Biotinylated geldanamycin

Article abstract of DOI:10.1021/jo049848m

Inhibition of the 90 kDa heat shock proteins (Hsp90) represents a promising new chemotherapeutic approach for the treatment of several cancers. Hsp90 is essential to the survival of cancer cells and is inhibited by members of the ansamycin family of antib

Full text of DOI:10.1021/jo049848m

Biotin yla ted Geld a n a m ycin
Randell C. Clevenger, J oseph M. Raibel, Angela M. Peck, and Brian S. J . Blagg*
Department of Medicinal Chemistry and The Center for Protein Structure and Function, The University
of Kansas, 1251 Wescoe Hall Dr., Malott Hall 4070, Lawrence, Kansas 66045-7564
bblagg@ku.edu
Received J anuary 26, 2004
Inhibition of the 90 kDa heat shock proteins (Hsp90) represents a promising new chemotherapeutic
approach for the treatment of several cancers. Hsp90 is essential to the survival of cancer cells
and is inhibited by members of the ansamycin family of antibiotics. In particular, the quinone-
containing antibiotics geldanamycin (GDA) and herbimycin A inhibit Hsp90 function in vitro at
low micromolar concentrations via interaction with an ATP binding domain. Many proteins bind
ATP, and the discovery of selective Hsp90 inhibitors requires the identification of other proteins
that bind GDA and may cause undesired effects. Biotinylated analogues of GDA with varying tether
lengths have been synthesized to elucidate other proteins that competitively bind GDA. Analogues
containing a photolabile tether have also been prepared as a complementary method for the removal
of GDA-bound proteins from neutravidin-containing resin. Preliminary studies indicate several
proteins other than Hsp90 are isolated with biotinylated GDA.
In tr od u ction
Heat shock proteins (Hsp’s) are molecular chaperones
responsible for protein transport, conformational activa-
tion, disaggregation, and maturation of nascent polypep-
tides.1 Recent studies have shown that a large number
of disease states occur as a consequence of Hsp function.2
The 90 kDa heat shock proteins (Hsp90) are overex-
pressed in cancer cells, and these increased levels are
essential for maintaining high intracellular concentra-
tions of active oncogenic proteins, including Raf-1, v-Src,
steroid hormone receptors, and many others.3 In fact,
proteins involved in all six hallmarks of cancer are
dependent upon Hsp90 for their conformational matura-
tion.4 Consequently, Hsp90 inhibition represents a prom-
ising new approach to cancer chemotherapeutic devel-
F IGURE 1. Inhibitors of Hsp90.
opment by the simultaneous inhibition of multiple
oncogenic targets.5
(1) (a) Rutherford, S. L.; Lindquist, S. Nature 1998, 396, 336-342.
(b) Toft, D. O. Trends Endocr. Metab. 1998, 9, 238-243. (c) Buchner,
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Biochem. 2001, 70, 603-649.
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1113.
Originally geldanamycin (GDA, 1, Figure 1) was
believed to be an inhibitor of Src kinase,6 but later studies
by Whitesell and Neckers demonstrated that GDA binds
to Hsp90.7 In their studies, they prepared affigel-10
covalently bound to GDA and were successfully able to
isolate Hsp90 by affinity purification. At the time, other
proteins were also observed, but the identity of those
proteins remained unknown. Subsequent studies have
shown Src kinase to be an Hsp90-dependent client
protein and that inhibition of Hsp90 leads to drastic
reduction in Src kinase activity.8 GDA and herbimycin
A (3, Figure 1) inhibit Hsp90’s inherent ATPase activity
(3) (a) Hartman, F.; Horak, E. M.; Cho, C.; Lupu, R.; Bolen, J . B.;
Stetler-Stevenson, M. A.; Pferundschuh, M.; Waldmann, T. A.; Horak,
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M. V.; Ingui, C.; Neckers, L. J . Biol. Chem. 1995, 270, 24585-24588.
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10.1021/jo049848m CCC: $27.50 © 2004 American Chemical Society
Published on Web 05/25/2004
J . Org. Chem. 2004, 69, 4375-4380
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Raible et al.
in vitro at low micromolar concentrations.9 However, in
vivo GDA is a 100 nM inhibitor of the Hsp90 multiprotein
complex.10 The poor bioavailability and high cytotoxicity
of GDA has led to the development of 17-allylamino
geldanamycin (17-AAG, 2), which recently entered Phase
I clinical trials for the treatment of several cancers.9a,11
Although better tolerated, 17-AAG exhibits toxicity un-
related to Hsp90 inhibition and has formulation difficul-
ties.4,12
F IGURE 2. Affinity purification of GDA-binding proteins.
have a predisposed bent conformation.14,16 The flexibility
observed in the GDA macrocycle is likely to allow GDA
to bind other ATP-dependent proteins in a similar
fashion. The toxicity of GDA and analogues unrelated to
Hsp90 inhibition may be the result of binding to similarly
shaped ATP binding motifs. The design of analogues with
selective affinity for Hsp90 requires the identification of
other proteins that bind GDA as a control for the design
of future Hsp90 inhibitors.
In an effort to identify GDA-binding proteins, biotin-
ylated derivatives of GDA have been prepared for affinity
purification of these proteins. Addition of neutravidin-
containing resin enables the isolation of GDA binding
proteins (Figure 2).
Examination of the cocrystal structure of GDA bound
to both bovine13a and yeast14a Hsp90 revealed that both
the free hydroxyl and carbamate group reside deep within
the ATP binding site and thus were not suitable moieties
for the incorporation of biotinylated linkers. Methoxy
quinones undergo nucleophilic substitution reactions,9c,d,18
and provide an alternative method for biotin introduction.
The cocrystal structure of GDA bound to Hsp90 shows
the quinone moiety to reside near the protein-solution
interface of the nucleotide binding domain, with the
methoxy group directed away from the interior of the
protein. Replacement of the methoxy group with an
appropriate tether19 provides a solvent exposed biotin
GDA and 17-AAG inhibit Hsp90 by competing with
ATP binding to a highly conserved nucleotide binding site
located near the N-terminus of the homodimeric pro-
tein.13 Unlike most ATP binding sites, the N-terminal
ATP binding site has a unique bent conformation, requir-
ing both ATP and GDA to adopt a folded or puckered
shape upon binding to Hsp90, as determined by cocrystal
structures.13,14 Although this bent shape may lead to
some selectivity of GDA for Hsp90 versus other ATP
binding proteins, it is likely that GDA shares a high
affinity for proteins other than Hsp90, as a consequence
of normal evolutionary processes.15 Furthermore, the bent
conformation of GDA differs significantly from its native
crystallographic form.14 It has been suggested that this
change in conformation results in an affinity of GDA for
Hsp90 lower than that of other Hsp90 inhibitors, which
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L.; Moyer, J . D.; DiOrio, C. I.; Barbacci, E. G.; Miller, P. E.; O’Brien,
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G.; Huezo, H.; Rosen, N.; Mimnaugh, E.; Whitesell, L.; Neckers, L.
Mol. Cancer Ther. 2003, 2, 123-129.
SCHEME 1. Syn th esis of GDA Am in es
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Biotinylated Geldanamycin
SCHEME 2. Syn th esis of Biotin yla ted GDA
SCHEME 3. P h otola bile Lin k er
the amino quinone as evidenced by the appearance of a
purple solution resulting from vinylagous amide forma-
tion. The reaction was complete within 30 min as
demonstrated by reverse-phase HPLC to provide the
desired products in >96% purity. Extended reaction
times resulted in the formation of a complex mixture of
products and low yields of the desired vinylagous amides.
In solution, these compounds decompose at -20 °C and
were either stored as a purple solid or used immediately
in subsequent reactions.
Coupling of the N-hydroxysuccinimide (NHS)-activated
ester 820 with 6 and 7 provided the biotinylated GDA
analogues 10 and 11, respectively (Scheme 2). The
reaction was complete within 20 min and provided the
easily separable purple product 10, in 89% yield. Like-
wise, 11 was prepared in an analogous fashion, providing
the desired product in 69% yield. A more hydrophilic
SCHEME 4. Con str u ction of P h otola bile Lin k er s
handle capable of interacting with neutravidin for affinity
purification. The incorporation of a photolabile group in
the GDA-biotin tether provided an alternative method
for removal of GDA binding proteins from the im-
mobilized complex. SDS-PAGE followed by peptide
sequencing serves to isolate and identify proteins other
than Hsp90 that bind GDA. Herein we describe the
synthesis of four photolabile and four nonphotolabile
derivatives of biotinylated GDA along with evidence
supporting the isolation of proteins other than Hsp90
with biotinylated GDA.
Resu lts a n d Discu ssion
Syn th esis of Biotin yla ted GDA Der iva tives. Meth-
oxy quinones undergo rapid Michael addition and â-
elimination with primary amines to furnish the corre-
sponding vinylagous amide products.18 Consequently,
GDA was treated with bis(alkylamino) ethyleneglycols
4 and 5 to afford 6 and 7, respectively (Scheme 1). Ten
equivalents of the diamine were added to a yellow
solution of GDA. The methoxy quinone was converted to
J . Org. Chem, Vol. 69, No. 13, 2004 4377

Raible et al.
SCHEME 5. Syn th esis of P h otola bile Biotin yla ted GDA
PEG-derivative, 12,20 was used for the preparation of two
additional GDA analogues, 14 and 15 (Scheme 2). These
reactions required the addition of 6 or 7 to a solution of
11 and provided both 14 and 15 in 89% yields, respec-
tively.
Syn th esis of P h otola bile Biotin yla ted GDA De-
r iva tives. Incorporation of a photolabile group between
the biotin and GDA portions of the affinity purification
handle provided an alternative method for removal of
GDA-bound proteins from the immobilized complex.
Alternatively, this linker may prove useful for isolation
of Hsp90 multiprotein complexes that are formed be-
tween the chaperone, client proteins, immunophilins,
partner proteins, and cochaperones. Holmes previously
reported the use of 16 as a photolabile linker for solid-
phase synthesis (Scheme 3).21 The half-life of the benzylic
C-N bond is only 40 s when exposed to 365 nm
ultraviolet light in buffered (pH 7.4) aqueous solution.
Photolysis of such functionality results in the cleavage
of the linker to form the corresponding amide and styrene
products (Scheme 3). Holmes has previously prepared 16
in six steps, and minor modification of this procedure
gave the unprotected amino acid 18, which was subse-
quently used to prepare photolabile analogues of biotin-
ylated GDA.
The biotinylated photolabile linker 19 was assembled
by coupling the unprotected amino acid 18 with 9.20
Treatment of LC-biotinic acid (9) with dicyclohexyl car-
bodiimide (DCC) in methylene chloride and dimethyl
formamide, followed by addition of 18 led to poor yields
of 19. Acceptable yields were obtained when the reaction
was sonicated for 5 min after the addition of DCC and
for an additional hour upon addition of 18. Photoinduced
cleavage of 19 occurred when the sample was exposed to
light for long periods of time. Consequently, purification
of photolabile compounds was accomplished using reverse-
phase HPLC to provide 19 in 55% yield (Scheme 4). In
contrast to the alkyl-derived product, 20 was prepared
by the treatment of 1320 with DCC, followed by addition
(20) Biotin-tethered compounds were provided as both the free acid
and activated esters by Quantabiodesign.
(21) Holmes, C. P. J . Org. Chem. 1997, 62, 2370-2380.
4378 J . Org. Chem., Vol. 69, No. 13, 2004

Biotinylated Geldanamycin
of 18. The desired product was purified via reverse phase
HPLC to give 20 in 44% yield.
The photolabile biotin intermediate 19 was coupled
with 6 and 7 using 1-[3-(dimethylamino)propyl]-3-ethyl-
carbodiimide hydrochloride (EDCI) and dimethyl amino
pyridine (DMAP, Scheme 5). Flash chromatography in
the absence of significant light provided the desired
compounds 21 and 22 in moderate yields. Likewise,
compounds 23 and 24 were synthesized by an analogous
procedure using 20 and either GDA-amine 6 or 7.
Isola tion of GDA-Bou n d P r otein s. To verify biotin-
ylated-GDA derivatives were capable of the isolation of
Hsp90, recombinant Hsp90 from yeast (Hsp82) was
overexpressed and purified to homogeneity following the
procedure of Buchner and co-workers.22 Purified Hsp82
was incubated with 14 for 1 h in a buffered solution at 4
°C. Neutravidin-containing resin was added to the mix-
ture and further incubated at 4 °C for 30 min. After
centrifugation and removal of the supernatant, the resin
was successively washed with buffer and resuspended in
a minimal amount of buffer. Increasing concentrations
of GDA were added to the mixture to competitively
displace Hsp82 from the neutravidin resin. SDS-PAGE
of the GDA competition experiments determined both
that Hsp82 was bound to the neutravidin resin and that
the protein could be displaced with unmodified GDA
(Figure 3).
F IGURE 4. Affinity isolation of GDA-binding proteins. J urkat
A3 lysate exposed to 14 (200 µg) was incubated with neutra-
vidin resin and centrifuged, and the supernatant was removed.
The washed resin was exposed to 1 (10 µg) for 15 min at 4 °C
with shaking and centrifuged. The supernatant was removed,
denatured in SDS sample buffer, and separated by SDS-
PAGE (lane 2). The experiment was repeated with the same
resin using 50 µg (lane 3), 100 µg (lane 4), and 300 µg (lane 5)
of 1. Lane 1 represents the molecular weight markers.
Visualization of the separated proteins with silver stain
showed increasing intensities of proteins with elevated con-
centrations of 1.
Affin it y P u r ifica t ion of GDA-Bin d in g P r ot ien s.
J urkat A3 cells are known to be sensitive to inhibition
with GDA (IC90 ) 100 nM).23 Lysate containing the
J urkat A3 proteome24 was incubated with 14 at 4 °C in
buffer for 1 h. Neutravidin resin was added to the
mixture and incubated for an additional 35 min at 4 °C.
The heterogeneous mixture was centrifuged, and the
nonbinding proteins were removed by successive washing
with an appropriate buffer. The washed resin was
resuspended in buffer and incubated with increasing
concentrations of GDA. As can be seen from Figure 4,
Hsp90 was competitively displaced with increasing
amounts of GDA. However, proteins other than Hsp90
were also competitively displaced with unmodified GDA.
These data indicate that proteins other than Hsp90 are
also isolable with biotinylated GDA and may play a
significant role in GDA’s affect on tumor cells. Studies
are now underway to determine the identity of these
proteins, as well as their affinity for GDA or Hsp90. The
results of these studies will be presented in due course.
F IGURE 3. Binding of biotinylated GDA with purified Hsp
82. Purified Hsp82 (300 µg) was exposed to 14 (200 µg),
incubated with neutravidin resin, and centrifuged, and the
supernatant was removed. The resin/14/protein complex was
washed with buffer. The second wash (lane 1) shows band
intensities higher than those of the third wash (lane 2),
indicating the removal of excess Hsp82. The resin was exposed
to 1 (10 µg) for 15 min at 4 °C with shaking and centrifuged.
The supernatant was removed and denatured in SDS sample
buffer before separation by SDS-PAGE (lane 4). The experi-
ment was repeated with the same resin using 50 µg (lane 5)
and 100 µg (lane 6) of 1. Molecular weight markers are in lanes
3 and 7. Increasing band intensity from lanes 4 to 6 show that
as increasing concentrations of 1 were added, more Hsp82 was
released from the neutravidin resin. The gel was visualized
with silver stain.
Con clu sion
The syntheses of eight biotinylated analogues of GDA
containing photolabile and nonphotolabile tethers have
been accomplished, incorporating both hydrophobic and
hydrophilic tethers. These molecules were prepared by
the treatment of diamines with GDA to provide amino
modified GDA products, which could be easily coupled
to a number of biotin-containing carboxylic acids. Incuba-
tion of 14 with purified recombinant Hsp90 from yeast
and affinity purification using neutravidin resin resulted
in the capture and release of Hsp90. Incubation of 14
with the J urkat A3 proteome resulted in the isolation of
several proteins, including Hsp90. Proteins isolated from
these experiments were sufficient for mass spectrometric
identification. Studies are now underway to determine
the identity of these proteins, their Kd’s for GDA, and
determination of a conserved ATP binding pocket to
which these molecules bind.
(22) (a) Richter, K.; Muschler, P.; Hainzl, O.; Buchner, J . J . Biol.
Chem. 2001, 276, 33689-33696. (b) Prodromou, C.; Piper, P. W.; Pearl,
L. H. Proteins: Struct., Funct., Genet. 1996, 25, 517-522.
(23) Losiewicz, M. D.; Kaur, G.; Sausville, E. A. Biochem. Pharm.
1999, 57, 281-289.
(24) Obtained from ATCC; catalog no. 30-2021.
J . Org. Chem, Vol. 69, No. 13, 2004 4379

Raible et al.
Ack n ow led gm en t. The project described was sup-
ported by NIH Grant RR-P20 RR17708 from the Insti-
tutional Development Award (IDeA) program of the
National Center for Research Resources and The Uni-
versity of Kansas Center for Research. GDA was gener-
ously provided by Conforma Therapeutics and biotin
derivatives by Quantabiodesign. Additionally, the au-
thors would like to thank Drs. Robert Hanzlik and
Michail Alterman for helpful discussions.
Su p p or tin g In for m a tion Ava ila ble: Spectra and experi-
mental procedures for all compounds. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
J O049848M
4380 J . Org. Chem., Vol. 69, No. 13, 2004