Synthesis and analysis of stabilizing ligands for FKBP-derived destabilizing domains

Article abstract of DOI:10.1016/j.bmcl.2007.11.044

We recently identified mutants of the human FKBP12 protein that are unstable and rapidly degraded when expressed in mammalian cells. We call these FKBP mutants destabilizing domains (DDs), because their instability is conferred to any protein fused to the

Full text of DOI:10.1016/j.bmcl.2007.11.044

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 759–761
Synthesis and analysis of stabilizing ligands
for FKBP-derived destabilizing domains
Joshua S. Grimley, Denise A. Chen, Laura A. Banaszynski and Thomas J. Wandless*
Department of Chemical & Systems Biology, Stanford University, 318 Campus Drive,
Clark Center W350A, Stanford, CA 94305-5441, USA
Received 4 October 2007; accepted 12 November 2007
Available online 17 November 2007
Abstract—We recently identified mutants of the human FKBP12 protein that are unstable and rapidly degraded when expressed in
mammalian cells. We call these FKBP mutants destabilizing domains (DDs), because their instability is conferred to any protein
fused to the DDs. A cell-permeable ligand binds tightly to the DDs and prevents their degradation, thus providing small molecule
control over intracellular protein levels. We now report the synthesis and functional characterization of a stabilizing ligand called
Shield-2. The synthesis of Shield-2 is efficient, and this ligand binds to the FKBP(F36V) protein with a dissociation constant of
29 nM.
Ó 2007 Elsevier Ltd. All rights reserved.
The ability to regulate the functions of specific proteins
DD
DNA Fusion
transcription
translation
POI
A
using cell-permeable small molecules is a powerful meth-
od to interrogate biological systems.1 Studies using sev-
eral natural products have proven the utility of this
approach,2–5 and the desire for additional reagents has
driven screening efforts to identify new, and hopefully
specific, perturbants.6,7 The fruits of these searches
may ultimately be a large collection of reagents that
might be used to interrogate many different proteins.
DD
POI
POI
degraded
fusion protein
stable
We have pursued an alternative strategy of creating a
‘one ligand-many proteins’ strategy to manipulate pro-
tein function. We recently developed an experimental
system in which the stability of a small protein domain
depends upon the presence of a synthetic, cell-permeable
molecule (Fig. 1A).8 The specificity of this method
comes from the genetic fusion of the destabilizing do-
main (DD) to any protein of interest, and the cell-per-
meable stabilizing small molecule provides the
elements of speed, reversibility, and tunability to regu-
late protein levels.
O
B
R
N
H
O
O
O
OMe
OMe
N
O
Shield-1, R=
Shield-2, R=
OMe
Me
MeO
MeO
Me
N
We started with a well-studied protein–ligand pair: the
human FKBP12 protein and a high-affinity, synthetic
H
Et
Figure 1. (A) Strategy for conferring ligand-dependent stability to any
protein of interest (POI) by fusing the DNA sequence encoding a
destabilizing domain (DD) to the gene encoding the POI. (B)
Structures of the ligands for FKBP(F36V).
Keywords: Protein engineering; Protein stability; Degradation.
*
Corresponding author. Tel.: +1 650 723 4005; fax: +1 650 725
4665; e-mail: wandless@standford.edu
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.11.044

760
J. S. Grimley et al. / Bioorg. Med. Chem. Lett. 18 (2008) 759–761
ligand called Shield-1 (Fig. 1B). Shield-1 possesses a
structural ‘bump’ and the FKBP partner harbors the
F36V mutation, which provides a complementary cavity
for the ligand.9,10 Because of these structural modifica-
tions, Shield-1 binds to the FKBP(F36V) mutant
approximately 1600-fold more tightly than to the wild-
type FKBP protein, and as expected, Shield-1 does not
elicit any detectable response when administered to cul-
tured mammalian cells.11
nished urea 3. The phenol was deprotected with TBAF,
and alkylation of the phenol with 4-(2-hydroxyethyl)mor-
pholine under Mitsunobu conditions provided Shield-2.
We used a fluorescence polarization-based competition
binding assay to measure the affinity of each ligand for
the FKBP(F36V) protein.13 A fluorescent FKBP ligand
was incubated with various concentrations of purified
FKBP(F36V), and this saturation binding experiment
provided the dissociation constant for this interaction
(KD = 3.6 nM, Supplementary data, Fig. S1). To mea-
sure the affinities of the ligands for FKBP(F36V), the
FKBP(F36V) can typically be fused to other proteins
without affecting the stability of the resulting fusion pro-
tein, so we screened a library of FKBP sequences to
identify mutants that are unstable in the absence of
Shield-1. Additional screening enriched for FKBP mu-
tants that are stabilized by Shield-1, and further charac-
terization of these mutants revealed that the most
destabilizing mutants cause a 50- to 100-fold reduction
in the levels of the proteins to which they are fused.8
FKBP-derived destabilizing domains (DDs) confer
instability to a variety of proteins, and these DDs desta-
bilize proteins that are expressed in cultured mammalian
cells as well as in living mice.
0.8
A
0.6
0.4
0.2
0
The stabilizing ligands are key elements to the success of
this technology. Biophysical properties (e.g., affinity,
kon, koff) as well as pharmacological properties will influ-
ence the potency and kinetic behavior of this system in
both cultured cells and living animals. We have synthe-
sized additional ligands for the FKBP(F36V) protein
and evaluated these molecules as stabilizing ligands for
the FKBP-derived DDs.11 Yang and colleagues recently
reported an FKBP ligand in which the chiral 2-arylbutyric
ester of Shield-1 is replaced with an achiral urea, signifi-
cantly simplifying the synthesis.12 The synthesis of a stabi-
lizing ligand called Shield-2 that incorporates this
modification is shown in Scheme 1. Alcohol 1 was acyl-
ated with Fmoc-protected pipecolinic acid, and treatment
with piperidine furnished amine 2. This intermediate was
treated with triphosgene and 3-aminopentane, which fur-
100
101
102
103
104
[FKBP ligand] (nM)
B
100
80
60
40
20
OH
1) Fmoc-HoPro-OH
TBSO
OMe
OMe
DCC 84%
2) piperidine 85%
1
HN
H
O
3-aminopentane
triphosgene
O
100
101
102
103
104
105
TBSO
OMe
OMe
[FKBP ligand] (nM)
Et3N 68%
2
Figure 2. (A) Binding analysis of Shield-1 and Shield-2. FKBP(F36V)
(20 nM) and the fluorescent tracer FL-SLF (1 nM) were incubated
with Shield-1 (h, 500–1 nM) or Shield-2 (s, 12.8 lM–1.6 nM). Data
were fitted as described in Ref. 12 to obtain dissociation constants of
2.4 nM (R2 = 0.98) and 29 nM (R2 = 0.99) for Shield-1 and Shield-2,
respectively. Error bars represent the standard deviation of four
repetitions. (B) NIH3T3 cells stably expressing the FKBP(F36V,
L106P)-YFP fusion protein were treated with Shield-1 (h, 10 lM–
Me
O
Me
1) TBAF 99%
N
H
N
2)
OH
N
O
H
O
O
Shield-2
TBSO
OMe
OMe
DIAD, Ph3P 88%
1 nM,
EC50 ꢀ 100 nM)
or
Shield-2
(s,
100 lM–10 nM,
EC50 ꢀ 1.2 lM), and YFP fluorescence was measured at 24 h using
flow cytometry. Error bars represent the standard deviation of three
repetitions.
3
Scheme 1. Synthesis of Shield-2.

J. S. Grimley et al. / Bioorg. Med. Chem. Lett. 18 (2008) 759–761
761
protein and fluorescent tracer were incubated at fixed
Supplementary data
concentrations along with various concentrations of
Shield-1 or Shield-2, and the fraction of bound tracer
is plotted as a function of competitor concentration
(Fig. 2A). Fitting these competition binding curves re-
veals that Shield-1 and Shield-2 bind to the F36V pro-
tein with affinities of 2.4 nM and 29 nM, respectively.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2007.11.044.
References and notes
To test the stabilizing ligands in cultured mammalian
cells, we used an MMLV-derived retrovirus to stably
transduce NIH3T3 cells with the FKBP(F36V/L106P)
DD fused to yellow fluorescent protein (YFP). In the
absence of stabilizing ligand the levels of the YFP-con-
taining fusion protein are 2–3% of ligand-stabilized con-
trol. Populations of these cells were then treated with
various concentrations of either Shield-1 or Shield-2,
and the YFP levels were quantitated using analytical
flow cytometry (Fig. 2B). Both ligands stabilize the
DD–YFP fusion protein, and Shield-1 is approximately
12-fold more potent than Shield-2.
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We have developed a general small molecule-dependent
strategy to regulate the stability, and thus function, of
specific proteins when expressed in mammalian cells.
The dose-dependence that is conferred by the stabilizing
ligand makes this a particularly attractive experimental
approach for studying biological processes in which
the cellular concentration of a particular protein exerts
a strong effect on biological function.14 Shield-2 is con-
siderably easier to synthesize than Shield-1, and contin-
ued studies with these ligands as well as others will likely
produce compounds that display a variety of biophysi-
cal and pharmacological properties. The choice of the
appropriate ligand will be governed by the experimental
strategy and biological context.
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Financial support was provided by the NIH
(GM073046) and the California Tobacco-Related
Disease Research Program (15DT-0015) to J.S.G.