A small molecule that preferentially binds the closed conformation of Hsp90

Article abstract of DOI:10.1016/j.bmcl.2011.09.096

Described is the synthesis of three different fluorescein-tagged derivatives of a macrocycle, and their binding affinity to heat shock protein 90 (Hsp90). Using fluorescence polarization anisotropy, we report the binding affinity of these fluorescein-labeled compounds to Hsp90 in its open state and ATP-dependent closed state. We show that the compounds demonstrate a conformation-dependent preference for binding to the closed state.

Full text of DOI:10.1016/j.bmcl.2011.09.096

Bioorganic & Medicinal Chemistry Letters 21 (2011) 7068–7071
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A small molecule that preferentially binds the closed conformation of Hsp90
Leslie D. Alexander b, James R. Partridge c, David A. Agard c, 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
c Howard Hughes Medical Institute and the Department of Biochemistry and Biophysics, University of California, San Francisco, 600 16th Street, San Francisco, CA 94158, 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 three different fluorescein-tagged derivatives of a macrocycle, and their
binding affinity to heat shock protein 90 (Hsp90). Using fluorescence polarization anisotropy, we report
the binding affinity of these fluorescein-labeled compounds to Hsp90 in its open state and ATP-depen-
dent closed state. We show that the compounds demonstrate a conformation-dependent preference
for binding to the closed state.
Received 13 August 2011
Revised 22 September 2011
Accepted 22 September 2011
Available online 29 September 2011
Published by Elsevier Ltd.
Keywords:
Sansalvamide A
Hsp90
Fluorescein
Fluorescence polarization
Anticancer
Anistropy
Molecules that bind to and inhibit the function of heat shock
protein 90 (Hsp90) have proven to be excellent clinical candidates
in oncogenic therapies.1–6 Hsp90 is an ATP-dependent chaperone
protein that helps fold and regulate more than 200 substrate ‘cli-
ent’ proteins that are involved in cell growth and signaling.4,7 Since
pathway-specific inhibitors can be problematic in drug-resistant
cancers, shutting down multiple pathways at once is a promising
approach when developing new therapeutics. Given Hsp90’s abil-
ity to modulate many growth and cell signaling pathways simulta-
neously,8 this protein has become an attractive target in the field of
cancer therapeutics.
Recently, we reported the mechanism of action of the cyclic
pentapeptide Sansalvamide A-amide (Fig. 1, compound 1).9–11
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.12 We recently reported that San A-amide binds
to and modulates Hsp909,11 as does compound 2.10 Compound 2
(Fig. 1) exhibited lower IC50 values than compound 1 and other
San A-amide structures, and has been shown to modulate the bind-
ing between numerous client proteins and Hsp90.10 Thus, it was
chosen as a tool for further investigation.
N, M, and C, respectively).13 It functions in conjunction with sev-
eral co-chaperones, progressing through a complex ATP-dependent
conformational cycle (Fig. 2). Without ATP, Hsp90 maintains an
open dimerized conformation (Fig. 2a). Upon ATP binding (2b), it
converts to a stabilized closed and twisted conformation (2c),
which is favorable for the subsequent hydrolysis of ATP. Upon
hydrolysis of ATP Hsp90 forms a more compact ADP-bound state
(2d). After ADP is released, Hsp90 reverts back to its open state
(2a).14–16
Prior to our work, two types of Hsp90 inhibitors had been re-
ported: those that bind to the ATP-binding pocket in the N-domain,
thus inhibiting Hsp90’s function,17and those that bind to the C-do-
main.18,19 All of the compounds currently in clinical trials bind to
the N-domain and most are closely related to the natural product
III
O
O
IV
O
O
N
N
H
H
II
HN
HN
N
NH
NH
O
O
HN
NH
O
NH
O
O
O
CbzHN
Hsp90 is a constitutive dimer that contains three domains per
monomer: the N-terminal domain, which houses an ATP-binding
site, the middle domain, and the C-terminal domain (known as
V
I
Compound 2
San A-amide
Compound 1
⇑
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. Sansalvamide A-amide and derivative.
0960-894X/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2011.09.096

L. D. Alexander et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7068–7071
7069
We placed the tags at positions I, III, and IV of compound 2
(Fig. 3). We have established that the presence of an N-methyl-
D-
AT P
N
N
AT P
phenylalanine is an important motif in our potent molecules, thus
we did not place a tag at position II as that would require replacing
this residue. In addition, our SAR has indicated that the Cbz-pro-
tected lysine at position V is also important for biological activity
hence we did not tag that position.
ADP
M
M
Pi
ADP
C
C
The tagged compounds were synthesized following a solid-
phase synthesis and tagging method (Scheme 1). Beginning with
pre-loaded 2-chlorotrityl-leucine resin, Fmoc-protected amino
acids were subsequently coupled and deprotected until the desired
linear pentapeptide was reached. A Boc-protected lysine was used
in place of the amino acid at positions I, III, and IV, respectively. The
peptide was cleaved from the resin in 50% trifluoroethanol in
methylene chloride followed by cyclization using optimized mac-
rocyclization conditions.11 After purification, the Boc-protecting
group of the lysine was removed, and fluorescein was coupled to
the macrocycle using activated NHS-fluorescein and 8 equiv of DI-
PEA. The final fluorescein-tagged compounds were purified by re-
verse-phase HPLC.
Using fluorescence polarization anisotropy, we determined that
all three tagged derivatives of compound 2 bind effectively to
Hsc82, the full-length yeast homolog of Hsp90, where their micro-
molar Kds were reasonably similar to our cytotoxicity data for the
non-tagged compounds (Fig. 4, and Table 1).22 In addition, we
found that the placement of the tag was very important, where
Hsp90 dimer:
Open state
Closed-twisted
conformation
2a
2b
2c
2d
Figure 2. Hsp90 open–close mechanism.
Geldanamycin (Kd ꢀ1
l
M).2 Compounds that bind to the C-domain
include the natural product antibiotic novobiocin and its deriva-
tives. The advancement of novobiocin as a chemotherapeutic agent
has been hampered by its high micromolar cytotoxic activity
(ꢀ700
lM) and poor binding affinity for Hsp90 (calculated Kds in
the millimolar range).20,21 Recent studies have reported novobiocin
derivatives with low micromolar affinities for Hsp90,17 but to date
no molecules that bind to the C-terminal site of Hsp90 have been
tested in clinical trials.21 Unlike the current inhibitors, we have
established that San A-amide and compound 2 bind to the N–M
domain and allosterically modulate the C-domain client protein
IP6K2, and co-chaperones FKBP52, and HOP.9,10
In an effort to further explore San A-amide’s unique mechanism
of action we synthesized three fluorescein-tagged derivatives of
compound 2 (Fig. 1). Using fluorescence polarization anisotropy,
we determined the binding affinities of these tagged derivatives
to Hsp90 in both the open (2a) and closed state (2c). We report
that compound 2 demonstrates preferential binding showing a sig-
nificant preference for the closed state (2c) over the open state
(2a). Compound 2’s enhanced affinity for the closed state likely
contributes to its increased potency over San A-amide
2-T-III had a Kd of 57 lM for the open apo state, 2-T-I and 2-T-IV
had Kds almost 4- and 2-fold higher, respectively (Table 1).
In addition to testing the affinity of compound 2 for Hsc82 in
the absence of nucleotide, we also examined compound binding
to Hsc82 pre-incubated with the non-hydrolyzable ATP analog,
AMPPNP. While AMPPNP only subtly shifts the equilibrium of hu-
man Hsp90 towards the closed state, the yeast homolog (Hsc82) is
quantitatively shifted towards the closed conformation (Fig. 5c).14
Thus, using Hsc82 allows us to probe conformational discrimina-
tion of our molecules. All three compounds, 2-T-I, 2-T-III, and 2-
T-IV prefer the closed conformation (conformation 5c) of Hsc82
over the open conformation (conformation 5a and Table 1). Previ-
ous data showed that compound 2 binds to a site on the N–M do-
main of Hsp90. The interface between N and M domains changes
(Kd = 100 lM using this approach and 20 lM using biotin-labeled
analogs)11 and suggests a novel mechanism of action. Although
current Hsp90 inhibitors disrupt closure by binding to Hsp90’s
ATP-binding site, this is the first report of a small molecule favor-
ing the closed state.
NHTag
NHTag
III
III
O
O
IV
O
O
O
IV
N
H
O
N
H
N
H
II
II
N
NH
N
O
NH
N
NH
O
O
HN
NH
O
HN
NH
O
HN
O
NH
O
O
O
CbzHN
CbzHN
V
CbzHN
I
NHTag
2-T-I
2-T-III
2-T-IV
O
O
O
OH
CO2H
Tag= Fluorescein
Figure 3. Fluorescein-tagged derivatives of compound 2.

7070
L. D. Alexander et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7068–7071
O
O
NRFmoc
(3 eq)
N
AT P
N
NRFmoc
(3 eq)
AT P
2
3
HRN
RN
AT P
HO
HO
R2
San
A
R3
AT P
R2
1) HOBT (3 eq)
DIC (6 eq),
NRH
R1
1) HOBT (3 eq)
DIC (6 eq),
M
M
O
DMF 0.2M
DMF 0.2M
R1
O
O
2) 20% piperidine/DMF
C
C
C-terminal
Binding region
2) 20% piperidine/DMF
1
1-2
C-terminal
Binding region
R = H or Me
O
O
R3
O
Hsp90 dimer:
Open state
Closed-twisted
conformation
R3
NRFmoc
R4
O
NRFmoc
4
HO
N
R
HO
(3 eq)
5
O
R3
NHR
RN
RN
R2
HRN
O
R5 (3 eq)
5a
5b
5c
1) HOBT (3 eq)
DIC (6 eq)
RN
RN
R1
1) HOBT (3 eq)
DIC (6 eq)
DMF 0.2 M
R2
O
Figure 5. Hsp90 open–close mechanism with San A.
DMF 0.2M
R1
O
2) 20% piperidine/DMF
2) 20% piperidine/DMF
O
1-2-3-4
1-2-3
dramatically during the ATPase cycle.10,11 We had also already re-
ported that allosteric modulation of Hsp90 by San A-amide and
compound 2 effectively inhibit the interactions between Hsp90
and C-terminal-interacting co-chaperones and client proteins.
The observed preference of compound 2 for the closed conforma-
tion is consistent with compound 2’s binding to the N–M domain,
and to its allosteric inhibition of C-terminal client proteins and co-
chaperones. That is, binding to a site that is sensitive to conforma-
tional rearrangements during the ATPase cycle of Hsp90, and hav-
ing a preference for a particular Hsp90 conformation, may block
client protein access to Hsp90’s C-terminus (Fig. 5). Although only
2-fold preference, this modification could be optimized by SAR,
constituting an approach that is unique among known Hsp90
inhibitors, and making compound 2 a useful tool in exploring
Hsp90 pathways.
O
R3
O
R3
R4
O
O
O
R4
N
N
R
1) 1:1 (v/v) TFE:CH2Cl2, 24 hr
(yields: 80-95%)
R
O
NR
NHR
RN
RN
R2
O
NR
RN
R2
O
2) TBTU (0.7 eq), HATU (0.7 eq)
DEPBT (0.7 eq), DIPEA (6 eq)
10:1 CH2Cl2:CH3CN
R5
NR RN
R5
R1
O
R1
(0.007-0.0007M) (yields: 5-76%)
O
San A derivative
Linear Pentapeptide*
1-2-3-4-5
1) 20% TFA, CH2Cl2 0.1M,
2eq anisole
Fluorescein-tagged
Sansalvamide A derivative
2)
1.2 eq NHS-fluorescein
8eq DIPEA, CH2Cl2 0.1M
(yields: 20-40%)
Scheme 1. Solid-phase synthesis and tagging of San A derivatives.
In summary, we report the synthesis of fluorescein-tagged
derivatives of a San A macrocycle, compound 2, which binds to
Hsp90 at the N–M domain. We used these compounds in fluores-
cent assays to determine their preference for binding to Hsp90 in
its open versus ATP-induced closed state and we show that com-
pound 2 binds preferentially to the closed state. This is the first
observation of preference of a non-nucleotide mimetic for the
closed state. Based on these findings and our previous reports,
compound 2 has an inhibitory profile unlike that of any Hsp90
inhibitor reported to date. It’s ability to bind to the N–M domain
of Hsp90, allosterically modulate C-terminal client proteins, and
a preference for Hsp90’s ATP-driven closed state will expand the
range of mechanistic tools useful in exploring Hsp90’s protein fold-
ing cycle. Further studies are currently underway to gain a deeper
understanding of this interaction.
Acknowledgments
We thank the University of New South Wales Sydney for sup-
port of S.R.M., the Frasch Foundation (658-HF07) (support of
L.D.A.), NIH 1R01CA137873 (support of S.R.M. and L.D.A.), NIH
MIRT (support of L.D.A.). We thank HHMI in support of J.R.P. and
D.A.A.
Supplementary data
Figure 4. Exemplary anisotropy experiments and corresponding graph with Hsc82
and San A compound 2 tagged with fluorescein at position 1 (red) or at position 3
(blue). Data fitting and analysis is described inside the Supplementary data.
Supplementary data (spectral data and experimental details for
compounds and fluorescence polarization methods) associated
with this article can be found, in the online version, at
doi:10.1016/j.bmcl.2011.09.096.
Table 1
Anisotropy data
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