Facile synthesis of CIDs: Biotinylated estrone oximes efficiently heterodimerize estrogen receptor and streptavidin proteins in yeast three hybrid systems

Article abstract of DOI:10.1021/ol0497537

We synthesized estrone oximes as chemical inducers of protein heterodimerization (CIDs). Estrone-17-(O-carboxymethyl)oxime coupled to biotinamidocaproic acid via N,N′-dimethylhexane-1,6-diamine efficiently heterodimerizes estrogen receptors (ERs) and stre

Full text of DOI:10.1021/ol0497537

ORGANIC
LETTERS
2004
Vol. 6, No. 9
1409-1412
Facile Synthesis of CIDs: Biotinylated
Estrone Oximes Efficiently
Heterodimerize Estrogen Receptor and
Streptavidin Proteins in Yeast Three
Hybrid Systems
Smita S. Muddana and Blake R. Peterson*
Department of Chemistry, The PennsylVania State UniVersity,
UniVersity Park, PennsylVania 16802
brpeters@chem.psu.edu
Received February 11, 2004
ABSTRACT
We synthesized estrone oximes as chemical inducers of protein heterodimerization (CIDs). Estrone-17-(O-carboxymethyl)oxime coupled to
biotinamidocaproic acid via N,N′-dimethylhexane-1,6-diamine efficiently heterodimerizes estrogen receptors (ERs) and streptavidin Y43A in
yeast three hybrid systems, activating gene expression over 100-fold at 10 µM. Related hexane-1,6-diamine and estradiol-6-(O-carboxymethyl)-
oxime derivatives were ineffective CIDs due to low affinity for ERs when bound to streptavidin. Estrone oximes bind ERs with submicromolar
affinity and effectively display small molecules to target proteins expressed in yeast.
Chemical inducers of protein dimerization (CIDs) are power-
ful molecular tools for probing diverse biological processes.
CIDs have been used to investigate signal transduction path-
ways,1-8 regulate gene expression,9-12 control protein secre-
tion,13 activate protein splicing,14 manipulate enzyme activ-
ity,15-17 and identify protein targets of small molecules.18-20
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10.1021/ol0497537 CCC: $27.50 © 2004 American Chemical Society
Published on Web 04/01/2004

The identification of protein targets of natural products
and other biologically active small molecules has been
traditionally pursued by affinity chromatography methods.
In this approach, a small molecule of interest is typically
immobilized on an insoluble support and treated with cellular
extracts. Noninteracting proteins can be washed from the
support, whereas specifically interacting proteins are retained,
enriched, and can be eluted with a specific competitor.
Peptide fragments of eluted proteins are analyzed by
sequencing, and this sequence information can be matched
with databases to identify genes encoding putative protein
targets. However, given the low abundance and low stability
of many proteins, this strategy can be quite technically
challenging.
Figure 1. Structures of novel (1) and previously reported (2) CIDs
that efficiently heterodimerize ER and SA proteins.
CIDs can be used to identify protein targets of natural
products by activating gene expression in living yeast
cells.18,19 In these yeast three hybrid systems, the natural
product is covalently linked to a cell-permeable ligand such
as the steroid dexamethasone.18,19 A receptor such as the
dexamethasone-binding glucocorticoid receptor is expressed
in yeast fused to a DNA binding domain (DBD). This DBD
(e.g., the bacterial LexA protein) anchors the receptor on
specific DNA sites of a reporter gene in the nucleus of yeast
cells. Addition of a cell-permeable CID results in binding
of the ligand element to the receptor and displays the linked
natural product to other target proteins expressed in yeast.
Genetic libraries (e.g., cDNA libraries) encoding tens of
thousands of potential target proteins each fused to a
transcriptional activation domain (AD, e.g., the B42 protein)
can be introduced and coexpressed in these recombinant
yeast. Because each yeast cell in theory expresses a unique
protein member of the library, yeast cells can be rapidly
screened to identify putative protein targets of the natural
product. Binding of a target-AD fusion protein to the natural
product displayed by the receptor-DBD fusion protein
reconstitutes a functional transcription factor by positioning
the AD in proximity of specific DNA sites of a reporter gene.
This transcription factor in turn recruits the cellular tran-
scriptional machinery to DNA to activate expression of a
reporter gene typically encoding a readily detected enzyme
such as â-galactosidase.21,22 A major potential advantage of
this genetic approach over affinity chromatography is the
ability to immediately sequence specific genes encoding
putative protein targets without requiring purification of
unstable protein products.
Estrone (7) is a high affinity ligand of estrogen receptor (ER)
proteins, and the appended natural product biotin provides
a simple model of complex natural products with unknown
protein targets. CID 1 resembles our previously reported
biotinylated 7R-substituted â-estradiol derivative 2, an ef-
ficient heterodimerizer of ERs with the biotin-binding protein
streptavidin (SA) in yeast three hybrid systems (Figure 2).23
Figure 2. Schematic of the ER/SA yeast three hybrid system
showing a previously reported23 model of a ternary ER-2-SA
complex. Heterodimerization of ER-LexA and SA-B42 by CIDs 1
or 2 activates expression of the lacZ (â-galactosidase) reporter gene.
Although 2 is an exceptionally active CID in yeast,23 the
routine use of 7R-substituted â-estradiol derivatives as CIDs
is limited by the lengthy 14-step synthesis required to prepare
We report here a simple two-pot synthesis of a novel,
highly active, and cell-permeable CID (1 Figure 1) compris-
ing an estrone (O-carboxymethyl)oxime linked to biotin.
23,24
these compounds.
As a dramatically streamlined alternative, a two-pot
synthesis of CID 1 and control compounds 3-5 (Figure 3),
is shown in Scheme 1. (O-Carboxymethyl)oxime derivatives
of estrone (7) and 6-ketoestradiol (9)25 were prepared in
quantitative yield from treatment with aminooxyacetic acid
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Org. Lett., Vol. 6, No. 9, 2004

fusion protein linked to either the ERR or ERâ ligand binding
domains (LBDs). The B42 AD28 was similarly coexpressed
fused to the C-terminus of SA Y43A.23 This SA mutant
exhibits low toxicity in yeast23 and binds biotin with an
affinity of Kd∼100 pM.29 Yeast were also transformed with
the commercially available reporter vector pSH18-34 (In-
vitrogen). This reporter contains the lacZ gene encoding
â-galactosidase under the control of four dimeric LexA DNA
binding sites. The nonbiotinylated analogue 4 and the
antiestrogen ICI 182,780 (6)30 were also examined in these
assays as negative control compounds. As shown in Figure
4, compounds 1 and 2 strongly activated dose-dependent
Figure 3. Structures of control compounds.
hydrochloride in pyridine.26,27 The resulting carboxylic acids
were converted to N-hydroxysuccinimidyl esters in situ and
treated with excess 1,6-hexanediamine or N,N′-dimethyl-1,6-
hexanediamine, and the resultant amine product was sub-
jected to a second acylation step to afford compounds 1 and
3-5 in good to moderate overall yields.
Figure 4. Dose-response curves from yeast three hybrid assays.
Panel A: ERR assays. Panel B: ERâ assays. Fold activation )
observed â-Gal activity/â-Gal activity without ligand.
reporter gene expression in yeast three hybrid systems
expressing ERR or ERâ fusion proteins. At concentrations
of 10 and 50 µM in the ERR assay, the previously reported
CID 2 was only slightly more active than CID 1, with both
compounds enhancing reporter gene expression by ∼300-
fold above basal levels (50 µM). In yeast expressing ERâ,
CID 1 proved to be the most active compound at 10 and 50
µM, activating gene expression 130-fold above basal levels
(50 µM), albeit with a 4-fold reduction in potency compared
with 2 (Table 1). Surprisingly, the structurally similar
Scheme 1. Two-Pot Synthesis of Compounds 1 and 3-5a
Table 1. Compilation of EC50 and IC50 Values Quantified by
Nonlinear Regression Analysis of the Data Shown in Figures 4
and 6a
a Reagents and conditions: (a) H2NOCH2CO2H‚HCl, pyridine;
(b) DCC, N-hydroxysuccinimide (NHS), MeHN(CH2)6NHMe,
CH2Cl2; (c) D-biotinamidocaproic acid NHS ester, DIEA, CH2Cl2,
MeOH; (d) acetic acid NHS ester, DIEA, CH2Cl2; (e) DCC, NHS,
H2N(CH2)6NH2, CH2Cl2.
ligand Y3H ERR + SA Y3H ERâ + SA FP ERR FP ERR + SA
1
2
3
4
5
6
7
5600 ( 2200
6600 ( 1500
4900 ( 2200
1200 ( 470
240 ( 130 720 ( 330
16 ( 10 330 ( 220
290 ( 170 2100 ( 1200
200 ( 80 370 ( 160
1400 ( 450 4200 ( 1600
14 ( 10
22 ( 10
3.4 ( 1.1
2.8 ( 1.5
To assess the effectiveness of 1 and related analogues 3
and 5 as CIDs, these compounds were compared with CID
2 in our ER/SA yeast three hybrid system.23 In these
experiments, the bacterial LexA DBD28 was expressed as a
a Y3H: yeast three hybrid EC50 values (nM). FP: competition fluores-
cence polarization IC50 values (nM). Errors correspond to 95% confidence
intervals.
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analogues 3 and 5 were much less effective CIDs, possibly
due in part for 5 to lower stability of this electron-rich
aromatic oxime in relatively acidic yeast media (pH ) 3 to
4). Furthermore, as expected, the control compounds lacking
biotin (4, 6) were essentially inactive in these assays.
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3454.
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Org. Lett., Vol. 6, No. 9, 2004
1411

To evaluate the specificity of CID 1, competition and
omission experiments were employed (Figure 5). These
Figure 6. Fluorescence polarization assays with recombinant ERR
protein. Panel A: Competition binding experiments with ERR.
Panel B. Competition binding experiments with ERR and equimolar
streptavidin (SA) protein.
examine the effects of this protein on binding of ligands to
ERR. As summarized in Table 1, 7R-substituted estradiol
derivatives such as 2 and 6 exhibited the highest affinities
for ERR (IC50 values <20 nM). However, added recombinant
SA protein, which binds biotin irreversibly with an affinity
of ∼100 fM,31 reduced the affinity of CID 2 for ERR by
21-fold (ERR/SA IC50 (2) ) 330 nM). In contrast, although
CID 1 bound more weakly to ERR alone (ERR IC50 (1) )
240 nM), the addition of SA reduced the affinity for ERR
by only 3-fold (ERR/SA IC50 (2) ) 720 nM). The similar
affinities of SA-bound 1 and SA-bound 2 for ERR (within
2-fold) explain why these compounds exhibit similar maxi-
mal activities and potencies in the yeast three-hybrid assays.
In contrast, in the presence of SA the affinities of 3 (rigidified
by secondary amides) and 5 were substantially lower for
ERR, dramatically reducing their activities as CIDs.
(O-Carboxymethyl)oximes derived from aliphatic ketones
are typically highly stable under physiological conditions.
Correspondingly, CID 1 showed no significant decomposition
to estrone (7) even after 16 h in relatively acidic (pH ) 3 to
4) yeast media (HPLC analysis provided in the Supporting
Information). These results demonstrate that readily synthe-
sized estrone oximes can provide efficient tools for the ER-
mediated display of small molecules to target proteins.
Figure 5. Yeast three hybrid competition experiments (panel A)
and omission experiments (panel B).
experiments confirmed that specific ER or SA ligands
blocked reporter gene expression. Omission experiments
confirmed the importance of each protein component of the
yeast three-hybrid system and compared the influence of
CIDs 1 and 2 on elements of this system. These experiments
revealed that both the ER and SA proteins were required
for maximal activation of gene expression. However, CID 1
promoted some one-hybrid (ER-dependent) activity not
observed (<1%) with CID 2 (Figure 5, panel B). These
differences in one-hybrid activity likely relate to subtle
changes in ER conformation induced upon CID binding. The
one-hybrid activity promoted by CID 1 (10 µM) comprised
16% of the total activity in the ERR assay but only 5% of
the total activity in the ERâ assay. This lower one-hybrid
activity in the ERâ assay provided a 20-fold dynamic range
for activation of gene expression by SA Y43A, suggesting
that ERâ is the best platform for the display of estrone-linked
small molecules to target proteins in yeast three hybrid
systems.
To understand the dramatic differences in activity observed
for CIDs 1 and 2 compared with the structurally similar 3
and 5, the affinities of compounds for recombinant ERR
protein were quantified with competition fluorescence po-
larization (FP) assays (Figure 6). These experiments com-
pared competition IC50 values for ERR both in the absence
and presence of equimolar recombinant SA protein to
Acknowledgment. We thank the American Cancer
Society (RSG-02-025-01) and National Institutes of Health
(R01-CA83831) for financial support.
Supporting Information Available: Experimental pro-
cedures, characterization data for new compounds, and HPLC
assays of the stability of CID 1. This material is available
free of charge via the Internet at http://pubs.acs.org.
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OL0497537
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