An Hsp90 modulator that exhibits a unique mechanistic profile

Article abstract of DOI:10.1016/j.bmcl.2012.03.012

Described is the synthesis of two biotinylated derivatives of a cytotoxic macrocycle. Pull-down assays indicate that this macrocycle targets the N-middle domain of Hsp90. Untagged compound can effectively compete away tagged compound-Hsp90 protein complex

Full text of DOI:10.1016/j.bmcl.2012.03.012

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3287–3290
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
An Hsp90 modulator that exhibits a unique mechanistic profile
Deborah M. Ramsey a, Jeanette R. McConnell a, Leslie D. Alexander b, Kaishin W. Tanaka a,
Chester M. Vera a, Shelli R. McAlpine a,
⇑
a Department of Chemistry, University of New South Wales, Sydney, NSW 2052, Australia
b Department of Chemistry and Biochemistry, San Diego State University, 5500 Campanile Dr., San Diego, CA 92182, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Described is the synthesis of two biotinylated derivatives of a cytotoxic macrocycle. Pull-down assays
indicate that this macrocycle targets the N-middle domain of Hsp90. Untagged compound can effectively
compete away tagged compound–Hsp90 protein complexes, confirming the binding specificity of the
macrocycle for Hsp90. The macrocycle is similar in potency to other structurally-related analogs of San-
salvamide A (San A) and induces apoptosis via a caspase 3 mechanism. Unlike other San A derivatives, we
show that the macrocycle does not inhibit binding between C-terminal client proteins and co-chaperones
and Hsp90, suggesting that it has a unique mechanism of action.
Received 1 February 2012
Revised 5 March 2012
Accepted 5 March 2012
Available online 11 March 2012
Keywords:
Hsp90
Sansalvamide A
Pull-down
Ó 2012 Elsevier Ltd. All rights reserved.
Caspase 3
Heat shock proteins
Recently, we reported the mechanism of action of the cyclic
pentapeptide Sansalvamide A-amide and two analogs (Fig. 1).1–7
The peptide structure is based on the natural product depsipeptide
Sansalvamide A (San A) that was isolated by the Fenical group from
a marine fungus of the genus Fusarium sp. and found to exhibit
anticancer activity.8 Our reports showed that these 3 molecules
bind to the N-middle domain of heat shock protein 90 (Hsp90)
and modulate its function. Hsp90 is a ubiquitous molecular chap-
erone that regulates over 200 client proteins involved in multiple
growth and signaling pathways.3,4,6,7 The roles of Hsp90 in these
pathways include: regulating the conformational folding of numer-
ous signal transduction molecules, and refolding denatured pro-
teins under stress conditions.9,10 Because many hormone
receptors, kinases and signaling molecules involved in these path-
ways are targets for chemotherapeutic strategies, Hsp90’s role as a
master regulator has made it an attractive candidate for drug
development.11,12
shown to bind to the N-middle region, in a pocket between the
two domains.4–6 Using their tetratricopeptide repeat (TPR) motif19
several cofactors interact with the MEEVD region of the 12-kDa C-
terminal domain of Hsp90. These C-terminal binding proteins are
Hsp90 exists as a homodimer in the cytosol, with three highly
conserved domains.13 The 25-kDa N-terminal domain contains an
ATP-binding site and is the target of the natural product geldana-
mycin and its derivative 17-AAG [17-(allylamino)-17-demethoxy-
geldanamycin]14,15 as well as other drugs under clinical
development that modulate Hsp90.16 The 35-kDa middle domain
binds to multiple client proteins and co-chaperones, including hu-
man growth factor-2 (HER2) and the anti-apoptotic factor Akt.17,18
Three Sansalvamide A analogs (Fig. 1, compounds 1–3) have been
⇑
Corresponding author. Tel.: +61 4 1672 8896; fax: +61 2 9385 6141.
E-mail address: s.mcalpine@unsw.edu.au (S.R. McAlpine).
Figure 1. San A-amide (compound 1)4 Hsp90 inhibitors (compounds 25 and 3,6 and
compound of interest (compound 4).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.03.012

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D. M. Ramsey et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3287–3290
unaffected by all drug candidates that are currently in clinical trials
that exploit this pathway.20 And while the C-terminal binder novo-
biocin was reported to inhibit the binding between Hsp90 and four
TPR-containing co-chaperones with an IC50 in the millimolar
range,21 all three of the previously reported San A-amide com-
pounds (compounds 1–3, Fig. 1) were recently shown to allosteri-
cally interfere with the binding interactions of four co-chaperones
that interact with Hsp90 via their TPR domains with IC50s in the
low micromolar range.4–7
During our investigation of San A-amide and its mechanism of
action, we came across a San A-amide derivative, compound 4,
which exhibits the same IC50 values as those that target Hsp90,
but displays a mechanism that is unique from the other San A-
amide analogs.3,4,6,7 Specifically, compound 4 (Fig. 1) has an IC50
of 2.9 lM in HCT-116 cells (colon cancer) but does not inhibit
the interaction between Hsp90 and the client/co-chaperone pro-
teins that bind via their TPR domains to the C-terminus of
Hsp90.2,3,6 Because compound 4 does not interfere with Hsp90–
TPR protein complexes, this raised a question as to whether com-
pound 4 binds directly to Hsp90 or exerts its cytopathic effect by
another mechanism. To better understand how the conformation
of the macrocycle contributed to cell death, we investigated the
cytotoxicity of compound 4 analogs.3 These analogs included (a)
replacing the chlorobenzyloxycarbonyl (2-Cl-Z) group on the lysine
residue (IV) with a carbobenzyloxy (Cbz) group, (b) replacing the
Lys(2-Cl-Z) with -Lys(2-Cl-Z) (IV), (c) moving the N-methyl from
the -Phe residue (V) to the -Lys(2-Cl-Z) (IV). All modifications
resulted in over 100-fold decrease in IC50 values. Interestingly
removing the N-methyl from the -phe residue (V) and adding it
to the valine residue (III) generated an active compound with an
M (2.6 fold greater than compound 4). Thus,
D-
L
D
D
D
IC50 value of ꢀ8
l
compound 4 has a very distinct conformation and 3-D structure
that induces a cytotoxic effect, yet its binding specificity for
Hsp90 was unknown.
Figure 2. Biotin-tagged derivatives of Compound 4.
Herein we report the synthesis of two biotinylated analogs of
compound 4 (Fig. 2), and the results from the pull-down assay
performed using these derivatives. Similar to other derivatives of
San A-amide, compound 4 binds to Hsp90 and forms stable inter-
actions within the N-middle domain. As previously published,
compound 4 does not interfere with binding interactions between
client proteins and the C-terminal domain of Hsp90.6 Further, we
show that: (a) compound 4 binds to the same site as compounds
1, 2, and 3 and competitively inhibits the binding of compound 3
to Hps90’s N-middle domain, (b) it displays a distinct phenotype,
which indicates it goes through an Hsp90-mediated mechanism,
and (c) it induces apoptosis via cleavage of poly (ADP-ribose) poly-
merase (PARP), which suggests a caspase 3-mediated pathway.
These results indicate that the mechanism of action of compound
4 is unique from other San A-amide derivatives or 17-AAG, in that
its interaction with Hsp90 leads to a pro-apoptotic effect but not
through the inhibition of the same co-chaperone or client protein
binding interactions that are observed with other Hsp90 inhibitors.
In an effort to explore compound 4’s unique mechanism of ac-
tion we synthesized two biotinylated derivatives (Fig. 2): 4-Tagged
at position II (4-T-II) and 4-tagged at position III (4-T-III). We
placed the tags at positions II and III as position I, IV, and V all
had phenyl moieties, which are important for activity.14,15 Specifi-
cally, through structure activity relationships (SAR) we had found
that the phenyl at position I was critical,2 the Chloro-Cbz-protected
Fmoc-protected amino acids and amine deprotection were per-
formed until the desired linear pentapeptide was reached. A Boc-
protected lysine was incorporated at the tagged position. After
cleaving the peptide from the resin with 50% trifluoroethanol in
dichloromethane, the linear pentapeptide was cyclized using mac-
rocyclization conditions. The macrocycle was then subjected to
20% TFA to remove the Boc from the lysine, and biotinylated using
NHS-peg-biotin and 8 equiv of DIPEA.
The biotinylated compounds were incubated with HCT116 cell
lysate, whereupon neutravidin bound agarose beads were added
to immobilize the compound along with the bound target pro-
tein(s) (Fig. S18). The beads were washed ten times to remove
non-specifically bound proteins, followed by elution of proteins
using sample buffer. The eluted proteins were run on an SDS–PAGE
gel and visualized with Coomassie blue staining (Fig. S18). Protein
targets were isolated from the binding assay containing compound
4-T-III, and five major proteins were visible. The most prominent
protein bound to compound 4-T-III was visualized as a band at
approximately 90–95 kDa. Four other major proteins appeared,
and protein sequencing of these five bands was performed using
a nano-LC/MS/MS followed by peptide identification using the
NCBI Eukaryotic database and fingerprinting software. The bands
were identified as: myosin-9, Hsp90, beta tubulin, actin and glyc-
eraldehyde 3-phosphate dehydrogenase (GAPDH). No protein tar-
gets were isolated using compound 4-T-II (Fig. 3).
D
-lysine at position IV was important for biological activity,1 and
the presence of an N-methyl-D-phenylalanine at position V was
an important motif.1,15 Thus we chose not to tag these positions.
These compounds were synthesized using a solid-phase protocol
(Scheme S1 and S2).1,3 Both 4-T-II and 4-T-III were synthesized
using a commercially available chlorotrityl resin pre-loaded with
It is well documented that myosin, b-tubulin, and actin are
commonly pulled down during these assays due to their hydropho-
bicity.16 Comparison of the band isolated from 4-T-III at 90–95 kDa
to that region in the gel for 4-Tag-II, the negative control (PEGylat-
ed biotin linker alone) or DMSO (Fig. 3) indicates that the
L-phenylalanine or L-leucine, respectively. Subsequent coupling of

D. M. Ramsey et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3287–3290
3289
Figure 3. Western blot of proteins isolated in pull down assay.
Figure 5. (a) Compound 4-T-III selectively pulls down the N-middle domain of
Hsp90. [11.5-fold above background] (b) Compound 4 and 3 compete off the tagged
analog 4-T-III (IC50 6.2 lM and IC50 1.9 lM, respectively).
interaction between Hsp90 and compound 4-T-III is specific to the
molecule. Given that Hsp90 is the only protein pulled down that is
associated with an oncogenic pathway, we verified the targets by
Western blot analysis (Fig. 3). In support of the LC/MS/MS data,
western blot analysis shows that compound 4-T-III pulls down
Hsp90, whereas compound 4-T-II and control reactions do not.
Activation of hsp90 gene expression can occur through a variety
of stress signals, including temperature, heavy metals, cytokines
and mitogenic agents.17 Once expressed, Hsp90 localizes mainly
to the cytoplasm due to a C-terminal cytosolic localization signal.18
To determine if treatment with compound 4 alters localization of
Hsp90 in colon cancer cells, immunofluorescence staining was per-
formed using a polyclonal antibody to Hsp90. Small, punctate areas
of Hsp90 staining intensify in brightness when the cells are treated
with the Hsp90 inhibitor 17-AAG, yet staining remains confined to
the cytosol (Fig. 4). Treatment with compounds 3 and 4 increased
the staining density of Hsp90 in the cytosol, and there was no
significant difference in the brightness intensity between cells
treated with either compound (Fig. 4). From these data, we see that
compound 4 affects Hsp90 localization in a similar manner to
compound 3, and the change in staining intensity over the
DMSO-treated control is likely due to an increase in treatment-in-
duced cellular stress.
Hsp90.4 In order to evaluate where compound 4 bound to Hsp90,
we used compound 4-T-III in a pull-down assay with purified do-
mains of Hsp90 (Fig. 5a). Similar to other derivatives of San A-
amide, compound 4-T-III bound preferentially to the N-middle do-
main of Hsp90. Significantly, compound 4 competes for the same
binding site as compound 3, our most potent analog to date
(Fig. 5b). That is, as you add increasing concentrations of com-
pound 4 to Hsp90 bound to 4-T-III, you are able to displace 50%
of the bound 4-T-III molecules using 6.2
ther, consistent with its binding affinity (3.6
l
M of compound 4. Fur-
M) is the fact that
l
compound 3 outcompetes 4-T-III with a significantly lower IC50
than compound 4. Thus, both compounds bind to the same binding
pocket of the N–M domain of Hsp90.
Given that compounds 1, 2, and 3 all induce apoptosis4and that
compounds 3 and 4 bind to the same site, it was likely that com-
pound 4 induces apoptosis. A well-known and reliable method
for detecting apoptosis is the evidence of cleaved poly(ADP-ribose)
polymerase (PARP) fragments. PARP is an enzyme activated in the
presence of DNA strand breaks, and it catalyzes the synthesis of
poly(ADP-ribose) groups, which it covalently attaches to itself
and to several nuclear proteins.22 Caspase 3 is largely responsible
for the cleavage of PARP during programmed cell death. Examina-
tion of whether a molecule triggers caspase-dependent apoptosis
can be measured by evaluating the levels of full-length PARP and
its corresponding apoptotic enzyme-cleaved fragments. An in-
crease in the degradation of full length PARP upon the addition
of increasing amounts of compound verifies that the compound
is inducing caspase-dependent apoptosis.23 Cell lysates from
HCT-116 treated for 24 hrs with increasing concentrations of com-
The three domains of Hsp90 form a unique scaffold that dynam-
ically responds to client/co-chaperone protein binding and ATP
hydrolysis. The N-terminal domain of Hsp90 is the frequent target
of drug development because of its ATP-binding pocket13 The mid-
dle domain contributes significantly to the maintenance and stabil-
ity of Hsp90’s conformation. Recent studies suggest that the
middle domain modulates the position of the c-phosphate group
of ATP prior to hydrolysis and directly affects the reaction rate.19
In addition, the middle domain may also play a key role in client
protein recognition and binding.20 Previous data showed that
derivatives of San A-amide bound to the N-middle domains of
pound 4 (0–50 lM) were analyzed by Western blot. We observed
Figure 4. Visualization of Hsp90 in HCT 116 cells treated with DMSO (72 h; 1%), 17-AAG (24 h; 200 nM), compound 3 (72 h; 5 lM), or compound 4 (72 h; 5 lM).

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D. M. Ramsey et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3287–3290
that full length PARP (ꢀ113 kDa) decreased in a dose-dependent
Compound 4 represents a unique tool that binds specifically to
Hsp90’s N-middle domain and allows co-chaperone and client
binding interactions with Hsp90, yet it inhibits cell growth by
potentially altering Hsp90’s conformational dynamics and pre-
venting release of client proteins from the Hsp90 scaffold. Further
studies are currently underway to gain a deeper understanding of
these interactions.
fashion upon the addition of increasing concentrations of com-
pound 4. For cells treated with 50 lM of compound 4, there was
a 48% decrease in full-length PARP compared to non-treated cells,
(Fig. S22). These data indicate that compound 4 causes apoptosis in
a caspase dependent manner.
In addition to the above reported data, we investigated the abil-
ity of our compounds to inhibit clients and co-chaperones from
binding to Hsp90. Previously, we had found that unlike compounds
1,2 2,3 and 3,4 compound 4 did not inhibit the interaction between
four client proteins and co-chaperones that bound to the C-termi-
nus of Hsp90, including: inositol hexakisphosphate kinase-2
(IP6K2), FKBP38, FKBP52, and Hsp organizing protein (HOP).6
Two other proteins of interest, which bind to the middle-domain
of Hsp90 are HER2 and Akt1. The protein kinase B (Akt) and phos-
phatidylinositol-3-kinase (PI3K) pathways are activated by growth
factors such as HER2, which leads to anti-apoptosis or cellular pro-
liferation of cancer cells.24 Hsp90 inhibitor 17-AAG prevents the
association of Akt1 with Hsp90, leading to ubiquitinization and
degradation of Akt1in the cell.25 To determine if compound 4’s
cytotoxic effect is due to the binding inhibition of these client
proteins to Hsp90, we compared the binding effects of compound
4 to 17-AAG. 17-AAG reduced the binding of Hsp90 to Akt by
80% and to HER2 by 47% (Fig. S23). Similar to our earlier results
with proteins that bound to the C-terminus of Hsp90, compound
4 had little to no effect on the binding of either client protein to
Hsp90 (Fig. S23). These data indicate that the cytotoxic effect of
compound 4 is not due to the disruption of HER2- or Akt-binding
interactions to Hsp90.
Acknowledgments
We thank the University of New South Wales (UNSW), as well
as NIH 1R01CA137873 for support of D.M.R. and S.R.M. We also
thank the Frasch Foundation (658-HF07) (support of L.D.A.) and
NIH MIRT (support of J.R.M and L.D.A.).
Supplementary data
Supplementary data (spectral data and experimental details for
compounds and biological assay conditions) associated with this
article can be found, in the online version, at http://dx.doi.org/
10.1016/j.bmcl.2012.03.012.
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In summary, compound 4 is a cytotoxic macrocycle that induces
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