3H), 1.72 (m, 1H), 1.03 (m, 6H). MS m/z 813.7 (M+H).
8. MCF-7 cells were serum starved for 24 h and stimulated
5
.19 (s, 1H), 4.76(bs, 2H), 4.28 (m, 2H), 4.03 (t, 2H,
J=5.2 Hz), 3.81 (m, 4H), 3.56(m, 2H), 3.45 (m, 6H ), 3.35 (s,
H), 3.29 (s, 3H), 2.71 (m, 2H), 2.37 (m, 1H), 2.05 (s, 3H), 1.77
m, 10H), 1.48 (m, 4H), 1.25 (m, 2H), 1.00 (d, 3H, J=6.9 Hz),
3
(
0
.96(d, 3H, J=6.5 Hz). MS m/z 897.6(M+Na).
1
LY5-GM: H NMR (400 MHz, CDCl ) d 9.16(s, 1H), 8.03
3
(d, 1H, J=8.8 Hz), 7.26(s, 1H), 6. 94 (d, 1H, J=11.7 Hz), 6.88
(dd, 1H, J=8.8 Hz, J=2.2 Hz), 6.71 (d, 1H, J=2.2 Hz), 6.57
(t, 1H, J=11.4 Hz), 6.28 (bt, 1H), 5.86 (m, 2H), 5.47 (s, 1H),
with 1 mM insulin. Cell extracts were made in PI3K lysis buffer
.
(137 mM NaCl, 20 mM Tris HCl, pH 5, 1 mM MgCl
2
, 1 mM
5
.18 (s, 1H), 4.82 (bs, 2H), 4.29 (d, 1H, J=9.9 Hz), 4.03 (t, 2H,
J=6.0 Hz), 3.81 (m, 4H), 3.56 (m, 2H), 3.50 (m, 5H), 3.34 (s,
H), 3.25 (s, 3H), 2.68 (m, 2H), 2.37 (m, 1H), 2.02 (s, 3H), 1.77
m, 2H), 1.76 (s, 3H), 1.68 (m, 6H), 1.25 (m, 2H), 1.00 (d, 3H,
J=6.9 Hz), 0.96 (d, 3H, J=6.5 Hz). MS m/z 883.8 (M+Na).
2
CaCl , 10% v/v glycerol, 1% v/v Triton X-100) and the
enzyme was immunoprecipitated using anti-p85 antibody
(Upstate Biotechnologies Inc. no. 06-195). The complex was
immobilized on Protein A beads (Amersham) and after several
3
(
.
washes (3ꢄ1% Triton X-100 in PBS, 2ꢄ0.1 M Tris HCl, pH
1
.
LY4-GM: H NMR (400 MHz, CDCl
3
) d 9.15 (s, 1H), 8.05
7.5, 0.5 M LiCl, 1ꢄ10 mM Tris HCl, pH 7.5, 100 mM NaCl,
(d, 1H, J=8.8 Hz), 7.27 (s, 1H), 6.94 (d, 1H, J=12.0 Hz), 6.89
(dd, 1H, J=8.8 Hz, J=2.2 Hz), 6.73 (d, 1H, J=2.2 Hz), 6.57
(t, 1H, J=11.3 Hz), 6.32 (bt, 1H), 5.86 (m, 2H), 5.42 (s, 1H),
1 mM EDTA) the immunoprecipitates were subjected to PI3-
kinase activity assays. The lipid mix in 10 mM Hepes pH 7,
1 mM EGTA (final concentration 0.5 mM/mL) was incubated
with the immobilized enzyme with or without drugs at room
temperature for 15 min. The reaction was started upon addi-
5
1
.19 (s, 1H), 4.76(bs, 2H), 4.30 (d, 1H, J=9.9 Hz), 4.24 (bs,
H), 4.06(t, 2H, J=5.0 Hz), 3.80 (m, 4H), 3.53 (m, 3H), 3.48
3
2
(
m, 5H), 3.31 (s, 3H), 3.26(s, 3H), 3.08 (m, 2H), 2. 67 (m, 2H),
tion of 10 mL of [g- P]ATP (5 mCi/mL) and was allowed to
proceed for 20 min at 37 C after which was quenched by
ꢀ
2
4
.39 (m, 1H), 2.02 (s, 3H), 1.99 (m, 4H), 1.78 (s, 3H), 1.68 (m,
H), 1.25 (m, 2H), 0.99 (d, 3H, J=6.9 Hz), 0.94 (d, 3H,
addition of 80 mL 1 M HCl. Lipids were extracted with 160 mL
J=6.5 Hz). MS m/z 869.8 (M+Na).
1
3 3
CHCl /CH OH (1:1). The organic layer was applied to a pre-
LY3-GM: H NMR (400 MHz, CDCl ) d 9.15 (s, 1H), 8.05
activated silica gel plate (EM Bioscience) and eluted with n-
propanol/methanol/2 M AcOH (50:15:35). Plates were visual-
ized by autoradiography and the lipids quantified using
BioRad Gel Doc 1000 software.
3
(
d, 1H, J=8.8 Hz), 7.27 (s, 1H), 6.92 (m, 2H), 6.82 (d, 1H,
J=2.2 Hz), 6.68 (bt, 1H), 6.56 (t, 1H, J=11.3 Hz), 5.87 (m,
H), 5.69 (s, 1H), 5.17 (s, 1H), 4.84 (bs, 2H), 4.29 (d, 1H,
J=9.9 Hz), 4.17 (t, 2H, J=5.3 Hz), 3.82 (m, 5H), 3.69 (m, 1H),
2
1
9. SignaTECT DNA-PK assay system from Promega was
3
2
.53 (m, 5H), 3.42 (m, 1H), 3.34 (s, 3H), 3.25 (s, 3H), 2.68 (m,
H), 2.42 (m, 1H), 2.21 (m, 2H), 2.00 (s, 3H), 1.71 (s, 3H), 1.69
used with small modifications from the manufacturer’s
instructions. To each drug in 2 mL DMSO or to 2 mL DMSO
were added 23 mL reaction mix [250 ng DNA from calf thymus
(Sigma#D-3664), 0.2 mM biotinilated peptide substrate, 20
(
m, 3H), 1.25 (m, 2H), 0.97 (m, 6H). MS m/z 855.8 (M+Na).
1
LY4OMe-GM: H NMR (400 MHz, CDCl
.96(d, 1H,= J=8.7 Hz), 7.25 (s, 1H), 6.94 (d, 1H,
3
) d 9.11 (s, 1H),
3
2
7
units of DNA-PK (Promega no. V5811), 1 mCi [g- P]ATP,

G. Chiosis et al. / Bioorg. Med. Chem. Lett. 11 (2001) 909–913
913
2
MgCl , 1 mM EGTA, 0.5 mM EDTA and 5 mM DTT and
mg BSA in 250 mM HEPES, pH 7.5, 500 mM NaCl, 50 mM
10. GM was immobilized on Affigel 10 resin (BioRad) as
ꢀ
described in ref 5. The GM-beads were blocked for 1 h at 4 C
.
2
ꢀ
the reaction mix was incubated for 10 min at 30 C. After
addition of 12.5 mL guanidine hydrochloride 7.5 M, each
with 0.5% BSA in TEN buffer (50 mM Tris HCl pH 7.4, 1 mM
EDTA, 1% NP-40) prior to use. Hsp90 a protein (Stressgen)
was incubated with or without drugs for 20 min on ice. To
each sample were added 20 mL GM-beads and the mixtures
1
reaction was spotted to a SAM2 Biotin capture membrane
(
and 1% H PO in 2 M NaCl, the samples were quantifies
Promega no. 2861). After several washes with 2 M NaCl
ꢀ
3
4
were rotated at 4 C for 1 h followed by two washes with
using a scintillation counter. For the interaction modulation
assays the drugs were pre-incubated for 17 min on ice with
200 ng hsp90 a in PBS prior to the addition of the kinase
mix.
500 mL ice cold TEN each. The GM-beads bound protein was
eluted from the solid phase by heating in 35 mL 1ꢄSDS, ana-
lyzed by SDS/PAGE and visualized by immunoblotting with
Hsp90 a (Stressgen no. SPA-840).