Inhibition of ErbB2(Her2) expression with small molecule transcription factor mimics

Article abstract of DOI:10.1016/j.bmcl.2009.08.090

Small molecules that mimic the transcriptional activation domain of eukaryotic transcriptional activators have the potential to serve as effective inhibitors of transcriptional processes. Here we show that one class of transcriptional activation domain mi

Full text of DOI:10.1016/j.bmcl.2009.08.090

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6233–6236
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Inhibition of ErbB2(Her2) expression with small molecule transcription
factor mimics
Lori W. Lee a, Christopher E. C. Taylor a, , Jean-Paul Desaulniers a, , Manchao Zhang c, Jonas W. Højfeldt b,
Quintin Pan c, Anna K. Mapp a,b,
*
a Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA
b Chemical Biology Doctoral Program, University of Michigan, Ann Arbor, MI 48109, USA
c Department of Otolaryngology, The Ohio State University Medical Center, Columbus, OH 43210, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Small molecules that mimic the transcriptional activation domain of eukaryotic transcriptional activators
have the potential to serve as effective inhibitors of transcriptional processes. Here we show that one
class of transcriptional activation domain mimics, amphipathic isoxazolidines, can be converted into
inhibitors of gene expression mediated by the transcriptional activator ESX through small structural
modifications. Addition of the small molecules leads to decreased expression of the cell surface growth
receptor ErbB2(Her2) in ErbB2-positive cancer cells and, correspondingly, decreased proliferation.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 29 July 2009
Revised 21 August 2009
Accepted 24 August 2009
Available online 1 September 2009
Keywords:
Tanscriptional inhibitor
ESX
Her2
ErbB2
Many human cancers are characterized by elevated levels of
proteins that regulate cell cycle progression and proliferation. In
approximately one-third of breast cancers, for example, the cell
surface receptor tyrosine kinase ErbB2(Her2) is overexpressed,
and this is correlated with increased metastasis and resistance to
chemotherapeutic agents.1 Such proteins are promising drug tar-
gets, with sales of the ErbB2(Her2) antibody/inhibitor Herceptin
(Trastuzumab) reaching $750 million in 2006.2 An emerging alter-
native strategy is to directly down-regulate the expression of rele-
vant proteins through blocking specific transcriptional activator–
coactivator interactions that are critical for activation of the en-
coded gene (Fig. 1a).3,4 The challenges of this strategy are consider-
able, in part because many transcriptional activators exhibit poorly
characterized, multi-partner binding profiles that are difficult to
reconstitute with a small molecule.5 We have previously described
a class of small molecules that serve as generic mimics of amphi-
pathic transcriptional activators.6–9 Here we show that this same
scaffold can be converted to a transcriptional inhibitor, a molecule
that effectively abrogates the expression of the growth receptor
ErbB2 at low micromolar concentrations and, correspondingly,
inhibits the proliferation of ErbB2-overexpressing cancer cells.
To block the interaction of an activator with its target in the
transcriptional machinery (the coactivator), a small molecule must
bind to either protein partner with sufficiently high affinity to
block the binding of the second protein. In the course of developing
small molecule-based transcriptional activators, we identified sev-
eral amphipathic isoxazolidines that mimic the transcriptional
activation domain (TAD) of endogenous amphipathic activators,
the domain that interacts directly with the transcriptional machin-
ery (Fig. 1b).6–8 When localized to DNA, the isoxazolidine TADs up-
regulate transcription in human cell culture up to 80-fold.9 Thus, in
the absence of a DNA-targeting moiety, we reasoned that this mol-
ecule could serve as a competitive inhibitor of activator–coactiva-
tor interactions (Fig. 1a). Supporting this idea, the isoxazolidine
TADs display a multi-partner binding profile consistent with their
natural counterparts; in addition, small structural changes alter
the binding pattern of the TAD mimics.6,12 We hypothesized that
this molecular scaffold would be an excellent starting point for
inhibitor development.
The activator chosen as a target for this study is ESX (ESE-1/ELF-
3/ERT/Jen), an epithelial-specific transcriptional activator that has
been shown to regulate expression of the ErbB2 oncogene.13,14
ESX interacts with multiple coactivator proteins; the most well-
characterized of these interactions is with Med23(Sur2/DRIP130/
CRSP130), a coactivator located in the mammalian mediator com-
plex.13 Several lines of evidence suggest that the ESX-Med23
interaction is a key regulator of ErbB2 expression. Furthermore,
* Corresponding author.
E-mail address: amapp@umich.edu (A.K. Mapp).
Equivalent experimental contributions.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.08.090

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L. W. Lee et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6233–6236
Figure 1. (a) Down-regulation of ErbB2(Her2) expression can be accomplished by blocking the activator–coactivator interactions responsible for initiating gene expression
with transcriptional activation domain (TAD) mimics.4,10,11 (b) Amphipathic isoxazolidines that mimic the function and mechanism of transcriptional activation domains
when attached to a DNA binding domain (‘DBD’).6–9
partial inhibition of ESX-Med23 complex formation has an inhibi-
tory effect on the proliferation of ErbB2-overexpressing cells.11,13
Within the minimal region of ESX(137-SWIIELLE-144) that binds
to Med23, tryptophan 138 is essential for the ESX-Med23 interac-
tion. NMR spectroscopic studies suggest that this residue along
with Ile139, Ile140, Leu142, and Leu143 form a hydrophobic sur-
face along an amphipathic helix that interacts with Med23.13
A fluorescein-tagged variant of 1 (1b) was assessed for its abil-
ity to interact with a region of Med23 (residues 352–625) that con-
tains the binding site for ESX by fluorescence polarization, and
binding was observed with a KD of 5.9 0.1 lM (Fig. S2 in Supple-
mentary data). Although low micromolar dissociation constants
are sufficient for function as a transcriptional activator, tighter
binding is likely necessary to inhibit the formation of a complex l
between a DNA-bound transcriptional activator and the transcrip-
tional machinery. Isoxazolidine 1 does not contain a large hydro-
phobic substituent similar to Trp138 in ESX; when this residue is
mutated to Phe, binding and ESX activity are attenuated.13 We thus
sought to enhance the affinity for Med23(352–625) and increase
the resemblance of the molecules to ESX by replacing the N2 ben-
zyl substituent with larger hydrophobic aryl groups ranging from
p-CF3Phe (2a) to biphenyl (4a) (Fig. 2).
Isoxazolidines 2a–4a were prepared via straightforward manip-
ulations of a previously reported isoxazoline (see Supplementary
data for details).8 For binding experiments with Med23(352–
625), the azide handle present in each of the structures was
reduced under Staudinger conditions and the resulting amine
was conjugated to FITC. Fluorescence polarization binding experi-
ments with each of the fluorescein-labeled isoxazolidines revealed
that increasing the size of the N2 substituent produced compounds
that bound an order of magnitude more strongly to the fragment of
Figure 2. Isoxazolidines 1a–4a were synthesized, and their fluorescein labeled
derivatives 1b–4b were evaluated for binding to MBP-Med23(352–625). FITC = 2-
(6-hydroxy-3-oxo-3H-xanthen-9-yl)-5-methanethioamidobenzoic acid. See Sup-
plementary data for details.
transcriptional level. ErbB2 mRNA transcript levels were assessed
via quantitative real-time polymerase chain reaction (qPCR) exper-
iments. BT-474 cells treated for 6 hours with compound 4a exhibit
reduced ErbB2 mRNA levels as compared to those treated with
compound 1a or vehicle (Fig. 3b). This is consistent with isoxazol-
idine 4a impacting ErbB2 expression at the transcriptional level.
The activity of isoxazolidine 4a was further examined in a sec-
ond ErbB2-dependent breast cancer cell line, SkBr3. Analogous to
the activity observed in BT-474 cells, low micromolar concentra-
tions of 4a were sufficient to down-regulate ErbB2 expression
(Fig. 4a). Isoxazolidine 1a showed no activity in that concentration
Med23 that interacts with ESX (2b: 0.59 0.06
lM; 3b:
1.0 0.2 M; 4b: 0.62 0.08 M; Fig. S2 in Supplementary data).
l
l
Isoxazolidines 1a–4a were tested for their ability to down-reg-
ulate ErbB2 expression in BT-474 cells, an ErbB2-overexpressing
breast cancer cell line. Consistent with binding data, isoxazolidine
1a did not significantly impact ErbB2 expression at concentrations
up to 50 lM (Fig. 3a) as compared to isoxazolidines 2a–4a, all of
range, although at concentrations above 25 lM reduced ErbB2
which produced a dose-dependent reduction in ErbB2 expression
after a 24-h treatment. Of the three modified isoxazolidines, com-
pound 4a was the most effective at downregulating ErbB2 expres-
sion, with an EC50 in the low micromolar concentration range. We
subsequently evaluated the effects of compound 4a at the
expression was observed. Previous studies have correlated lowered
ErbB2 activity with attenuated cell proliferation;15 therefore we
examined growth effects of isoxazolidine 4a in SkBr3 cells. Consis-
tent with the results of Figure 4a, isoxazolidine 4a inhibited cell

L. W. Lee et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6233–6236
6235
Figure 3. (a) Effect of isoxazolidines 1a–4a on ErbB2 expression in BT-474 breast
cancer cells as assessed by Western blot analysis. Cells were treated with
compounds dissolved in DMSO for 24 h before analysis. Lane 1: DMSO; lane 2:
50
l
M 1a; lanes 3–6: 3.2, 6.25, 12.5, 25
l
M of 2a; lane 7: DMSO; lanes 8–11: 3.2,
Figure 4. (a) Effect of isoxazolidines 1a and 4a on ErbB2 expression in SkBr3 breast
cancer cells as assessed by Western blot analysis. Cells were treated with
compounds dissolved in DMSO for 6 h before analysis. Lane 1: DMSO; lanes 2–3:
6.25, 12.5, 25
lM of 3a; lane 12–15: 3.2, 6.25, 12.5, 25
l
M of 4a. (b) Effect of
isoxazolidines 1a and 4a on ErbB2 transcript levels as assessed by quantitative real-
time polymerase chain reaction. Cells were treated with compounds dissolved in
DMSO (10 lM) for 24 h before analysis. Graph shows the average (16 experiments
done in quadruplicate) of ErbB2 mRNA levels relative to DMSO and normalized to
GAPDH. Error represents SEM and p value obtained from Student’s t-test; right:
Western blot analysis of BT-474 cells from qPCR experiments. See Supplementary
data for details.
10, 50 lM of 1a; lanes 4–10: 0.625, 1.25, 2.5, 5, 10, 25, 50 lM of 4a. (b) Effect of
compounds 1a and 4a on the viability of ErbB2-positive SkBr3 cells. Viability was
measured 24 h after dosage via WST-1 assay. Data points are median values of
experiments done in triplicate with error bars representing standard deviation. See
Supplementary data for details.
growth with an IC50 of 14
which did not affect growth (Fig. 4b). Furthermore, the effects of
isoxazolidine 4a in the non-ErbB2 over-expressing cell line MCF-
1 lM, in contrast to isoxazolidine 1a
application of this strategy to the development of a repertoire of
transcriptional inhibitors will be reported in due course.
7 are reduced (IC50 27
4 lM; Fig. S3 in Supplementary data), in
agreement with previous findings.13,16 In addition to its role in cer-
tain breast cancers, ErbB2 over-expression is a hallmark of head
and neck cancers.17 Towards this end, we assessed the ability of
biphenyl 4a to inhibit the growth of CAL27, SCC-25, and SCC-15
cell lines and found that while isoxazolidine 1a had no impact on
cell proliferation, 4a dose-dependently inhibited cell growth
(Fig. S4 in Supplementary data). In the course of these experiments,
we observed that increasing serum concentrations lead to attenu-
ated effects of the small molecule. This may be attributable to ser-
um binding, a well-known phenomenon for lipophilic molecules,18
an additional contributor may be serum-dependent effects on cell
signaling.19 Differentiating these contributions will be the subject
of future investigations.
Acknowledgments
A.K.M. is grateful to the NIH (CA140667), Novartis (Novartis
Young Investigator Award), and the NSF (PECASE) for support of
this work. J.-P.D. thanks the American Cancer Society for a postdoc-
toral fellowship (PF-06-116-01). We would like to thank Taocong
Jin at the Molecular Biology Core Laboratory (School of Dentistry,
University of Michigan) for helpful discussions and qPCR assistance
and Kenneth Guire at the Center for Statistical Consultation and
Research (CSCAR, University of Michigan) for statistical analysis
of qPCR data.
Supplementary data
Taken together, these data suggest that isoxazolidine 4a is a
much improved mimic of the transcriptional activation domain
of ESX relative to the generic TAD mimic 1a, validating our strategy
for using an artificial TAD as a scaffold for the design of transcrip-
tional inhibitors. Treatment of ErbB2-positive cell lines with isoxa-
zolidine 4a attenuated ErbB2 protein levels as well as ErbB2 mRNA
transcripts. Additionally, isoxazolidine 4a inhibits the growth of
several ErbB2-overexpressing cell lines. This improved ESX
mimicry does not, however, correspond to improved activity as a
transcriptional activation domain. When localized to DNA, isoxa-
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.08.090.
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