Targeted inhibition of Snail family zinc finger
transcription factors by oligonucleotide-Co(III)
Schiff base conjugate
Allison S. Harney, Jiyoun Lee, Lisa M. Manus, Peijiao Wang, David M. Ballweg, Carole LaBonne1,
and Thomas J. Meade1,2
Departments of Chemistry, Biochemistry, Molecular and Cell Biology, Neurobiology and Physiology, and Radiology, Northwestern University,
Evanston, IL 60208
Communicated by Brian M. Hoffman, Northwestern University, Evanston, IL, June 9, 2009 (received for review February 27, 2009)
A transition metal complex targeted for the inhibition of a subset
of zinc finger transcription factors has been synthesized and tested
in Xenopus laevis. A Co(III) Schiff base complex modified with a
17-bp DNA sequence is designed to selectively inhibit Snail family
transcription factors. The oligonucleotide-conjugated Co(III) com-
plex prevents Slug, Snail, and Sip1 from binding their DNA targets
whereas other transcription factors are still able to interact with
their target DNA. The attachment of the oligonucleotide to the
complex. Studies demonstrate that neither the oligo, or the Co(III)
Schiff base complex alone, are sufficient for inactivation of Slug at
concentrations that the conjugated complex mediates inhibition.
Slug, Snail, and Sip1 have been implicated in the regulation of
epithelial-to-mesenchymal transition in development and cancer.
prove valuable as an experimental tool and a cancer therapeutic.
gene expression ? transition metal complex
including bone, lung, liver, and brain (1). The zinc finger
transcription factors Slug, Snail, and Sip1 have been implicated
in tumor metastasis through the regulation of epithelial-to-
mesenchymal transitions (EMTs) (2–5). Much of what is known
about the molecular mechanism via which these transcriptional
regulators mediate EMT are from developmental studies, par-
ticularly the induction of vertebrate neural crest migration
(6–11). Xenopus laevis embryos have been used as a model
system to study the molecular activities of Snail family members
(9–12). During EMT, these transcriptional repressors down-
regulate the expression of proteins involved in cell–cell adhe-
sions characteristic of epithelial cells. Proteins involved in inva-
sion, such as matrix metalloproteinases, are up-regulated, and
cells lose their epithelial characteristics becoming invasive mes-
enchymal cells (2, 13–17).
Snail family transcription factors interact with DNA through
zinc fingers of the C2H2type with each finger coordinating 1 zinc
Sip1 contains C-terminal and N-terminal zinc finger domains,
each of which is composed of 3 tandem zinc fingers. Slug, Snail,
and Sip1 bind to the Ebox consensus sequence CAGGTG in the
promoter region of target genes with high specificity to mediate
transcriptional repression. These transcription factors are
emerging as targets for cancer therapeutics because of their role
in EMT, and consequently, metastasis (19).
A zinc ion coordinated to 2 histidine and 2 cysteine residues
is integral to the structure and function of zinc finger domains
(20). It is well known that transition metals, such as Zn, Fe, and
Co have a high affinity for histidines and cysteines. The affinity
been used to generate transition metal inhibitors of enzymatic
activity and DNA-protein interaction (21–24). Displacement of
reast cancer is a heterogeneous disease with fatalities re-
sulting from recurrence and metastasis to distant sites
the zinc ion by a Co(III) Schiff base complex [Co(III)-sb] has
been shown to inhibit DNA binding of at least 1 other zinc finger
transcription factor (25) and enzymatic function of non-DNA
binding proteins (25–28).
In the present study, oligonucleotide-linked Co(III)-sb with
axial ammine ligands are used to target Ebox binding zinc finger
domains and inhibit DNA binding (Fig. 1). The mechanism
involves dissociative ligand exchange where the substitutionally
active axial ammines are displaced by water to form the di-aquo
species (28). The lone pair of electrons on the nitrogenous
donors of the imidazole ring of histidines present in a zinc finger
coordinate to the Co(III) agent irreversibly disrupting the
coordination environment of Zn(II) (25, 27).
To target the Co(III)-sb to Snail family factors, and not other
zinc-containing proteins, the acetyl acetonate ethylenediimine
(acacen) backbone is linked to a modified oligonucleotide. This
oligo contains the Ebox consensus sequence, CAGGTC, recog-
nized by Snail factors (14). This modification does not alter the
secondary structure of the DNA, enabling it to be readily
coordinated by Snail proteins in a concentration dependent
manner. The oligonucleotide confers DNA binding specificity
such that only proteins that contain a zinc finger domain and
bind the Ebox sequence are inhibited. A biochemical investiga-
tion of the functional role of the oligo and the Co(III) chelate of
Co(III)-Ebox in the inhibition of DNA binding of Snail family
transcription factors is presented using X. laevis as a model
system. The specific inhibitory effects of Co(III)-Ebox for Slug,
Snail, and Sip1 suggests that Co(III)-Ebox can be used as a tool
to study EMT in vertebrate development and epithelial tumor
metastasis (Fig. 1).
Design and Synthesis of a Transcription Factor-Targeted Co(III)-sb. A
Co(III)-sb conjugated to a DNA oligonucleotide that targets
Ebox-binding zinc finger transcription factors was synthesized
and characterized (Scheme 1). The Co(III) complex is prepared
by the reaction of 6-heptenoic acid with sodium aizde and iodine
monochloride to give 6-azido-7-iodo-heptanoic acid, compound
2, in 92% yield. The diazide, compound 3, was formed upon
reaction of compound 2 with excess sodium azide in 23% yield.
Compound 2 was reduced by hydrogenation to produce the
diamine. Crude diamine was condensed with 2,4-pentadione to
obtain the Schiff base ligand, compound 4, in 42% yield.
Author contributions: A.S.H., J.L., C.L., and T.J.M. designed research; A.S.H., J.L., L.M.M.,
The authors declare no conflict of interest.
1C.L. and T.J.M. contributed equally to this work.
2To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
www.pnas.org?cgi?doi?10.1073?pnas.0906423106 PNAS ?
August 18, 2009 ?
vol. 106 ?
no. 33 ?
Band intensities were quantified on a PhosphorImager. Slug-bound band
intensity values were background-subtracted using the uninjected lane
and normalized to the background signal in each individual lane. The
percentage of residual complex bound to Slug is the normalized intensity
at 100-fold excess unlabeled Ebox divided by the normalized intensity in
the lane where no competitor was added. These values for Co(III)-Ebox and
Ebox were averaged over three replicates and reported with the standard
The presence of 3 shifted complexes for Slug have been reported (38).
Co(III)-Ebox inhibits all of the complexes as seen in Figs. 3, 4, and 5.
Western Blot Analysis. For Western blots, embryo lysates from EMSA were used.
Samples were denatured and resolved on SDS/PAGE. Proteins were detected
(Biology), Core Research of Evolutional Sciences and Technology, Japan Science
and Technology Corporation, University of Tokyo, Tokyo, Japan] for providing
reagents and D. Mastarone for aid with NMR characterization. This work is
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www.pnas.org?cgi?doi?10.1073?pnas.0906423106Harney et al.