Chemically accessible hsp90 inhibitor that does not induce a heat shock response

Article abstract of DOI:10.1021/ml500114p

Recent cancer therapies have focused on targeting biology networks through a single regulatory protein. Heat shock protein 90 (hsp90) is an ideal oncogenic target as it regulates over 400 client proteins and cochaperones. However, clinical inhibitors of hsp90 have had limited success; the primary reason being that they induce a heat shock response. We describe the synthesis and biological evaluation of a new hsp90 inhibitor, SM253. The previous generation on which SM253 is based (SM145) has poor overall synthetic yields, low solubility, and micromolar cytotoxicity. By comparison SM253 has relatively high overall yields, good aqueous solubility, and is more cytotoxic than its parent compound. Verification that hsp90 is SM253s target was accomplished using pull-down and protein folding assays. SM253 is superior to both SM145 and the clinical candidate 17-AAG as it decreases proteins related to the heat shock response by 2-fold, versus a 2-4-fold increase observed when cells are treated with 17-AAG.

Full text of DOI:10.1021/ml500114p

Letter
pubs.acs.org/acsmedchemlett
Chemically Accessible Hsp90 Inhibitor That Does Not Induce a Heat
Shock Response
Yen Chin Koay, Jeanette R. McConnell, Yao Wang, Seong Jong Kim, Laura K. Buckton, Flora Mansour,
and Shelli R. McAlpine*
The University of New South Wales, Sydney, NSW 2052, Australia
S
* Supporting Information
ABSTRACT: Recent cancer therapies have focused on
targeting biology networks through a single regulatory protein.
Heat shock protein 90 (hsp90) is an ideal oncogenic target as
it regulates over 400 client proteins and cochaperones.
However, clinical inhibitors of hsp90 have had limited success;
the primary reason being that they induce a heat shock
response. We describe the synthesis and biological evaluation
of a new hsp90 inhibitor, SM253. The previous generation on
which SM253 is based (SM145) has poor overall synthetic
yields, low solubility, and micromolar cytotoxicity. By
comparison SM253 has relatively high overall yields, good
aqueous solubility, and is more cytotoxic than its parent
compound. Verification that hsp90 is SM253’s target was accomplished using pull-down and protein folding assays. SM253 is
superior to both SM145 and the clinical candidate 17-AAG as it decreases proteins related to the heat shock response by 2-fold,
versus a 2−4-fold increase observed when cells are treated with 17-AAG.
KEYWORDS: SM253, heat shock protein 90, heat shock response, SM145, macrocycle, conformation, peptide, natural product
ancer cells utilize numerous pathways in order to
maintain their propagation. Such rapid oncogenic
tumors and indicates poor patient prognosis in breast, colon,
and lung cancer.13,14 Since cancer cells have an abundance of
mis-folded and mutated proteins, they are also significantly
more dependent on these hsps than normal cells, making them
prime anticancer targets.15
C
proliferation produces an abundance of mutated and mis-
folded proteins, which require molecular chaperones such as
heat shock proteins (hsps) to fold and stabilize these proteins.
Specifically, hsps 90, 70, 40, and 27 are required to maintain the
cell’s function under stressed conditions by deaggregating and
folding over 400 proteins (Figure 1).1,2 Heat shock factor 1
Current clinical hsp90 inhibitors bind to the ATP binding
pocket in the amino terminus of hsp90. Over 40 clinical trials
were initiated using amino terminal inhibitors; however, the
entire class of compounds induces a significant HSR,16 which
led to antiapoptotic and chemo-resistance mechanisms and
their subsequent removal from the clinic as a monotherapy.17,18
In an attempt to circumvent the HSR, clinical hsp90 inhibitors
have been combined with alternative cancer therapies, which
has proven successful in preclinical and clinical settings.19−23
Finding an hsp90 inhibitor that does not induce a HSR would
be a significant achievement. The development of specific C-
terminal hsp90 inhibitors has been reported as a promising
alternative to the N-terminal clinical hsp90 inhibitors, as they
do not induce the heat shock response.24,25 However, these
inhibitors have micromolar and millimolar IC50 values, and only
one reported inhibitor had data discussing the HSR.25
Figure 1. Heat shock response involves HSF1 up-regulating HSF1
protein, hsp90, hsp70, hsp40, and hsp27.
(HSF1) transcribes and up-regulates the protein levels of
hsp90, 70, 40, and 27 under stressed conditions, and the
increase in HSF-1 and hsp levels is a primary feature of many
cancers (Figure 1).3−8 High levels of hsps and HSF-1 are
usually referred to as a heat shock response (HSR), whereby
these up-regulated proteins block apoptosis, promote onco-
genic growth, and reverse effects of a chemotherapeutic drug all
Our previous work describes the hsp90 inhibition activity of
SM145 (Figure 2).26−30 This molecule binds to a unique site
on hsp90 and blocks hsp90 from interacting with multiple
Received: March 17, 2014
Accepted: May 13, 2014
Published: May 13, 2014
by maintaining and stabilizing proteins in the cell.3,6,7,9−12
A
high level of HSF-1 is also associated with highly malignant
© 2014 American Chemical Society
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ACS Medicinal Chemistry Letters
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The inclusion of ortho-, meta-, and para-pyridal groups
(compounds 2−4) decreased the ClogP value (an estimate for
solubility); however, they have dramatically reduced potency
compared to compound 1 (>15 μM, Table 1). In addition,
when a tryptophan or methylated tyrosine was placed at
position V (compounds 5 and 6), the cytotoxicity was
improved over compound 1, but the aqueous solubility of
these two molecules is poor (<2 mg/mL, Table 1). One
derivative, compound 7, which incorporated a thiazole moiety,
had both improved cytotoxicity and solubility over compound 1
(Table 1).
Overall, compound 7 (from here on referred to as SM253)
shows improved properties over the parent compound SM145.
SM253, which contains a D-3-(4-thiazoyl)alanine at position V
and the biphenyl moiety at position I, is (1) significantly easier
to synthesize than SM145 (overall yields = 40%); (2) more
soluble than both SM145 and compound 1 (9.5 mg/mL); (3)
has improved cytotoxicity against 2 cancer cell lines relative to
both SM145 and compound 1 (Table 1); and (4) reduces the
HSR relative to the clinical inhibitor 17-AAG and SM145
(Figure 4).
All the analogues were made via a combination of solid-phase
and solution phase chemistry (Scheme 1).27−30,36 Using a
preloaded 2-chlorotrityl-leucine resin (CTC) (Supporting
Information) and then sequentially coupling fluorenylmethy-
loxy carbonyl (Fmoc) protected amino acids followed by
deprotection of the amine produced the desired linear
pentapeptide. The peptide was cleaved from the resin with
50% trifluoroethanol in dichloromethane, which generated the
double deprotected linear pentapeptide. The linear peptide was
then cyclized using three coupling reagents under dilute
conditions (Scheme 1 and Supplemental Schemes 1 and 4).37
To explore the role of SM253 in inducing cytotoxicity, we
examined its effects in two human cancer cell lines, HCT116
colon carcinoma and MiaPaCa-2 pancreatic carcinoma cells.
The cytotoxicity of compound 1 and SM253 toward HCT-116
and MiaPaCa-2 cells were analyzed using a cell-counting kit 8
(CCK8). Compound 1 demonstrated similar cytotoxicity to
that of SM145, while SM253 had a lower IC50 value compared
to SM145 in both HCT-116 and MiaPaCa-2 cells lines (Table
1 and Supplemental Figure S7). In general, hsp90 inhibitors
have been reported to display remarkable selectivity for tumor
cells as compared to healthy cells, and the inhibitors have been
shown to accumulate in tumor tissue while being rapidly
cleared from circulation and normal tissue.15,38,39 Analogues of
this series have shown an average of 3-fold greater selectivity of
cancer cells over normal cells (WS1) (Supplemental Figure
S7).31,33,35,40,41 In addition to its enhanced potency, SM253 is
2-fold more soluble than SM145 and 5-fold more soluble than
Figure 2. Structures of SM145 and new compounds 1−7.
proteins associated with its C-terminus. Furthermore, SM145
does not induce the HSR survival mechanism.26−30 However,
as noted in our previous work, there are three significant issues
associated with SM145: its low yielding synthesis (overall final
yield 1−7%), its hydrophobicity (ClogP = ∼9), and its
micromolar potency.
Herein we describe the design, synthesis, and character-
ization of 7 new molecules, compounds 1−7, derived from the
hsp90 inhibitor SM145 (Figure 2). The first analogue involved
a substitution of a biphenyl moiety in place of the benzylated
phenyl serine (compound 1), which reduced synthetic
complexity and increased the synthetic yield dramatically,
while not disrupting the cytotoxicity or binding affinity for
hsp90 (Table 1, Figure 2, and Supplemental Figures S5 and
S7). This scaffold was used to create a series of analogues. Our
early structure−activity relationship (SAR) work indicated that
position V did not have significant impact on binding affinity
for hsp90; thus, we modified this position in order to improve
the pharmacological properties.31−35 The modifications in-
volved incorporating aromatic heteroatom containing side
chains with the goal of increasing hydrogen-bonding
capabilities, improving solubility, and improving cytotoxicity
while not impacting the macrocycles overall conformation.
Table 1. Comparison of SM145 and Compounds 1−7
compound
IC50 HCT-116 (μM)
IC50 MiaPaCa-2 (μM)
% yield
ClogP
saturated solution of 80% EtOH/20% H2O
SM145
7.4 0.5
7.6 0.4
18.1 0.4
20.8 2.5
15.7 2.9
3.9 0.2
5.1 0.6
5.0 0.5
7.8 0.5
12.8 2.3
23.6 2.5
23.0 2.8
18.5 5.3
4.5 0.9
4.4 0.7
5.5 0.2
1−7%
40%
25%
28%
30%
32%
30%
40%
9.02
9.09
7.60
7.60
7.60
9.02
9.09
7.44
5.2 mg/mL
2.1 mg/mL
1
a
2
N.D.
3
N.D.
4
N.D.
5
1.9 mg/mL
1.5 mg/mL
9.5 mg/mL
6
7 (SM253)
a
N.D.: not determined.
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ACS Medicinal Chemistry Letters
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a
a
Scheme 1. Synthesis of SM253 (Compound 7)
Scheme 2. Synthesis of SM253T-II
a
Abbreviations: 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azaben-
zotriazole (HOAt), dimethylformamide (DMF), N,N′-diisopropylcar-
bodiimide (DIC), 2,2,2-trifluoroethanol (TFE), 2-(1H-7-azabenzo-
triazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
(HATU), O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetra-
fluoroborate (TBTU), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-
morpholin-4-ium chloride (DMTMM), N,N-diisopropylethylamine
(DIPEA); sphere indicates CTC resin.
a
Abbreviations: tert-butoxycarbonyl (Boc), trifluoroacetic acid (TFA),
N-hydroxysuccinimide ester (NHS), poly(ethylene glycol) (PEG).
compound 1, which is attributed to the inclusion of the D-3-(4-
Thiazoyl)alanine. This improved solubility and cytotoxicity
warranted further mechanistic studies of SM253.
Since the parent molecule SM145 was already identified as
an hsp90 inhibitor,26,28 we used the same approach to
determine whether SM253 bound to hsp90. Our previous
work with SM145 had shown that tagging the molecule at
position II did not impact its ability to bind to hsp90.26,42,43
Using the same conditions as described in Scheme 1, SM253
was synthesized with a tag at position II (Scheme 2) via the
Boc-lysine derivative of SM253.
Briefly, using a pre-loaded 2-chlorotrityl-D-leucine resin and
then sequentially coupling the Fmoc protected amino acids
followed by removal of the Fmoc group and cleavage from the
resin with trifluoroethanol produced the linear precursor
(Scheme 2).44 After cyclization of the linear precursor, the
lysine was deprotected using 25% trifluoroacetic acid, and then
coupled to N-hydroxysuccinimydyl-D-biotin-15-amino-
4,7,10,13-tetraoxapentadecylate (NHS-Peg-4-biotin), thus com-
pleting the synthesis of SM253TII.28,29,36,45,7
The biotinylated SM253TII was then used in a pull-down
assay.28 Briefly, 0.1 mg of the biotinylated analogue was
incubated in HCT-116 or MiaPaCa-2 lysates, and then the
compound was retrieved from the mixture using the biotin tag
by adding NeutrAvadin beads. Proteins bound to the
compound were then eluted off the beads with sample buffer
and visualized using coomassie blue or Western blotting and
were sequenced using nano-LC/MS/MS (Figure 3A and
Figure 3. (A) Pull-down with SM253TII in HCT-116 and MiaPaCa-2
cell lysates, representative image shown of 3 independent assays. (B)
Luciferase refolding assay. Graph of mean SEM, n = 3.
Supplemental Figures S4 and S8). Like its parent compound
SM145, SM253 bound to hsp90 in both HCT-116 and
MiaPaCa-2 cells lysates as demonstrated by the dark band
identified as hsp90 in the SM253TII lane (Figure 3A). In
addition, sequencing of the proteins bound to SM253 revealed
that hsp90 was bound to SM253 (Supplementary Figure S8).
These data together demonstrate that SM253 interacts with
hsp90 in both HCT-116 and MiaPaCa-2 cells.
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Figure 4. (A) Protein levels of HSR proteins in HCT-116 and MiaPaCa-2 cells after 24 h with the indicated treatment; density values were
normalized to actin and then compared to DMSO. Graph of mean SEM, n = 3. (B) Representative Western blots of protein levels in HCT-116
and MiaPaCa-2 cells (black arrow indicates HSF1 band that was quantitated, *p < 0.02, **p < 0.005, ***p < 0.0005, ****p < 0.0001). 17-AAG
Western blots for MiaPaCa-2 appear in Supplemental Figure S6.
Since SM253 binds to hsp90 like its parent compound, it
should inhibit the function of hsp90 in a similar manner to
SM145.
and MiaPaCa-2) showed that SM253 decreased these HSR
proteins (Figure 4). In contrast to SM253, treatment with the
IC50 value of 17-AAG induced a significant heat shock response
increasing the antiapoptotic proteins HSF-1, hsp70, and hsp27
by greater than 2-fold in both cells lines (Figure 4 and
Supplemental Figure S6).26 Thus, SM253 is clearly acting via a
different mechanism to 17-AAG, as treatment of cells with 10-
fold over the IC50 value for SM253 not only failed to induce a
HSR, but significantly decreased all levels of these heat shock
proteins by approximately 2−4-fold (Figure 4).
Our previous work shows that 10-fold over 17-AAG’s IC50
value (1 μM) induces hsp90 by 3-fold, hsp70 by 2.5-fold, and
HSF1 by 4-fold.26 Perhaps the most remarkable aspect of
SM253 is the complete erasure of hsp27 protein in HCT-116
cells (Figure 4A) versus the 2.5-fold increase seen in cells
treated with 17-AAG. Since hsp27 is linked to the development
of chemo-resistance, the complete removal of this protein by
SM253 is novel and highlights that hsp90 can be inhibited
without inducing a resistance protein. Interestingly, SM145 has
a similar although less desirable profile to that of SM253. The
two molecules specifically diverge in their effect on hsp40.
Although hsp40’s role in cancer is poorly understood, it is
known to play a key role in delivering the unfolded proteins to
hsp70, which are then transferred to hsp90 for final folding and
activation. Therefore, SM253 may prove a highly relevant tool
for delineating the activity of hsp40 in colon and pancreatic
cancer.49 In summary, SM253’s ability to block hsp90 activity
and inhibit the HSR makes it a novel hsp90 inhibitor that
Confirmation that SM253 inhibits the function of hsp90 was
accomplished using a protein-folding assay, involving rabbit
reticulocyte lysate (RRL, Promega) and denatured firefly
luciferase. Denatured luciferase was incubated in RRL, which
was pretreated with the approximate IC50 values of 17-AAG (a
clinical hsp90 inhibitor), SM145, and SM253.46,47 After 2 h of
incubation, SM253 had the greatest impact on inhibiting the
refolding activity of the hsp90 machinery, reducing the
luciferase activity by 42% relative to control (Figure 3B). In
contrast, 17-AAG and SM145 only reduced luciferase activity
by 17% and 25%, respectively. Significantly, SM253 demon-
strates a greater ability to inhibit hsp90 machinery protein
folding over SM145 and prevents hsp90 from completing its
role as a molecular chaperone.
A key factor to consider when designing new hsp90
inhibitors is the ability to block hsp90 function without
inducing the HSR since it initiates a cascade that can protect
the cell from chemotherapy drugs and induces cell survival
mechanisms.16,48 Like the other clinical candidates, the HSR
induced by 17-AAG was a major reason behind its failure in the
clinic.26 Given the success of SM253 at blocking the protein
folding function of hsp90, we explored whether SM253 did so
without inducing the HSR. Treating the cells with the IC50
value of SM253 and evaluating the protein levels of HSF1,
hsp90, hsp70, hsp40, and hsp27 in two cell lines (HCT-116
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ACS Medicinal Chemistry Letters
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overcomes the resistance mechanisms associated with current
and past clinical hsp90 inhibitors.
It is already established that inhibition of hsp90 leads to cell
death via apoptosis and that both 17-AAG and SM145 induce
apoptosis.28 Using annexin V and 7-AAD we demonstrated that
SM253 also induces apoptosis (Figure 5). After 24 h of
ASSOCIATED CONTENT
* Supporting Information
Synthetic procedures, characterization data, and biological assay
procedures. This material is available free of charge via the
Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*(S.R.A.) E-mail: s.mcalpine@unsw.edu.au. Phone: +61 2 9385
4712.
■
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank C. Brownlee and Biological Resources Imaging
Laboratory (BRIL) and UNSW for support of S.R.M. Y.C.K.
thanks the Australian government for providing the Endeavour
scholarship fund. Finally we thank James Gardiner from CSIRO
for helpful discussions.
Figure 5. Apoptosis analysis of HCT-116 cells treated with SM253,
SM145, and 17-AAG. Graph shows the average of 3 separate assays (p
= 0.004 for all apoptotic cells of SM253 (50 μM) compared to DMSO;
raw data and table of mean SD appears in Supplemental Figure S2).
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