Hsp90 inhibitors identified from a library of novobiocin analogues

Article abstract of DOI:10.1021/ja0535864

Novobiocin is a C-terminal inhibitor of the Hsp90 protein folding machinery, which is responsible for the conformational maturation of numerous proteins involved in cancer growth and survival. Due to novobiocin's poor inhibitory activity (～700 μM), very little attention has been paid toward the development of novobiocin analogues for Hsp90 inhibition. In this study, a parallel library of 20 novobiocin derivatives was prepared and the biological activity of each evaluated by Western blot analysis of Hsp90 client proteins. A4 was found to be a potent inhibitor of Hsp90 as determined by its ability to cause the degradation of several Hsp90 client proteins in both breast and prostate cancer cell lines. In the presence of 1 μM A4, several Hsp90 client proteins were degraded, including AKT, Her2, Hif-1α, and the androgen receptor. Copyright

Full text of DOI:10.1021/ja0535864

Published on Web 08/25/2005
Hsp90 Inhibitors Identified from a Library of Novobiocin Analogues
Xiao Ming Yu,† Gang Shen,† Len Neckers,§ Helen Blake,§ Jeff Holzbeierlein,‡ Benjamin Cronk,‡ and
Brian S. J. Blagg*,†
Department of Medicinal Chemistry and The Center for Protein Structure and Function, The UniVersity of Kansas,
1251 Wescoe Hall DriVe, Malott 4070, Lawrence, Kansas 66045-7582, Urologic Oncology Branch, National Cancer
Institute, NIH, RockVille, Maryland 20850, and Department of Urology, The UniVersity of Kansas Medical Center,
Kansas City, Kansas 66103
Received June 1, 2005; E-mail: bblagg@ku.edu
Scheme 1
Multiple signaling pathways are upregulated or constituitively
activated in malignant cells leading to (1) self-sufficiency in growth
signals, (2) insensitivity to antigrowth signals, (3) evasion of
apoptosis, (4) limitless replicative potential, (5) sustained angio-
genesis, and (6) tissue invasion/metastasis.1 Several anticancer
agents target individual enzymes/proteins involved in the above
processes, but no clinically available antitumor agent is capable of
simultaneously inhibiting all six hallmarks of cancer.1
The 90 kDa heat shock proteins (Hsp90) are essential for
refolding denatured proteins as well as for the conformational
maturation of nascent polypeptides into biologically active three-
dimensional structures.2 Several Hsp90-dependent client proteins
have been identified, including Raf-1, HER2, Src-family kinases,
steroid hormone receptors, polo-1-kinase, death domain kinase,
protein kinase B, focal adhesion kinase, telomerase, hypoxia
inducible factor, and MET kinase.2 Consequently, Hsp90 has
emerged as a promising biological target for the development of
cancer therapeutics because signaling nodes regulating all six
hallmarks of cancer can be simultaneously disrupted by inhibition
of the Hsp90 protein folding machinery.3
substituent (A),7 removal of both the 4-hydroxy and amide linker
(B), steric replacements of both the 4-hydroxy and benzamide ring
(C), and 1,2-positional isomers of the noviosyl linkage (D and E).
These selected coumarin rings were coupled with trichloroace-
Hsp90 contains two nucleotide-binding sites; the N-terminal ATP
binding site is the region to which geldanamycin (GDA) and
timidate of noviose carbonate8 in the presence of boron trifluoride
radicicol bind4 and the C-terminus, which was recently shown to
etherate, as shown in Scheme 1.9 The resulting cyclic carbonates
bind novobiocin, a coumarin-containing DNA gyrase inhibitor.5
(1) were treated with methanolic ammonia to provide 2′-carbamoyl
Although other DNA gyrase inhibitors may also inhibit Hsp90, the
(2), 3′-carbamoyl (3), and descarbamoyl products (4) in good yields.
coumarin antibiotics have so far proven the most promising of this
Inhibition of Hsp90 results in the degradation of Hsp90-
type. Unfortunately, novobiocin’s ability to cause degradation of
dependent clients via ubiquitination of the unfolded client followed
Hsp90 clients is relatively weak (∼700 µM in SKBr3 cells)6 and
by proteasome-mediated hydrolysis.2,3 To test whether Hsp90 client
requires further scientific investigation for the development of more
proteins were degraded in the presence of these novobiocin
potent compounds.
analogues, each member of the library was incubated with SKBr3
breast cancer cells at a concentration of 100 µM. Western blot
analysis of the protein lysates demonstrated that several of the
compounds were capable of causing the degradation of the Hsp90-
dependent client protein, phospho-AKT as represented in Figure
1. Phospho-AKT was chosen as a client protein for this assay
because of previous reports indicating that phospho-AKT is a more
sensitive indicator of Hsp90 inhibition than AKT.10 Geldanamycin
(GDA, 0.5 µM)3 was used as a positive control to determine the
relative ratio of phospho-AKT in the same assay.
In this communication, we report the synthesis and identification
of a novobiocin analogue that is substantially more active than the
parent compound. These results clearly indicate that novobiocin
derivatives represent a promising, unexplored class of anticancer
agents that act by inhibition of the Hsp90 protein-folding machinery.
A library of novobiocin analogues was prepared that included
shortening of the amide side chain and removal of the 4-hydroxy
The most active compound identified in this assay was A4, which
contains an N-acetyl side chain in lieu of the benzamide, lacks the
4-hydroxyl of the coumarin moiety, and has an unmodified diol.
Structure-activity relationships for these compounds suggest that
attachment of the noviose moiety to the 7-position of the coumarin
ring is important for biological activity (B vs D and E). Incorpora-
tion of the amide linker (A) resulted in greater inhibitory activity
than the unsubstituted derivative, B. It is likely that the diol (4)
mimics the ribose ring in the normal substrate (ATP) and may
† The University of Kansas.
§ National Cancer Institute.
‡ The University of Kansas Medical Center.
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J. AM. CHEM. SOC. 2005, 127, 12778-12779
10.1021/ja0535864 CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
Figure 1. Relative ratios of phospho-AKT by Western blot analyses. Total
protein concentration of each lysate was determined, and equal amounts of
protein were run in each lane of the gels. For the graphs shown in Figure
1, the O.D.’s (optical density) of the Western bands for phospho-Akt were
measured, as were the O.D.’s for actin probed as controls on the same blots.
To obtain the graphed values, all specific O.D.’s (for Hsp90 clients) were
normalized to the respective actin O.D.
Figure 3. (Top) Western blot analysis of LNCaP cells treated with A4.
(Bottom) Western blot analysis of LAPC-4 cells incubated with A4. Actin
was used as a control in both assays.
provide compounds with increased inhibitory activity and may
ultimately provide clinically useful alternatives to GDA. Studies
are underway to provide improved analogues of A4 and to
determine its activity in vivo. The results from such studies will
be reported in due course.
Figure 2. The chemical structure of A4.
explain why replacement with a cyclic carbonate (1) or 2′-carbamate
(2) resulted in a loss of activity.
Acknowledgment. The authors gratefully acknowledge support
of this project by the NIH COBRE RR017708, The University of
Kansas Center for Research, and The University of Kansas New
Faculty General Research Fund. G.S. is the recipient of a Susan
G. Komen Dissertation Award.
The steroid hormone receptors are also dependent upon the
Hsp90 protein folding machinery for activation and hormone
binding.2 To determine whether A4 had similar effects on the
androgen receptor, A4 was tested in both a mutated androgen
receptor-dependent prostate cancer cell line (LNCaP) and a wild-
type androgen receptor prostate cancer cell line (LAPC-4). As can
be seen in Figure 3, A4 had a dramatic effect on the concentrations
of the mutant androgen receptor, AKT, and Hif-1R at ∼1 µM in
the LNCaP cell line. A4 drastically reduced levels of the androgen
receptor at lower concentrations in the wild-type androgen receptor
prostate cancer cell line (LAPC-4). To verify that A4 was not
affecting other transcriptional or translational processes that could
account for decreased protein, Hsp90 levels were determined. Under
normal conditions, Hsp90 binds heat shock factor 1 (HSF1), but in
the presence of Hsp90 inhibitors, this interaction is lost and HSF1
is able to induce Hsp90 expression. In this work, we showed that
Hsp90 levels were significantly increased in a manner dependent
upon the concentration of A4, consistent with similar results
previously obtained by incubation with geldanamycin and radicicol.
Both of these data are in contrast to actin, which is not an Hsp90
client protein and, thus, remains unaffected by Hsp90 inhibitors.11
In conclusion, we have prepared and evaluated a small library
of novobiocin analogues, from which we have identified A4 as a
potent inhibitor of the Hsp90 protein-folding process. Further
structure-activity relationships of A4 with Hsp90 are likely to
Supporting Information Available: Full experimental procedures
and characterization for all compounds. This material is available free
of charge via the Internet at http://pubs.acs.org.
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