Dexamethasone-methotrexate: An efficient chemical inducer of protein dimerization in vivo [18]

Full text of DOI:10.1021/ja9941532

J. Am. Chem. Soc. 2000, 122, 4247-4248
4247
We chose to build a heterodimeric CID based on the well-
characterized ligand-receptor pairs dexamethasone (Dex)-
glucocorticoid receptor (GR) and methotrexate (Mtx)-dihydro-
folate reductase (DHFR). Both Dex and Mtx present chemical
functionality that can be modified readily without disrupting
receptor binding.12-15 Because the interactions of both Dex and
Mtx with their respective receptors are well characterized,9,10,14,15
future characterization and optimization of the Dex-Mtx system
should also be facilitated. The rat glucocorticoid receptor (rGR)
binds Dex with a KD of 5 nM, and mutants of rGR with increased
affinity for Dex have been isolated.9 The steroid dexamethasone
has been used extensively as a cell-permeable small molecule to
regulate the activity and nuclear localization of GR fusion proteins
in vivo.10 Recently, a yeast “three-hybrid” system has been
reported in which a GR-fusion and an FKBP12-fusion could be
dimerized by the small molecule Dex-FK506.11 Likewise, DHFR
fusion proteins have been used for a variety of biochemical
applications16,17 due to Mtx’s picomolar affinity for DHFR.18 Both
Dex and Mtx are commercially available, and Mtx can be
synthesized readily from simple starting materials.
The retro-synthetic analysis of the Dex-Mtx heterodimer is
shown in Scheme 1. The synthesis is based on previous syntheses
of Dex and Mtx derivatives.12,19-21 The synthesis is designed to
allow the chemical linker between the two ligands to be varied
readily. Both ligands were introduced as thiol derivatives to a
di-halo linker. Following oxidative cleavage with periodate, Dex
was derivatized with cystamine using standard peptide coupling
reagents. The γ-carboxylate in Mtx was replaced with a thiol
simply by replacing glutamate with homocysteine. Homocysteine,
protected as the tert-butyl ester and disulfide, was coupled to
4-methylaminobenzoic acid. The resulting Dex and Mtx disulfide
derivatives were reduced to their corresponding thiols using tri-
n-butylphosphine, and the two thiols were coupled to a di-bromo
linker in a one-pot reaction. The 2,4-diamino-6-bromomethyl
pteridine was added after introduction of the dibromo linker to
simplify purification of the intermediates, and the final step was
cleavage of the tert-butyl ester. Thus, the Dex-Mtx heterodimer
was prepared from two components in eight steps in 2% overall
yield. The modular synthesis of the Dex-Mtx heterodimer will
facilitate future studies to evaluate the influence of the linker on
CID activity in different systems.
Dexamethasone-Methotrexate: An Efficient
Chemical Inducer of Protein Dimerization In ViWo
Hening Lin,† Wassim M. Abida,† Robert T. Sauer,§ and
Virginia W. Cornish*,†
Department of Chemistry, Columbia UniVersity
New York, New York 10027
Department of Biology
Massachusetts Institute of Technology
Cambridge, Massachusetts 02139
ReceiVed NoVember 29, 1999
Cell-permeable small molecules that can control cellular
processes in ViVo are integral to basic research in biochemistry
and cell biology. Isopropyl-â-D-thiogalactoside (IPTG), for
example, has been used for decades to activate gene transcription
by stabilizing a conformation of lac repressor with reduced
operator affinity. A clever extension of this approach involves
the use of dimeric ligands or “chemical inducers of dimerization”
(CIDs) to manipulate protein-protein interactions in ViVo.1-6 The
inspiration for CIDs were studies of the mechanism of action of
the natural immunosuppressants FK506 and rapamycin.7,8 It was
found that the immunosuppressant activity of both compounds
results from the fact that they dimerize two proteins that otherwise
do not interact. To generalize this molecular mechanism, Spencer
et al. showed in 1993 that two FK506 molecules tethered via
their C21 allyl groups could oligomerize proteins fused to FKBP12.
CIDs are particularly powerful because the same dimeric ligand
can be used over and over again simply by fusing the proteins of
interest to the receptors for the CID. The majority of CIDs
described to date are dimers of FK5061,3,4 or FK506-analogues,5,6
although other ligands have been reported also.2 Given the broad
utility of CIDs, we sought to design an optimized CID that could
be prepared readily from commercially available materials and
that was based on different ligand-receptor pairs. Here, we report
such a compound: a heterodimeric dexamethasone-methotrexate
molecule that can dimerize proteins efficiently in ViVo (Figure
1).
(3) Belshaw, P.; Ho, S.; Crabtree, G.; Schreiber, S. Proc. Natl. Acad. Sci.
U.S.A. 1996, 93, 4604-4607.
(4) Diver, S.; Schreiber, S. J. Am. Chem. Soc. 1997, 119, 5106-5109.
(5) Amara, J.; et al. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 10618-10623.
(6) Clackson, T.; et al. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 10437-
10442.
(7) Liu, J.; Farmer, J., Jr; Lane, W.; Friedman, J.; Weissman, I.; Schreiber,
S. Cell 1991, 66, 807-815.
(8) Brown, E.; Albers, M.; Shin, T.; Ichikawa, K.; Keith, C.; Lane, W.;
Schreiber, S. Nature 1994, 369, 756-758.
(9) Chakraborti, P.; Garabedian, M.; Yamamoto, K.; Simons, S. J. Biol.
Chem. 1991, 266, 22075-22078.
(10) Picard, D.; Yamamoto, K. EMBO J. 1987, 6, 3333-3337.
(11) Licitra, E.; Liu, J. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 12817-
12821.
Figure 1. Yeast three-hybrid assay. A heterodimeric ligand (Dex-Mtx)
bridges a DNA binding protein-receptor protein chimera (LexA-DHFR)
and a transcription activation protein-receptor protein chimera (B42-
GR), effectively reconstituting a transcriptional activator and stimulating
transcription of a lacZ reporter gene. The levels of lacZ transcription
serve as an indicator of the efficiency of Dex-Mtx-induced protein
dimerization.
(12) Govindan, M.; Manz, B. Eur. J. Biochem. 1980, 108, 47-53.
(13) Manz, B.; Heubner, A.; Kohler, I.; Grill, H.-J.; Pollow, K. Eur. J.
Biochem. 1983, 131, 333-338.
(14) Bolin, J.; Filman, D.; Matthews, D.; Hamlin, R.; Kraut, J. J. Biol.
Chem. 1982, 257, 13663-13672.
(15) Benkovic, S.; Fierke, C.; Naylor, A. Science 1988, 239, 1105-1109.
(16) Ryan, M.; Muller, H.; Pfanner, N. J. Biol. Chem. 1999, 274, 20619-
20627.
(17) Pelletier, J.; Campbell-Valois, F.-X.; Michnick, S. Proc. Natl. Acad.
U.S.A. 1998, 95, 12141-12146.
* To whom correspondence should be addressed.
† Columbia University.
(18) Sasso, S.; Gilli, R.; Sari, J.; Rimet, O.; Briand, C. Biochim. Biophys.
Acta 1994, 1207, 74-79.
§ Massachusetts Institute of Technology.
(1) Spencer, D.; Wandless, T.; Schreiber, S.; Crabtree, G. Science 1993,
262, 1019-1024.
(19) Piper, J. R.; Montgomery, J. A. J. Org. Chem. 1977, 42, 208-211.
(20) Hart, B. P.; Haile, W. H.; Licato, N. J.; Bolanowska, W. E.; McGuire,
J. J.; Coward, J. K. J. Med. Chem. 1996, 39, 56-65.
(21) Kokuryo, Y.; et al. J. Med. Chem. 1997, 40, 3280-3291.
(2) Farrar, M. A.; Alberola-lla, J.; Perlmutter, R. M. Nature 1996, 383,
178-181.
10.1021/ja9941532 CCC: $19.00 © 2000 American Chemical Society
Published on Web 04/13/2000

4248 J. Am. Chem. Soc., Vol. 122, No. 17, 2000
Communications to the Editor
Table 1. Small-Molecule Induced Activation of â-Galactosidase Synthesisa
hydrolysis of ONPGb (µmol/min/mg total protein)
no
CID
0.01 µM
Dex-Mtx
0.1 µM
Dex-Mtx
1 µM
Dex-Mtx
10 µM
Dex-Mtx
10 µM Mtx
1 µM Dex-Mtx
protein chimerasc
LexA-DHFR,
B42-GR
LexA, B42
0.06 ( 0.01
0.1 ( 0.05
1.3 ( 0.1
10.9 ( 2.0
12.3 ( 2.0
0.30 ( 0.10
0.25 ( 0.01
ND
ND
0.33 ( 0.04
ND
ND
a â-Galactosidase is encoded by the lacZ gene. Small-molecule-dependent protein dimerization was quantified in duplicate for cells grown in
synthetic complete liquid media (containing 2% galactose and 2% raffinose, lacking uracil, histidine, and tryptophan) based on levels of â-galactosidase
synthesis. The error estimates for the rates of o-nitrophenyl-â-D-galactopyranoside (ONPG) hydrolysis correspond to the standard error between the
two measurements. b â-Galactosidase synthesis levels were determined from the rate of hydrolysis of ONPG. c Plasmids encoding the protein chimeras
were introduced into S. cereVisiae FY250 containing a lacZ-reporter gene under control of 4 tandem LexA operators.
Scheme 1. Retro-synthetic Analysis of Dex-Mtx
It is difficult to compare the effectiveness of different CIDs,
as CID activity is assay-dependent and is not linear. It is
interesting to note, however, that in the yeast three-hybrid assay
the absolute levels of â-galactosidase synthesis are 150-fold higher
for Dex-Mtx than for Dex-FK506 at 1 µM CID concentration
based on liquid culture lacZ transcription assays (data not shown).
Although demonstrated using the yeast three-hybrid assay for
protein dimerization, the heterodimeric Dex-Mtx molecule in
principle can dimerize any two proteins fused to the hormone-
binding domain of GR and DHFR. To extend the Dex-Mtx CID
to organisms where Mtx is toxic, we are in the process of
modifying Mtx so that it binds selectively to a DHFR mutant.
This approach is well precedented in the AP-FKBP CID series.6
Given the ease with which Dex-Mtx and potential analogues
can be prepared and Dex-Mtx’s high efficiency in protein
dimerization, we anticipate that this Dex-Mtx protein dimeriza-
tion system will find broad use in biochemistry and cell biology.
To demonstrate that Dex-Mtx can dimerize proteins in ViVo,
we employed the yeast “three-hybrid” system,11,22,23 which consists
of a dimeric small molecule, two protein chimeras, and a reporter
gene (Figure 1). The small molecule (Dex-Mtx) bridges the
DNA-binding protein chimera (LexA-DHFR) and the transcrip-
tion activation protein chimera (B42-GR) effectively reconstitut-
ing a transcriptional activator and stimulating transcription of a
lacZ reporter gene.
Using standard â-galactosidase activity assays both on plates
and in liquid culture,25 we showed that Dex-Mtx can activate
lacZ transcription in ViVo (Figure 2 and Table 1). On the basis of
previous studies showing that lacZ transcription levels correlate
with the strength of protein-protein interactions in the yeast two-
hybrid assay,26 we expect â-galactosidase activity to be a good
indicator of Dex-Mtx induced protein dimerization. In these
assays, the extracellular concentration of Dex-Mtx ranged from
0.01 to 10 µM. Control experiments established that lacZ
transcription was dependent on Dex-Mtx (Figure 2 and Table
1). Only background levels of â-galactosidase activity were
detected when Dex-Mtx was omitted. A 10-fold excess of Mtx
reduced Dex-Mtx-dependent lacZ transcription to near back-
ground levels. A 10-fold excess of Dex, however, did not effect
Dex-Mtx-dependent lacZ transcription, and higher concentra-
tions of Dex were toxic to the yeast cells. This result may be due
to differences in cell permeability between Dex and Dex-Mtx
or may suggest that LexA-DHFR, but not B42-GR, is the
limiting reagent. In addition, when either or both receptors were
deleted, only background levels of lacZ transcription were
detected.
Figure 2. X-gal plate assay of Dex-Mtx-induced lacZ transcription.
Yeast strains containing a lacZ reporter gene and different LexA- and/or
B42-chimeras were grown on X-gal indicator plates with or without Dex-
Mtx. Columns 1-6 on each plate correspond to yeast strains containing
different LexA- and/or B42-chimeras: 1, LexA-Sec16p, B42-Sec6p;
2, LexA-Sec13, B42-Sec6p. 1 and 2 are direct protein-protein
interactions used as positive controls.27 3, LexA-DHFR, B42-GR; 4,
LexA-DHFR, B42; 5, LexA, B42-GR; 6, LexA, B42. X-gal plates A-C
have different small molecule combinations: A, no Dex-Mtx; B, 1 µM
Dex-Mtx; C, 1 µM Dex-Mtx, and 10 µM Mtx.
Acknowledgment. We are grateful for financial support for this work
from the NSF, Grant CHE 99-77402. V.W.C. is a Camille and Henry
Dreyfus New Faculty Awardee. V.W.C. initiated this project as an NSF
Postdoctoral Fellow in Professor Sauer’s laboratory. We thank Professors
J. Liu and J. Hu and Dr. S. Kopytek for helpful discussions about the
development of new CIDs. We are grateful to Professor H. Madhani,
Dr. D. McNabb, and members of the Kaiser Lab for their yeast expertise.
We thank Professor J. K. Coward and Dr. W. G. Bornmann for helpful
suggestions on the synthesis of Dex-Mtx. We thank D. Pincus and S.
Goldberg for making the protein constructs and J. Mahal for technical
assistance.
(22) Fields, S.; Song, O. Nature 1989, 340, 245-246.
(23) Gyuris, J.; Golemis, E.; Chertkov, H.; Brent, R. Cell 1993, 75, 791-
803.
(24) Watson, M.; Buckholz, R.; Weiner, M. Biotechniques 1996, 21, 255-
259.
(25) Adams, A.; Gottschling, D. E.; Kaiser, C. A.; Stearns, T. Methods in
Supporting Information Available: Experimental procedures for the
synthesis of Dex-Mtx 1, for the construction of the protein chimeras,
and for the lacZ assays (PDF). This material is available free of charge
via the Internet at http://pubs.acs.org.
Yeast Genetics, 1997 ed.; Cold Spring Harbor Laboratory Press: Plainview,
1998; pp 123-125.
(26) Estojak, J.; Brent, R.; Golemis, E. Mol. Cell. Biol. 1995, 15, 5820-
5829.
(27) Espenshade, P. Ph.D. Thesis, Massachusetts Institute of Technology,
1997.
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