A highly selective Hsp90 affinity chromatography resin with a cleavable linker

Article abstract of DOI:10.1016/j.bmc.2012.03.043

Over 200 proteins have been identified that interact with the protein chaperone Hsp90, a recognized therapeutic target thought to participate in non-oncogene addiction in a variety of human cancers. However, defining Hsp90 clients is challenging because interactions between Hsp90 and its physiologically relevant targets involve low affinity binding and are thought to be transient. Using a chemo-proteomic strategy, we have developed a novel orthogonally cleavable Hsp90 affinity resin that allows purification of the native protein and is quite selective for Hsp90 over its immediate family members, GRP94 and TRAP 1. We show that the resin can be used under low stringency conditions for the rapid, unambiguous capture of native Hsp90 in complex with a native client. We also show that the choice of linker used to tether the ligand to the insoluble support can have a dramatic effect on the selectivity of the affinity media.

Full text of DOI:10.1016/j.bmc.2012.03.043

Bioorganic & Medicinal Chemistry 20 (2012) 3298–3305
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A highly selective Hsp90 affinity chromatography resin with a cleavable linker
Philip F. Hughes a, Jared J. Barrott a, David A. Carlson a, David R. Loiselle a, Brittany L. Speer a,
Khaldon Bodoor a, Lauretta A. Rund b, Timothy A.J. Haystead a,
⇑
a Department of Pharmacology and Cancer Biology, C119 LSRC, Research Drive, Duke University, Durham, NC 27710, United States
b Department of Animal Sciences, University of Illinois, 382 ERML, 1201 W. Gregory Dr., Urbana, IL 61801-4734, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Over 200 proteins have been identified that interact with the protein chaperone Hsp90, a recognized
therapeutic target thought to participate in non-oncogene addiction in a variety of human cancers. How-
ever, defining Hsp90 clients is challenging because interactions between Hsp90 and its physiologically
relevant targets involve low affinity binding and are thought to be transient. Using a chemo-proteomic
strategy, we have developed a novel orthogonally cleavable Hsp90 affinity resin that allows purification
of the native protein and is quite selective for Hsp90 over its immediate family members, GRP94 and
TRAP 1. We show that the resin can be used under low stringency conditions for the rapid, unambiguous
capture of native Hsp90 in complex with a native client. We also show that the choice of linker used to
tether the ligand to the insoluble support can have a dramatic effect on the selectivity of the affinity
media.
Received 18 January 2012
Revised 14 March 2012
Accepted 21 March 2012
Available online 29 March 2012
Keywords:
Hsp90
Proteomics
Affinity chromatography
Chaperone
Ó 2012 Elsevier Ltd. All rights reserved.
Cleavable linker
1. Introduction
expression level and through posttranslational modifications for
example phosphorylation, acetylation and methylation. These pro-
Heat shock protein 90 (Hsp90) regulates cellular homeostasis
by chaperoning the folding and intracellular trafficking of protein
‘clients’ and is highly up regulated in response to stress.1,2 The
N-terminal domain contains an ATP binding site and ATPase activ-
ity is necessary for all of its cellular functions.3 To date, over 200
Hsp90 clients have been identified and many of these are involved
in signal transduction.4–6 The mechanisms by which Hsp90 pro-
motes protein folding/stability is under active investigation7,8
and although several solution structures of the N terminal ATP
binding domain have been solved, there are no native co-solution
structures of full length Hsp90 with its native clients.9,10 The stron-
gest evidence for its role in protein folding/stability comes from
studies with selective inhibitors that bind competitively to its
ATP binding domain.11–14 Typically 6–12 h exposure of a cell/xeno-
graft tumor model to an Hsp90 inhibitor results in degradation of
Hsp90 clients. To date, over 10 Hsp90 inhibitors have been clini-
cally evaluated in cancer patients for multiple indications.12,15,16
In vivo, Hsp90 does not function alone but acts in concert with
co-chaperones such as Sba1/p23 and Cdc37.17 Interactions with co-
chaperones are thought to be important to direct Hsp90 function
for specific physiological processes for example cell cycle progres-
sion, apoptosis, or signaling cascades.5 Hsp90 is regulated at the
cesses control its ATPase activity, its ability to interact with clients
and co-chaperones and its degradation.4,18–20 Glucose-regulated
protein 94 (GRP94) and tumor necrosis factor receptor-associated
protein 1 (TRAP1) share homology with Hsp90 and both proteins
possess ATPase activity.21,22 Client proteins selectively interacting
with these homologs have been less studied. Although there is se-
quence divergence between all three proteins in their N terminal
domains, none of the current Hsp90 inhibitors completely discrim-
inate between the chaperones in vitro.21,23
Approaches to defining Hsp90 clients typically involve immu-
noprecipitation or affinity purification of Hsp90 with immobilized
inhibitors. Client proteins are inferred by co-isolation.24,25 How-
ever, in vivo, many clients undergoing folding are thought to be
weakly associated with Hsp90, potentially complicating their iden-
tification using these approaches. Therefore, it seemed one could
benefit from the use of a highly selective affinity probe that would
allow for efficient Hsp90 release under mild conditions while
discriminating against non-specifically associated proteins that
typically bind to resin surfaces.26 Such a probe might enable isola-
tion of native Hsp90 in quantities suitable for crystallographic
studies. Also, an Hsp90 affinity resin could be used to remove the
protein when its absence is desirable. We describe here the devel-
opment of such a probe for Hsp90 that appears to discriminate the
protein from GRP94 and TRAP1, and its use as an affinity ligand
with an orthogonally cleavable linker. When incubated with
cellular extracts, the ligand binds Hsp90 in complex with novel
⇑
Corresponding author.
E-mail address: hayst001@mc.duke.edu (T.A.J. Haystead).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.03.043

P. F. Hughes et al. / Bioorg. Med. Chem. 20 (2012) 3298–3305
3299
client proteins as well as established clients such as HER2. Impor-
tantly, client selectivity can be unambiguously established by prior
quenching of the extracts with an Hsp90 inhibitor.
molecule which are solvent exposed and suggested a variety of
substitutions likely to be tolerated on the aniline nitrogen
(Fig. 1).27,28 Further analyses suggested that replacing the trime-
thoxyphenyl group with a linker on a 3-methylpyrazole analog
would provide a good balance of potency and ease of synthesis.
Therefore, fluoro-compound 2 was initially synthesized. This
intermediate provided a reactive center for the attachment of link-
ers and allowed for facile synthesis of a potent inhibitor 527 for
quenching studies. Thus (Scheme 1), dimedone was acylated with
acetic anhydride to give acetyldimedone 3, and separately, 2,4-dif-
luorobenzonitrile reacted with hydrazine to give a mixture of aryl-
hydrazines with the 4-substitution product 4 predominating. After
aqueous workups, the crude reaction products were combined in
methanol with acetic acid to give a mixture of regio-isomeric pyr-
azoles. The desired isomer was purified to give intermediate 2 in
50% yield from starting dimedone. For quenching experiments,
the known Hsp90 inhibitor 5 was made from 2 by fluoride dis-
placement with trans-4-aminocyclohexanol followed by peroxide
mediated hydrolysis of the nitrile to the amide (91%). Constructs
for affinity resins were prepared in a similar fashion except that
a greater excess of diamine was used to reduce, though not elimi-
nate, the formation of the doubly capped linkers. After hydrolysis
of the nitrile to the amide, ligand–inhibitor constructs 6–9 were
purified by silica gel chromatography.
2. Results
2.1. Resin synthesis and evaluation
A challenge to developing an affinity resin lies in choosing an
appropriate ligand and devising chemistry that allows linker
attachment without disrupting binding affinity. The structure
(pdb 3MNR) of the 2-aminobenzamide inhibitor 1 in complex with
the N-terminal domain of human Hsp90 revealed the parts of the
Affinity resins were prepared by reaction of the inhibitor–
linker construct with activated affinity media, CNBr-activated
Sepharose™ 4B according to the manufacturer’s instructions (www.
gelifesciences.com). Ligand was added at 1–10 lmol/gram of resin
in minimal methanol. We first prepared affinity resin A using the
short PEG-like 8 atom linker-construct 6 and analyzed its ability to
selectively capture Hsp90 from pig mammary gland extract, a tissue
shown to be high in ATP binding proteins including native forms of
Hsp90, GRP94 and TRAP1 (Supplementary Fig. S1). The resin was
incubated in the protein solution then washed with a high salt
buffer. The bound proteins were removed via an SDS boil procedure,
separated by SDS–PAGE electrophoresis, located by silver staining
and identified using mass spectrometry (MS) sequencing.29
A large number of proteins, including Hsp90, were retained
(Fig. 2, lane A). However, the initial hypothesis that proteins other
than Hsp90 might be clients was negated when a competition
experiment, performed by pre-incubating the protein solution
with known ligand 5, showed clean exclusion of Hsp90, but not
the other proteins.
We then tried linker-construct 7 that has the more hydrophobic
n-decane linker to prepared affinity resin B. This resin proved capa-
ble of capturing Hsp90 and was even better at capturing additional
Figure 1. (A) View of 2-aminobenzamide inhibitor 1, bound in Hsp90 and (B)
possible solvent side attachment points numbered.
Scheme 1. Synthesis of Hsp90 binding constructs. Reagents and conditions: (a) Acetic anhydride, DMAP, NEt3; (b) MeOH, N2H4ꢀH2O; (c) MeOH, acetic acid; (d)
chromatography; (e) (i) DMSO, excess amine or diamine, 90 °C, (ii) EtOH, 50% NaOH, (iii) 30% H2O2 dropwise, 90 °C.

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P. F. Hughes et al. / Bioorg. Med. Chem. 20 (2012) 3298–3305
proteins, particularly in the quenching experiments with ligand 5
(Fig. 2, lane B). This may be the result of enhanced and nonspecific
hydrophobic interactions of proteins with the linker.
Finally, we investigated another intermediate length PEG-like
13 atom linker with construct 9 and prepared affinity resin D.
Although it was more selective than resins A and B, some nonspe-
cific binding was observed (Fig. 2, lane D). All further experiments
were therefore conducted with linker-construct 8.
We next examined longer, more hydrophilic linkers that al-
lowed the ligand to extend farther away from the Sepharose sur-
face (Supplementary Fig. S2). Affinity resin C was prepared using
the polyethylene glycol (PEG)-like 19-atom linker-construct 8.
The capture experiment was repeated and an intense band of
Hsp90 was eluted with SDS (Fig. 2, lane C). Importantly, quenching
with 5 showed complete blocking of Hsp90 binding as well as sev-
eral N-terminal fragments. MS analysis demonstrated that all of
the recovered proteins were Hsp90 or proteolytic fragments of
the protein, suggesting that resin C is selective for Hsp90 over
GRP94 and TRAP-1. As mentioned previously (Supplementary
Fig. S2), the pig mammary gland tissue contained adequate levels
of GRP94 and TRAP-1 to be detected by MS when eluted from an
ATP resin.
2.2. Cleavable linker
An important goal of our studies was to derive a probe that could
be used to convincingly isolate Hsp90 under mild physiological
conditions in association with potentially weakly associated client
proteins. Although we can block Hsp90 recovery on our resins by
quenching with 5 or other Hsp90 inhibitors (e.g., geldanamycin30
or PU-H7131), we were unable to elute Hsp90 bound to the affinity
media without denaturing the protein. A linker that could be
broken under non-denaturing conditions was needed. Recently,
Verhelst et al.32 reintroduced33–35 an azo linker 10 (Scheme 2),
which can be cleaved under mild non-denaturing conditions with
sodium dithionite solution.36 This linker was synthesized using
the published procedure and coupled to our ligand-construct to
give compound 11. The FMOC group was removed with neat piper-
idine and ligand 12 was reacted to give affinity resin E. Following
exposure to pig mammary gland extract and treatment of the resin
with 25 mM sodium dithionite, the eluted proteins were identified
by MS as described earlier. The retained protein profile (Fig. 2, lane
E) is essentially identical to resin C, except that the proteins are
recovered under non-denaturing conditions.
In our hands, synthesis of the azo-linker 10 was not without
challenges. We observed production of numerous colored byprod-
ucts, possibly caused by difficulties in adequately controlling the
reaction pH. Analysis of the byproducts by NMR suggested prob-
lems with reactions of the free amine. When the azo forming reac-
tion was run with the BOC amide, using excess solid sodium
bicarbonate in the reaction, we were able to obtain azo-linker 13
as the only colored product. Acidification of the reaction, followed
by filtration gave clean azo-linker 13 (74%) as an orange powder.
Affinity resin E was made (Scheme 2) as before except that the
amide intermediate 14 was de-protected with TFA to give the iden-
tical linker construct 12 for bead attachment.
Figure 2. Directed chemical evolution of a selective affinity resin for Hsp90. SDS–
PAGE silver stain showing the effects of different side chain modifications on Hsp90
recovery and recovery of non-specifically bound proteins. Selectivity towards
Hsp90 was demonstrated by inclusion of 1 mM 5 (+) in the tissue extract prior to
mixing with affinity resin. Mass spectrometry was used to identify the bound
proteins. In lane E bound proteins were eluted with 25 mM sodium dithionite in
phosphate buffered saline. Numbers indicate bands that were sequenced by MS; (1)
fatty acid synthase (2) Hsp90 (3) Hsp90 (4) Hsp90 proteolytic fragments.
2.3. Nonspecific binding
One observation in these experiments (Fig. 2) is that the binding
of Hsp90 to each of the resins seems to lower the level of non-
Scheme 2. Preparation of a cleavable linker affinity resin. Reagents and conditions: (a) EDC, HOBT, CH2Cl2; (b) piperidine for 11, TFA for 14; (c) CNBr-activated Sepharose 4B.

P. F. Hughes et al. / Bioorg. Med. Chem. 20 (2012) 3298–3305
3301
specific binding, effectively protecting the resin. With each of the
2.5. GRP94 purification
affinity resins (A–E), addition of compound 5 to the protein mix-
ture completely blocked Hsp90 binding allowing recovery of other
proteins, especially abundant proteins such as fatty acid synthase
(240 kDa). Although binding of these proteins is clearly not
Hsp90 related, their recovery illustrates the challenges of using
affinity media to study interactions with Hsp90. Even with a highly
optimized ligand, our results illustrate the extent to which affinity
based strategies in defining Hsp90 clients can lead to misleading
conclusions. To define physiologically relevant associations with
Hsp90, it is necessary to demonstrate competitive binding with a
free ligand. Quenching experiments should be carried out using a
selective inhibitor of Hsp90 in the cell/tissue extract prior to expo-
sure to the affinity resin. Under these conditions, recovery of Hsp90
and any associated proteins should be selectively blocked. Proteins
recovered in the presence of the quenching agent are likely to be
non-specific.
It was possible to use this differential selectivity to purify
GRP94. Pig mammary gland extract was passed through resin E,
efficiently removing Hsp90, directly onto the ATP-resin. Elution
of the ATP resin with PU-H71 gave clean GRP94 when analyzed
by SDS–PAGE, silver staining and MS (Supplementary Fig. S4).
3. Proteomic studies
To test resin E as a proteomics tool we surveyed mouse organs
to determine both Hsp90 expression levels between tissues and
to define novel client proteins. Physiological conditions were used
to preserve weak binding interactions with Hsp90. To demon-
strate specificity, extracts were mixed with resin E +/ꢁ inhibitor
5. As seen in Figure 4A, each mouse tissue yielded a prominent
protein at 90 kDa of varying abundance as well as a distinct
pattern of proteins of varying molecular weight. In most in-
stances, inclusion of 5 in the extract blocked recovery of the
90 kDa protein as well as the additional proteins (Fig. 4B). As
2.4. Selectivity evaluation
expected, the 90 kDa protein was identified by MS as Hsp90 (
a
The finding that neither GRP94 nor TRAP-1 was recovered by re-
sin C suggests that construct 8 is highly selective for Hsp90. To ex-
plore this selectivity in a somewhat reverse fashion, we tested the
elution of pig mammary gland proteins from gamma phosphate
linked ATP Sepharose with compound 5, known potent Hsp90
inhibitors: 17-AAG,37 PU-H71,31 and SNX2112,27 and ligand con-
structs 6, 8 and 9 (Supplementary Fig. S3). This ATP resin has been
used in our lab for several years to study purine-binding proteins38
and to discover novel Hsp90 inhibitors.27 It can be used to test li-
gand binding selectivity against all other purine utilizing enzymes
expressed in cells.14 The ATP resin was charged with the pig mam-
mary gland extract and aliquots were distributed into individual
wells. Proteins eluted from the wells with increasing amounts of
the indicated compound were analyzed as described earlier (
Fig. 3). Compound PU-H71 demonstrates strong potency towards
both Hsp90 and GRP94, whereas SNX2112 shows weaker affinity
for GRP94 and ligand 8 shows essentially no elution of GRP94.
Examination of the crystal structures of Hsp9027 and GRP9439 with
various ligands provided no obvious clue as to why ligand 8 would
display a greater binding selectivity for Hsp90 over GRP94 com-
pared to the other known Hsp90 inhibitors. It may be that the
hydration of the PEG linker attached to 8 gives rise to a relatively
larger, more sterically demanding structure, which precludes bind-
ing to GRP94.
and b isoforms). Surprisingly, many of the other proteins in the
gel were either N-terminal fragments of Hsp90 or dimers of the
holoenzyme (Fig. 4A). Some proteins do appear to be potential
clients of Hsp90 and show tissue specific associations including
delta(3,5)-delta(2,4)-dienoyl-CoA isomerase, NADPH-dependent
retinol dehydrogenase/reductase, acetyl-CoA acyltransferase, and
glycogen debranching enzyme. Two proteins, epoxide hydroxy-
lase and glutaryl-CoA dehydrogenase are tentatively identified
as novel clients, since recovery of these proteins was not com-
pletely quenched by 5. Their recovery may be explained by the
presence of residual Hsp90 co-elution. Figure 4B also shows, as
observed earlier, a few proteins were still non-specifically recov-
ered from some tissues, even in the presence of 5, including albu-
min, CAZ-associated structural protein 1 and Ankrd11 protein.
Their recovery underscores the necessity of including a quenching
control with the free ligand to directly demonstrate Hsp90 depen-
dence of the co-isolation.
As a positive control, we confirmed that we could use resin E to
find HER2, a known client of Hsp90. Extract from the breast cancer
cell line (BTB-474) was added to the resin +/ꢁ inhibitor 5. Cleavage
of the linker with dithionite and analysis by SDS–PAGE and silver
staining showed only Hsp90. However, Western blot analysis
clearly showed the presence of HER2 (Fig. 5). Neither Hsp90 nor
HER2 were seen in the sample quenched with 5.
4. Discussion and conclusions
We report the development of a novel affinity probe, resin E, for
the selective recovery of Hsp90 in native complex with its physio-
logically relevant client proteins. Our ligand is the first reported
affinity reagent that only binds Hsp90 and shows no affinity for
GRP94 and TRAP1 at the limits of silver staining and MS analysis.
Therefore, resin E enables the study of native client proteins selec-
tive for Hsp90 independent of other chaperones. The development
of resin E illustrates the complexities of utilizing affinity ap-
proaches to study native protein–protein interactions. Clearly, lin-
ker choice is critical to avoid artifacts. Study of Hsp90 is
particularly challenging because interactions with its clients may
be weak and readily disrupted under stringent, non-physiological
conditions. Before assigning a particular protein as a client or co-
chaperone of Hsp90, it is essential to also perform a co-isolation
experiment with the appropriate quenching control. Any proteins
recovered under quenching conditions are probably artifacts and
unlikely to be a client or co-chaperone of Hsp90.
Figure 3. Elution of pig mammary gland protein from ATP-Sepharose resin with
SNX-2112, PU-H71 and ligand construct 8. ATP resin was charged with pig
mammary gland extract and washed in bulk. Then, 20
ll of charged resin was
placed in each well of 96-well filter plate and eluted with the indicated
a
compounds in parallel. The eluates were characterized by SDS–PAGE, silver staining
and mass spectrometry.

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P. F. Hughes et al. / Bioorg. Med. Chem. 20 (2012) 3298–3305
Figure 4. Proteomic survey of mouse tissues with azo-resin E. Tissue extracts were prepared from the indicated tissues and applied to a fixed volume of resin E (100
absence (A) and presence (B) of 5 (100 M). Following washes at physiological ionic strength the bound proteins were eluted with 30 mM dithionite and characterized by
SDS–PAGE (4–15% acrylamide), silver staining and mass spectrometry. Key: 1–22. Full length Hsp90 /b or N terminal fragments of Hsp90 /b; 23. Epoxide hydroxylase; 24.
Glutaryl-CoA dehydrogenase; 25. and 26. Hsp90 /b; 27. Hsp70; 28. Epoxide hydroxylase; 29. Delta(3,5)-delta(2,4)-dienoyl-CoA isomerase; 30. 3,2-trans-Enoyl-CoA
isomerase/NADPH-dependent retinol reductase; 31 and 32. Hsp90 /b; 33. Hsp70; 34. Hsp90 /b; 35. Glycogen debranching enzyme; 36 and 37. Hsp90 /b; 38. Epoxide
hydroxylase; 39. 3-Ketoacyl-CoA thiolase/3-ketoacyl-CoA thiolase; 40–42. Hsp90 /b; 43. Hsp70; 44. Epoxide hydroxylase/liver carboxylesterase 31; 45–47 Hsp90 /b; 48.
epoxide hydroxylase; 49–51. Hsp90 /b; 52. Albumin; 53. Hsp90 /b; 55. Albumin; 56. CAZ-associated structural protein 1/Ankrd11 protein; 56. Hsp90 /b; 57. Albumin; 58.
Epoxide hydroxylase; 59. Glutaryl-CoA dehydrogenase; 60. Hsp90 /b. Combined consisted of a mixture of striated muscle, liver, testis and adipose tissue. The lane w/o
ll) in the
l
a
a
a
a
a
a
a
a
a
a
a
a
dithionite was eluted with SDS. This gel is a representative example of using resin E against tissue extracts. (A larger view is shown in Supplementary Fig. S5).
In proteomic studies of mouse tissues using cleavable resin E,
Hsp90 was recovered either alone or in association with novel cli-
ent proteins. Recovery of the putative clients was authenticated by
quenching control experiments with 5. Interestingly, the majority
of the recovered clients are associated with the metabolism of
various lipids namely delta(3,5)-delta(2,4)-dienoyl-CoA isomerase,
acetyl-CoA acyltransferase, NADPH-dependent retinol reductase
and epoxide hydroxylase 2. Delta(3,5)-delta(2,4)-dienoyl-CoA
isomerase and acetyl-CoA acyltransferase are peroximal enzymes
participating in b oxidation of long chain fatty acids. Delta(3,5)-
delta(2,4)-dienoyl-CoA isomerase isomerizes 3-trans,5-cis-die-
noyl-CoA to 2-trans,4-trans-dienoyl-CoA functioning as a auxiliary
step of the fatty acid beta-oxidation pathway, enabling the metab-
olism of unsaturated fatty acids in mammals. NADPH-dependent
retinol reductase (RDH12) has activity toward 9-cis and all-trans-
retinol and is involved in the metabolism of short-chain aldehydes.
The enzyme forms 11-cis-retinal from 11-cis-retinol during regen-
eration of the cone visual pigments. Mutations in the RDH12 are
associated with retinitis pigmentosa type 53.40,41 Retinitis pigmen-
tosa is characterized by retinal pigment deposits as well as loss of
rod photoreceptor cells with some secondary cone photoreceptor
loss. The condition is typified by night vision blindness and re-
duced peripheral visual field. If RDH12 is a client for Hsp90, loss
of expression of the protein in the eye could explain some of the
idiosyncratic visual side effects anecdotally reported in some pa-
tients treated with a variety of Hsp90 inhibitors in clinical tri-
als.42,43 This would require that these drugs cross the blood–
brain barrier since the retina is contiguous with the central ner-
Figure 5. Proteomic analysis for HER2 in breast cancer cell line (BTB-474) with azo-
vous system. Epoxide hydroxylase 2 and its immediate family
resin E. W—whole cell lysate (BTB474); R—BTB474 lysate on Hsp90 resin (resin E);
members have roles in the metabolism of arachidonic and linoleic
R+—BTB474 lysate pretreated with free drug, compound 5 (1 mM), before passing
over Hsp90 resin (resin E). ⁄Hsp90, ⁄⁄HER2.
acid epoxides, as well as, xenobiotics.44 The finding that epoxide

P. F. Hughes et al. / Bioorg. Med. Chem. 20 (2012) 3298–3305
3303
hydroxylase 2 is a client of Hsp90 could explain some of the anti-
inflammatory effects of Hsp90 inhibitors in addition to their indi-
cations in cancer.45,46
The mouse study was used primarily to demonstrate the use
of the affinity resin for initial identification of potential client
proteins. Further studies are needed to confirm that these proteins
are true clients of Hsp90. As shown with the breast cancer cell line,
actual client proteins may be present below the detection limits of
silver staining or MS detection but still be detectable by western
blot analysis.
A final observation relates to the impact of the linker choice on
the performance of an affinity resin. There is a radical difference in
the amount of nonspecific binding between using the n-decane lin-
ker (resin B) and PEG-6 linker (resin C). We were initially disap-
pointed with the number of clients identified with our affinity
media, although we now believe the approach of using a ‘clean’
cleavable linker along with a quenching agent gives rise to fewer
experimental artifacts. It may be worthwhile to re-examine other
affinity experiments where hydrophobic linkers are used, beyond
the field of chaperone proteins. Judicious linker replacement may
show some affinity experimental conclusions to be premature
and may yet yield positive results in other previously failed exper-
iments. We continue to explore this interesting observation.
and treated with acetic acid (1 mL) and stirred at RT for 3 days.
The mixture was concentrated and then dissolved in methylene
chloride (20 ml). The mixture was chromatographed in batches,
with the best approach to load a DCE solution onto a dry column,
wait (15–20 min) then elute with 10% ethyl acetate in hexanes to
remove the yellow (isomeric product) followed by elution with
20% to get the product. The chromatographed products were
recrystallized from ethyl acetate/hexanes to give 2 (10.4 g, 49%).
TLC (hexane/EtOAc: 60/40) Rf = 0.47; 1H NMR (CDCl3) d 7.73 (dd,
J = 7, 8.5 Hz, 1H), 7.49 (dd, J = 2, 9.8 Hz, 1H), 7.45 (dd, J = 2,
8.5 Hz, 1H), 2.90 (s, 2H) 2.54 (s, 3H) 2.43 (s, 2H), 1.14 (s, 6H); MS
(ESI): m/z 298.2 [M+H]+.
5.1.42.-(((1r,4r)-4-Hydroxycyclohexyl)amino)-4-(3,6,6-trimethyl-
4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-yl)benzamide (5)
A mixture of nitrile 2 (200 mg, 0.67 mmol) and trans-4-amino-
cyclohexanol (232 g, 2.0 mmol), Hunig’s base (117
lL) and DMSO
(300 L) were heated to 90 °C for 30 m. The mixture diluted with
l
ethanol (2 mL) and treated with 50% NaOH (5 drops) and then,
very slowly, a drop at a time, with hydrogen peroxide. After each
drop, the reaction foamed up a bit. After adding 5 drops over
10 m, the mixture was diluted with water (18 mL) and allowed
to cool slowly with rapid stirring. After stirring overnight, the so-
lid was filtered off to give the product 5 (251 mg, 91%) as a white
powder. TLC (EtOAc) Rf = 0.25; 1H NMR (CDCl3)
d 8.07 (d,
5. Experimental section
5.1. General
J = 7.2 Hz), 7.44 (d, J = 8.6 Hz, 1H), 6.75 (d, J = 2 Hz, 1H), 6.60
(dd, J = 2, 8.6 Hz, 1H), 5.6 (br s, 2H), 3.71 (m, 1H), 3.35 (m, 1H),
2.80 (s, 2H) 2.53 (s, 3H) 2.38 (s, 2H), 2.13 (m,2H), 2.02 (m, 2H),
1.64 (br s, 1H), 1.41 (m, 4H), 1.09 (s, 6H); MS (ESI): m/z 411.3
[M+H]+.
Reagents were obtained from commercial sources and used
without further purification. 1,19-Diamino-4,7,10,13,16-pentao-
xanonadecane was obtained from Berry and Associates. Proton
NMR spectra were obtained on Varian 400 and 500 MHz spectrom-
eters. LC/MS were obtained on an Agilent ion-trap LC/MS system.
HRMS results were obtained on an Agilent 6224 LCMS-TOF and
are reported as an average of four runs. PU-H71 and 17-AAG were
purchased from commercial sources. SNX2112 was synthesized
using published methods. Spectral data (NMR etc.), descriptions
of tissue collection, cell culture, tissue extraction and proteomic
analysis are included in Supplementary data.
5.1.5. 2-((2-(2-(2-Aminoethoxy)ethoxy)ethyl)amino)-4-(3,6,6-
trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-yl)benzamide
(6)
A mixture of 2 (500 mg, 1.7 mmol) and 2,20-(ethylenedioxy)
bis(ethylamine) (1.2 g, 8.4 mmol) in DMSO (1 mL) were and heated
to 90 °C for 30 min. The mixture was diluted with ethanol (2 mL)
and, still at 90 °C, treated with 50% NaOH (20 drops) and very
slowly with 30% hydrogen peroxide (40 drops). The reaction mix-
ture was diluted with methylene chloride and methanol and ad-
sorbed onto silica gel. The mixture was chromatographed (silica
gel, 1.5 cm ꢃ 20 cm) and eluted with 9/1 CH2Cl2/MeOH, followed
by 9/1/0.1 CH2Cl2/MeOH/NH3 to give 6 (670 mg, 89%) as a clear
glass. TLC (4/1/0.1 CH2Cl2/MeOH/NH3) Rf = 0.14; 1H NMR (CDCl3)
d 8.17 (t, J = 5 Hz), 7.47 (d, J = 8.4 Hz, 1H), 6.77 (d, J = 2 Hz, 1H),
6.60 (dd, J = 2, 8.4 Hz, 1H), 6.1 (br s, 2H), 3.73 (t, 3H), 3.65 (m,
4H), 3.50 (t, 2H), 3.36 (m, 2H), 2.85 (t, 2H), 2.77 (s, 2H) 2.51 (s,
3H) 2.36 (s, 2H), 1.07 (s, 6H); HRMS (ESI) [M+H]+ calcd for
5.1.1. 2-Acetyl-5,5-dimethylcyclohexane-1,3-dione (3)
Dimedone (10 g, 71.3 mmol) was dissolved in methylene chlo-
ride (200 mL) and treated with Hunig’s base (9.7 g, 74.9 mmol)
and DMAP (440 mg, 3.6 mmol) followed by slow addition of acetic
anhydride (7.65 g, 74.9 mmol). After 24 h, the mixture was concen-
trated, partitioned between hexanes (150 mL) and 1 N HCl (70 mL),
then washed with brine (50 mL), treated with Norit A and then
dried (MgSO4), filtered and concentrated to give 2-acetyl-5,5-
dimethylcyclohexane-1,3-dione 3 (ꢂ11+ g) as a yellow oil. The en-
tire product was used in the next step.
C23H34N5O4, 444.2605; found 444.2607.
5.1.6. 2-((19-Amino-4,7,10,13,16-pentaoxanonadecyl)amino)-
4-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-
yl)benzamide (8)
5.1.2. 2-Fluoro-4-hydrazinylbenzonitrile (4)
2,4-Difluorobenzonitrile (10 g, 72 mmol) was dissolved in
methanol (100 mL) and treated drop wise with hydrazine hydrate
(18 g, 0.36 mol) and stirred at rt. After 16 h, the reaction mixture,
containing a mixture of isomers, was concentrated then parti-
tioned between ethyl acetate (100 mL), water (70 mL) and 1 N
NaOH (30 mL). The organic layer was washed with brine (40 mL)
then concentrated to give 4 in a mixture of isomers as a white
solid.
A
mixture of 2 (482 mg, 1.62 mmol) and 1,19-diamino-
4,7,10,13,16-pentaoxanonadecane (1 g, 3.24 mmol), diisopropyl-
ethylamine (628 mg, 4.8 mmol) and DMSO (1 mL) were heated to
90 °C for 20 m. Still at 90 °C, the mixture diluted with ethanol
(2 mL) and treated with 50% NaOH (10 drops) and then, very
slowly, a drop at a time, with hydrogen peroxide. After each drop,
the reaction foamed up substantially. After about 10 drops over
10 m, the reaction mixture was diluted with ethanol and added
to silica gel (6 g), concentrated to a powder, added to a silica gel
column (2.5 ꢃ 20 cm) and chromatographed with CH2Cl2
(300 mL), CH2Cl2/MeOH/NH3 19/0.9/0.1 (300 mL), 9/0.9/0.1
(300 mL) and 4/0.9/0.1 (500 mL). The cleanest fractions were
5.1.3. 2-Fluoro-4-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-
indazol-1-yl)benzonitrile (2)
All of 3 (assume 13 g, 71.8 mmol) and 4 (assume 10.9 g,
71.8 mmol) were combined, then dissolved in methanol (40 mL)

3304
P. F. Hughes et al. / Bioorg. Med. Chem. 20 (2012) 3298–3305
combined to give 8 (600 mg, 61%) as a lightly yellow glass. TLC (4/
1/0.1 CH2Cl2/MeOH/NH3) Rf = 0.30; 1H NMR (CDCl3) d 7.98 (t,
J = 4 Hz), 7.47 (d, J = 8.4 Hz, 1H), 6.77 (d, J = 2 Hz, 1H), 6.60 (dd,
J = 2, 8.4 Hz, 1H), 6.0 (br s, 2H), 3.61 (m, 16H), 3.28 (m, 2H), 2.85
(t, 2H), 2.79 (s, 2H), 2.52 (s, 3H) 2.37 (s, 2H), 2.28 (br s, 2H), 1.94
(m, 2H), 1.76 (m, 2H), 1.07 (s, 6H); MS (ESI): m/z 604.4 [M+H]+;
HRMS (ESI) [M+H]+ calcd for C31H50N5O7, 604.3705; found
604.37155.
5.2. Resin synthesis (GE healthcare instructions 71-7086-00
AFA)
Buffers and solutions
Swelling solution
Coupling buffer
Capping solution
Low buffer
High buffer
Storage buffer
1 mM HCl
0.1 M NaHCO3, 0.5 M NaCl, pH 8.3
1 M ethanolamine
0.1 M AcOH/NaAcOH, 0.5 M NaCl pH 4
0.1 M TRIS–HCl, 0.5 M NaCl pH 8
0.1 M KH2PO4, pH 7.4 w/200 mg NaN3/L
5.1.7. 2-((3-(2-(2-(3-Aminopropoxy)ethoxy)ethoxy)propyl)-
amino)-4-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-
indazol-1-yl)benzamide (9)
Compound 9 was prepared in the same way as compound 6. TLC
(4/1/0.1 CH2Cl2/MeOH/NH3) Rf = 0.39; 1H NMR (CDCl3) d 8.00 (t,
J = 5 Hz, 1H), 7.46 (d, J = 8.2 Hz, 1H), 6.77 (s, 1H), 6.60 (d,
J = 8.4 Hz, 1H), 6.1 (br s, 2H), 3.59 (m, 8H), 3.29 (m, 2H), 2.82 (t,
2H), 2.79 (s, 2H) 2.59 (br s, 2H), 2.51 (s, 3H), 2.37 (s, 2 h), 1.93
(M, 2 h), 1.73 (m, 2H), 1.07 (s, 6H); MS (ESI): m/z 516.4 [M+H]+;
HRMS (ESI) [M+H]+ calcd for C27H42N5O5, 516.3180; found
516.3191.
Ligand 14 (25 mg 25.74 lmol) was dissolved in trifluoroacetic acid
(1 mL). TLC (9/1/0.1:CH2Cl2/MeOH/NH3) showed loss of starting
material and formation of a lower product 12. The mixture was con-
centrated, then dissolved in ethanol (5 mL) and concentrated again.
The residue was then dissolved in ethanol (5 mL) for addition to the
resin.
In a big 275 mL column, CNBr-activated Sepharose™ 4B (25 g)
was swelled in 1 mM HCl (450 ml) and then washed with 1 mM
HCl (5 L). The resin was washed with coupling buffer (125 mL)
and then slurried with coupling buffer (125 mL). The mixture
was then treated with the linker-PEG-6 compound described
above. The mixture was tumbled at rt for 4 h. The resin was then
drained and washed with coupling buffer (5 ꢃ 125 mL), diluted
with more coupling buffer (ꢂ125 mL) and treated with capping
solution (2 mL) and rotated at rt for 2 h. The solution was drained
and washed with 3 rounds of high buffer/low buffer (250 mL ea.)
and finally washed with water (250 mL) and transferred in storage
buffer (125 mL) to a bottle and stored at 4 °C.
5.1.8. (E)-4-((5-(2-((tert-Butoxycarbonyl)amino)ethyl)-2-
hydroxyphenyl)diazenyl)benzoic acid (13)
4-Aminobenzoate (500 mg, 3.6 mmol) was slurried in 6 N HCl
(10 mL), cooled to 0 °C and treated slowly with sodium nitrite
(629 mg, 9.11 mmol). After stirring for 20 m, the mixture was
added slowly to an ice-cooled solution of N-Boc tyramine
(865 mg, 3.6 mmol) in saturated sodium bicarbonate solution
(40 mL) with added sodium bicarbonate (4 g) and a bit of acetone
(ꢂ5 mL). The orange reaction slurry was left to stir overnight.
The reaction mixture was treated 1 N HCl (100 mL) until acidic
and then stirred an additional 2 h. The solids were filtered off,
washed with water and air dried, then dried under vacuum to
give 13 (1.03 g, 73%) as a reddish-orange solid. The product
was used as is though it contained a minor impurity by NMR.
TLC (9/1 CH2Cl2/MeOH) Rf = 0.20; 1H NMR (DMSO-d6) d 13.19
(br s, 1H), 10.82 (s, 1H), 8.11 (d, J = 8.5 Hz, 2H), 8.05 (d,
J = 8.5 Hz, 2H), 7.55 (s, 1H), 7.28 (d, J = 8.3 Hz, 1H), 7.00 (d,
J = 8.3 Hz, 1H), 6.88 (t, 1H), 3.12 (m, 2H), 2.67 (t, J = 6.8 Hz, 2H),
1.33 (s, 9H); MS (ESI): m/z 384.2 [M]ꢁ, 791.4 [2 M+Na]ꢁ;
HRMS (ESI) [M+Na]+ calcd for C20H23N3O5Na, 408.1536; found
408.1518.
Acknowledgements
This work was funded by 1R01-AI089526-01 and 1R01-
AI090644-01 to T.A.J.H. K.B. is supported by a postdoctoral fellow-
ship from the King Hussein Institute for Biotechnology and Cancer,
Amman, Jordan and is currently on a sabbatical leave from the
Department of Applied Biology-Faculty of Science and Arts at the
Jordan University of Science and Technology, Irbid 21110, Jordan.
Supplementary data
5.1.9. (E)-tert-Butyl 3-((4-((19-((2-carbamoyl-5-(3,6,6-trimethyl-
4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-yl)phenyl)amino)-4,7,
10,13,16-pentaoxanonadecyl)carbamoyl)phenyl)diazenyl)-4-
hydroxyphenethylcarbamate (14)
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.03.043.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
Amine 8 (483 mg, 800
lmol), acid 13 (308 mg, 800
lmol), EDC
(161 mg, 840 mol) and HOBT (113 mg, 840
l
l
mol) and 2 chips of
DMAP and were dissolved in CH2Cl2 (10 mL) and stirred at RT for
2 h. The reaction mixture was added to a column and chromato-
graphed (2.5 ꢃ 20 cm, silica gel, CH2Cl2 (300 mL), 9/1 CH2Cl2/
MeOH (300 mL), 4/1 CH2Cl2/MeOH (300 mL). The active fractions
were combined and concentrated to a frothy glass to give 14
(560 mg, 72%). The hard glass was scraped out to give an orange
powder (460 mg). TLC (9/0.9/0.1 CH2Cl2/MeOH/NH3) Rf = 0.44; 1H
NMR (DMSO-d6) d 10.85 (s, 1H), 8.60 (br t, 1H), 8.40 (br t, 1H),
8.02 (br s, 4H), 7.92 (b s, 1H), 7.73 (d, J = 8.1 Hz, 1H), 7.55 (s, 1H),
7.27 (d, J = 8.1 Hz, 1H), 7.00 (d, J = 8.1 Hz, 1H), 6.88 (br t, 1H),
6.76 (s, 1H), 6.66 (d, J = 8.1 Hz, 1H), 3.47 (m, 20H), 3.19 (m, 1H),
3.12 (m, 1H), 2.90 (s, 2H), 2.67 (t, 2H), 2.52 (m under DMSO, 2H),
2.38 (s, 3H), 2.31 (s, 2H), 1.77 (m, 4H), 1.33 (s, 9H), 0.99 (s, 6H);
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