Targeting the FKBP51/GR/Hsp90 Complex to Identify Functionally Relevant Treatments for Depression and PTSD

Article abstract of DOI:10.1021/acschembio.8b00454

Genetic and epigenetic alterations in FK506-binding protein 5 (FKBP5) have been associated with increased risk for psychiatric disorders, including post-traumatic stress disorder (PTSD). Some of these common variants can increase the expression of FKBP5, the gene that encodes FKBP51. Excess FKBP51 promotes hypothalamic-pituitary-adrenal (HPA) axis dysregulation through altered glucocorticoid receptor (GR) signaling. Thus, we hypothesized that GR activity could be restored by perturbing FKBP51. Here, we screened 1280 pharmacologically active compounds and identified three compounds that rescued FKBP51-mediated suppression of GR activity without directly activating GR. One of the three compounds, benztropine mesylate, disrupted the association of FKBP51 with the GR/Hsp90 complex in vitro. Moreover, we show that removal of FKBP51 from this complex by benztropine restored GR localization in ex vivo brain slices and primary neurons from mice. In conclusion, we have identified a novel disruptor of the FKBP51/GR/Hsp90 complex. Targeting this complex may be a viable approach to developing treatments for disorders related to aberrant FKBP51 expression.

Full text of DOI:10.1021/acschembio.8b00454

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Article
Targeting the FKBP51/GR/Hsp90 complex to identify
functionally relevant treatments for depression and PTSD
Jonathan J. Sabbagh, Ricardo A Cordova, Dali Zheng, Marangelie Criado-Marrero, Andrea Lemus,
Pengfei Li, Jeremy D. Baker, Bryce A. Nordhues, April L. Darling, Carlos Martinez-Licha, Daniel A Rutz,
Shreya Patel, Johannes Buchner, James W. Leahy, John Koren, Chad A. Dickey, and Laura J. Blair
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00454 • Publication Date (Web): 12 Jun 2018
Downloaded from http://pubs.acs.org on June 13, 2018
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Jonathan J. Sabbagh1,2, Ricardo A. Cordova1,2, Dali Zheng1,2, Marangelie Criado-Marrero1,2,
Andrea Lemus3, Pengfei Li1, Jeremy D. Baker1,2, Bryce A. Nordhues1,2, April L. Darling1,2, Carlos
Martinez-Licha1,2, Daniel A. Rutz4, Shreya Patel3, Johannes Buchner4, James W. Leahy1,3,5
John Koren III1,2, Chad A. Dickey1,2§, Laura J. Blair1,2*
,
1Department of Molecular Medicine, University of South Florida, Tampa, Florida, United States of America. 2USF
Health Byrd Institute, University of South Florida, Tampa, Florida, United States of America. 3Department of Chem-
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istry, University of South Florida, Tampa, Florida, United States of America. Department Chemie, Technische
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Universität München, 85748 Munich, Germany. Center for Drug Discovery and Innovation, University of South
Florida, Tampa, Florida, United States of America. §Deceased.
KEYWORDS: FKBP5, FKBP51, HPA axis, glucocorticoid receptor, Hsp90, stress response, PTSD
Abstract: Genetic and epigenetic alterations in FK506-binding protein 5 (FKBP5) have been associated with increased
risk for psychiatric disorders, including post-traumatic stress disorder (PTSD). Some of these common variants can in-
crease the expression of FKBP5, the gene that encodes FKBP51. Excess FKBP51 promotes hypothalamic-pituitary-
adrenal (HPA) axis dysregulation through altered glucocorticoid receptor (GR) signaling. Thus, we hypothesized that GR
activity could be restored by perturbing FKBP51. Here, we screened 1280 pharmacologically active compounds and
identified three compounds that rescued FKBP51-mediated suppression of GR activity without directly activating GR.
One of the three compounds, benztropine mesylate, disrupted the association of FKBP51 with the GR/Hsp90 complex in
vitro. Moreover, we show that removal of FKBP51 from this complex by benztropine restored GR localization in ex vivo
brain slices and primary neurons from mice. In conclusion, we have identified as a novel disruptor of the
FKBP51/GR/Hsp90 complex. Targeting this complex may be a viable approach to develop treatments for disorders re-
lated to aberrant FKBP51 expression.
Introduction: The hypothalamic-pituitary-ad-
renal (HPA) axis is the central regulator of stress
response in the body. The HPA axis is controlled
by hormones called glucocorticoids (CORT) that
are produced by the adrenal cortex in response to
other stress hormones secreted from the brain.
Glucocorticoid receptors (GR) are the main cyto-
plasmic receptors for CORT1. These receptors are
widely distributed throughout the brain. The
51kDa FK506-binding protein (FKBP51) is ubiqui-
tously expressed throughout the brain, but is en-
riched in the hippocampus, cortex, hypothalamus,
and amygdala2-5 and is a central regulator of this
stress response system6. Together with the 90kDa
heat shock protein (Hsp90), FKBP51 suppresses
glucocorticoid receptor (GR) activity by decreas-
ing its affinity for CORT7-12. Once bound to CORT,
GR enters the nucleus and alters the expression of
many genes including upregulation of FKBP5, the
gene that encodes FKBP51; thus creating a short
negative feedback loop9. Therefore, higher intrin-
sic levels of FKBP51 can prolong the duration of
the stress response and, consequently, cause in-
with depression and anxiety3–5. Since GR defi-
ciency also correlates with HPA hyperactivity, it
has been proposed that persistent elevated CORT
in the limbic system contributes to the etiology of
depression and anxiety. However, the inverse has
been reported in individuals with PTSD6–10, that is
these patients commonly present with HPA hypo-
activity. One possible explanation for the blunted
cortisol response could be the desensitization of
the HPA axis after prolonged stress. A recent
study showed a positive correlation between HPA
hypoactivity (low cortisol) and fear-potentiated
startle14,15. These inconsistencies may also result
from the high comorbidity with mood disorders,
gender differences, types of trauma, and subtypes
of PTSD profiles11. While there are discrepancies,
overall these data suggest that there is a careful
balance of HPA regulation and that disturbances
in this regulation, such as FKBP51-mediated sup-
pression of GR activity, will have negative conse-
quences.
FKBP51 has been implicated in several deleteri-
ous processes and diseases including depression,
PTSD, anxiety disorders, Alzheimer’s disease, and
even cancer19-22(see review for additional de-
tails20). FKBP5 has not been identified as a risk
gene by genome-wide association studies (GWAS).
However, chromosome 6, which houses the FKBP5
gene, was associated with psychosis by GWAS23.
creased the levels of circulating CORT8, 13-15
.
Dysfunctional and dysregulated GR signaling
mechanisms have been associated with CORT re-
sistance as well as mental health disorders16-18
.
Most, but not all, studies have shown a positive
association between HPA hyperactivity in patients
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Results
Instead, candidate gene studies, which use a hy-
1
pothesis drive approach, have identified common
FKBP5 single nucleotide polymorphisms (SNPs)
that combine with environmental factors to in-
crease risk for multiple neuropsychiatric disor-
ders24-26. In PTSD, multiple studies have reported
that FKBP5 SNPs combine with early life stress to
increase risk; however, GWAS studies have not
supported this27. These discrepancies may be due
to the limitations of either the candidate studies
or the GWAS or both. Candidate gene studies are
limited because they are based on what we know
of the disease and have not often been replicated
in follow-up studies28. Whereas, GWAS studies are
limited because they often use DNA extracted
from peripheral tissue29, require a large effect
size to be above the limit of detection30, and do not
sufficiently include gene-environmental interac-
tions20. Still, FKBP5 may be one of the most well-
studied candidate genes and, while there are dis-
crepancies in certain SNPs being associated with
specific psychiatric disorders, there is a growing
amount of complementary data which has identi-
fied SNPs that have been repeatable and demon-
A cell-based compound library screen reveals
modulators of FKBP51. We sought to identify
compounds that mitigated the suppression of GR
activity by FKBP51. A high throughput screen us-
ing the LOPAC library was performed in HeLa cells
(see Figure 1A for schematic), which have high
endogenous GR expression38. The ability of com-
pounds to rescue this suppression was normalized
within each plate and converted to Z scores. Four
compounds were identified that restored GR ac-
tivity in the presence of FKBP51 overexpression
(Figure 1B). Each of these compounds were tested
in a secondary screen for their ability to directly
increase GR activity independent of FKBP51. Only
one of these molecules, BVdU, was found to di-
rectly increase GR activity (Figure 1C), suggesting
its effect was not specific to FKBP51 suppression.
The effect of the other three compounds, 3-AB,
resveratrol, and benztropine, was dependent on
FKBP51.
Benztropine targets the FKBP51/GR/Hsp90
complex. Next, we used an in vitro flow cytometry
protein interaction assay (FCPIA) to validate the
disruption of the FKBP51/GR/Hsp90 complex (see
Figure 2A for schematic) by 3-AB, resveratrol, or
benztropine. A biotinylated recombinant ligand-
binding domain of GR (GR-LBD) was complexed
with full-length recombinant Hsp90α and a fluo-
rescently-labeled FKBP51 in the presence of dexa-
methasone. These complexes were then incubated
with 3-AB, resveratrol, or benztropine. Benztro-
pine, but not 3-AB or resveratrol, disrupted
FKBP51 from the complex, as determined by meas-
uring median bead-associated fluorescence (Fig-
ure 2B). To ensure the effects of benztropine were
not unique to our screening model, we validated
our initial findings in a neuroblastoma cell line,
M17 (Figure 2C). Importantly, these data demon-
strate that benztropine rescued FKBP51-mediated
suppression of GR activity without affecting en-
dogenous GR signaling pathways.
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strate mechanisms for the increased risk25, 26, 31-33
.
Some of these disease-associated SNPs work by
boosting the intrinsic levels of FKBP531, suggest-
ing that higher levels of FKBP5 are deleterious in
humans. These SNPs function through a process
involving FKBP5 de-methylation, which allows for
higher levels of FKBP51 protein to be made follow-
ing stress31. This is a similar mechanism through
which FKBP51 is upregulated in aging26, 34. Thus,
decreasing FKBP51 activity or levels may have
therapeutic benefits for patients afflicted with af-
fective disorders.
In fact, Fkbp5-/- mice are viable and present age-
dependent anti-depressive-like phenotypes, en-
hanced stress resiliency, and accelerated stress
response feedback without impaired cognitive or
immune function5, 35-37. The viability of Fkbp5-/-
mice suggests that inhibition of FKBP51 may be
well-tolerated.
Herein, we performed a high-throughput assay
to identify ligands that could block FKBP51-
mediated suppression of GR activity. These stud-
ies revealed a single compound, benztropine me-
sylate, which disrupted the FKBP51/GR/Hsp90
heterocomplex in vitro. Follow-up analyses in cell
culture models, primary neurons, and ex vivo
brain slices confirmed the effects of this com-
pound. These results demonstrate a novel mecha-
nism for inhibiting FKBP51 by a small molecule.
These findings also highlight the feasibility of
high-throughput screening for modulators of ster-
oid hormone receptor activity and suggest
benztropine may be a useful scaffold upon which
to develop a specific FKBP51 inhibitor.
Benztropine selectively inhibits FKBP51. We
then examined if benztropine was selectively
binding FKBP51. To accomplish this, we synthe-
sized benztropine with an exposed biotin group.
In designing a benztropine probe compound (see
Figure 3A), we wanted to retain the tertiary
amine functionality, which precluded the use of
an amide functionality. We chose propargyl ana-
log 4, which would fully retain the basicity of the
parent benztropine. Demethylation of benztro-
pine 2 (synthesized from tropine and benzhydryl
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Figure 1. A cell-based screen identified compounds that suppress FKBP51 repression of GR activity. (A) Schematic depicting the
design of the cell-based compound library screen. HeLa cells were transfected as shown. Cells were then treated with hormone and
compounds from the LOPAC library. Luciferase assays were performed to evaluate compound efficacy. (B) Quantification of Luciferase
activity following Lopac library screen as described in A. Chemiluminescence was normalized within each plate and converted to Z scores.
Four compounds significantly increased GR activity: 3-aminobenzamide (3-AB; green), (E)-5-(bromovinyl)-2’-deoxyuridine (BVdU; or-
ange), benztropine (blue), and resveratrol (red). (C) Quantification of Luciferase activity in HeLa cells transfected with a glucocorticoid
response element (GRE) luciferase reporter and treated with hormone and indicated drugs. ***p<0.001 by one-way ANOVA. Rel = relative
chloride)39 was achieved with chloroethyl chlo-
roformate, isolated as the corresponding methyl
carbamate. Following liberation of the secondary
amine, alkylation with propargyl bromide gave
the requisite acetylene, and click cyclization with
a biotinylated azide afforded the probe compound
5.
Streptavidin beads were incubated with fluores-
cently labeled FKBP51, BSA, or FKBP52 (FKBP4 –
an FKBP51 homologue) in the presence or absence
of the biotinylated-benztropine (see diagram in
Figure 3B). Benztropine selectively interacted
with FKBP51, but not to BSA, or to the highly ho-
mologous FKBP52 (Figure 3C-D). The lack of
benztropine interaction with FKBP52 was surpris-
ing given the high homology with FKBP51. How-
ever, this specificity is preferred, since ablation of
FKBP52 may have some negative impacts40, 41. To
learn more about how benztropine was interact-
ing with FKBP51, we performed a peptidyl-prolyl
isomerase (PPIase) activity assay (Supplemen-
tary Figure 1). This assay confirmed that benztro-
pine does not inhibit the PPIase activity of
FKBP51, and therefore acts through a mechanism
that is independent of this activity.
Figure 2. Benztropine disrupts the FKBP51/GR/Hsp90 com-
plex and restores GR signaling. (A) Schematic depicting the
flow cytometry protein interaction assay (FCPIA) using labeled re-
Benztropine restores GR activity by disrupt-
ing a hormone-dependent FKBP51/GR/Hsp90
complex. Since benztropine did not directly affect
GR activity and was able to interact directly with
combinant
protein
to
measure
disruption
of
the
FKBP51/GR/Hsp90 complex. (B) Median bead-associated fluo-
rescence was measured for each compound at the indicated
range of doses. (C) GR activity was measured by luciferase assay
in M17 cells treated as shown. *p < 0.05 by one-way ANOVA.
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Figure 3. Benztropine is a selective inhibitor of FKBP51. (A) Scheme of biotinylated-benztropine synthesis (B) Schematic depicting
the flow cytometry benztropine-interaction assay using recombinant labeled protein to determine if benztropine interacts with FKBP51,
FKBP52, or BSA. (C) Event counts and fluorescence intensity for bead associated BSA, FKBP51, and FKBP52, as indicated. (D) Quan-
tification of the median fluorescence intensity. FCPIA = flow cytometry protein interaction assay; *Benz = biotinylated benztropine; Protein
= BSA, FKBP51, and FKBP52. n.s. = not significant, *p < 0.05 by one-way ANOVA.
FKBP51 and remove it from an Hsp90/GR com-
presence of hormone increases the effects of
benztropine on disrupting the FKBP51/GR com-
plex. To test this latter hypothesis, we used the
FCPIA (see Figure 2A) to examine whether the in-
hibitory effect of benztropine was dependent on
hormone. Interestingly, benztropine was more ef-
plex, we speculated that benztropine was target-
ing FKBP51 or a transient FKBP51/GR/Hsp90 com-
plex enriched in the presence of hormone. To as-
sess this, we performed co-immunoprecipitations
of GR, in the presence and absence of both hor-
mone (CORT) and benztropine. Though the addi-
tion of hormone did not enhance the interaction
between GR and FKBP51, the amount of FKBP51
found associating with GR was significantly de-
creased exclusively in the presence of both hor-
mone and benztropine (Figure 4A-B). Im-
portantly, changes were not observed in overall
GR levels from the inputs (Figure 4A), indicating
the interaction difference was not a result of in-
creased GR translation. These findings suggest
that, when in complex with Hsp90 and GR, the
fective
at
disrupting
FKBP51 from the
Hsp90/GR/FKBP51 complex in the presence of
hormone (Figure 4C-D). This increase may be due
to a conformation change in the heterocomplex
that has been previously described upon ligand
binding42-45
.
Benztropine mechanism is structurally and
mechanistically distinct. Benztropine is a known
antagonist of muscarinic acetylcholine (ACh) re-
ceptors, histamine receptors, and dopamine
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Figure 5. The unique structure of benztropine is responsi-
ble for its inhibitory effect. (A) HeLa cells were transfected
with GRE luciferase, and FKBP51 or 6TR. Cells were treated
with benztropine, biperiden, brompheniramine, atropine, or di-
phenhydramine as well as hormone. GR activity was measured
using a luciferase assay. ***p < 0.01, n.s. = not significant by
one-way ANOVA. The structures of (B) benztropine, (C) biperi-
den and brompheniramine, and (C) atropine and diphenhydra-
mine are shown. GR = glucocorticoid receptor GRE = gluco-
corticoid response element
Figure 4. Hormone-dependent FKBP51/GR/Hsp90 complex
disruption. (A) HeLa cells were transfected with FKBP51 and
treated with the indicated doses of the hormone hydrocortisone
(CORT) and/or benztropine prior to immunoprecipitation of GR
and Western blotting. (B) Quantification of the Western blot is
shown. *p < 0.05 by one-way ANOVA. (C) Schematic depicting
the flow cytometry protein interaction assay (FCPIA) using la-
beled recombinant protein to measure disruption of the
FKBP51/GR/Hsp90 complex in the presence or absence of dex-
amethasone. (D) Median bead-associated fluorescence was
measured for benztropine at the indicated range of doses in the
presence or absence of dexamethasone.
benztropine (Figure 5A, D). Thus, benztropine
uniquely rescued the suppression of GR by
FKBP51, which correlates with restored expres-
sion of GRE regulated genes (Supplementary Fig-
ure 2).
Benztropine reverses FKBP51 inhibition of GR
nuclear trafficking. Next, we measured GR local-
ization using a RFP-GR reporter plasmid in
HEK293T cells, to determine if benztropine was
regulating GR activity by altering GR localization.
As expected, overexpression of FKBP51 signifi-
cantly reduced GR translocation (Figure 6A).
Benztropine rescued this effect (Figure 6B),
demonstrating that benztropine can abrogate
FKBP51-mediated suppression of GR transloca-
tion, an important step in GR activation47.
Benztropine did not affect GR nuclear localization
in the absence of hormone. Moreover, benztropine
only affected hormone-mediated GR translocation
when FKBP51 was overexpressed, reinforcing its
specificity for FKBP51.
transporters (DATs)46; interestingly, other com-
pounds from the LOPAC library targeting these
same mechanisms, including the M1 muscarinic
antagonist, biperiden, and the H1 histaminergic
antagonist, brompheniramine, did not ablate the
FKBP51-mediated suppression of GR (Figure 5A-
C). This suggested that the effects on GR signaling
were not related to the activity of a muscarinic re-
ceptor antagonist; possibly through a novel mech-
anism. We also assessed the selectivity of the
benztropine scaffold by comparing small mole-
cules with moieties and structural similarities to
benztropine. Neither atropine nor diphenhydra-
mine rescued FKBP51-mediated suppression of GR
activity, despite being the component scaffolds of
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Figure 6. Benztropine rescued FKBP51-mediated GR inhibition. (A-B) Confocal microscopy images of HEK293T cells transfected
with RFP-GR (A) or RFP-GR and FKBP51 and stained with FKBP51 and DAPI. Cells were treated as indicated for 90 minutes. Scale bar
= 5 µm. (B) Quantification of z-stack images of percent GR nuclear colocalization. ***p<0.001 by two-way ANOVA.
To corroborate these results, we prepared acute
ex vivo brain slices from aged, wild-type mice to
examine whether benztropine enhanced GR trans-
location in the brain. We used aged mice since we
have previously shown that FKBP51 levels in-
crease with age in both mouse and human brain34,
48. Therefore, ex vivo slices from aged mice were
treated for 4 hours with benztropine and/or dex-
amethasone prior to subcellular fractionation to
isolate nuclear and cytosolic fractions. Western
blot analysis revealed that benztropine enhanced
dexamethasone-induced GR nuclear translocation
(Figure 7A-B), suggesting that benztropine re-
stored FKBP51-mediated GR suppression in these
slices. These results were further confirmed in
primary neurons. To do this, primary neurons
were transduced with eGFP or FKBP51 adeno-as-
sociated virus serotype 9 (AAV9) for 10 days and
then treated with dexamethasone and/or benztro-
pine for 3 hours. The neurons were lysed, and nu-
Figure 7. Benztropine enhanced GR translocation in the
presence of high FKBP51. (A) Western blots of acute ex vivo
slices from aged wild-type mice (N=3) treated as indicated for 4
hours. (B) Quantification of the GR translocation ratio. *p<0.05
by one-way ANOVA. (C) Western blots from wild-type primary
neurons (N=3) transduced with GFP or FKBP51 AAV for 10
days and treated with dexamethasone (0.5 μM) and/or benztro-
pine (10 μM) for 3 hours. (D) Quantification of the GR transloca-
tion ratio. *p<0.05, ***p<0.001 by two-way ANOVA.
clear and cytosolic fractions were collected. West-
ern blot data revealed a decrease in the GR trans-
location ratio following FKBP51 overexpression,
which was rescued by benztropine (Figure 7C-D).
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Taken together, these data confirm that benztro-
drug treatment for Parkinson’s disease52. It works
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pine restores GR translocation in the presence of
high FKBP51.
through decreasing the imbalance between neuro-
transmitters acetylcholine and dopamine to re-
duce tremors and rigidity53. It is possible that
some of these effects may be in part related to this
newly identified mechanism of altering in GR sig-
naling through FKBP51. For example, benztropine
increases dopamine levels by blocking reuptake
and storage53 as well as improves motility through
reduced rigidity53. Studies have shown that CORT
levels play an important role in controlling dopa-
mine balance54, 55 and locomotor activity56, 57. One
of the known side effects of benztropine is im-
paired long-term memory58. It has been well-doc-
umented that CORT affects learning and
memory59-61. Interestingly, benztropine was re-
cently shown to be beneficial in reversing deficits
in myelination caused by α-synuclein62, similar
white matter deficits have been previously meas-
ured in PTSD for patients with FKBP5 risk SNPs32.
While there are many parallels in the effects of
benztropine and CORT regulation, additional
studies are needed to determine if each of these
described effects or any of the other side effects,
including coma, mania, anticholinergic toxicity,
and delirium52, 63-65, are mediated through an
FKBP51-dependent mechanism.
Discussion
Here, we have identified benztropine mesylate
as a novel FKBP51-specific inhibitor. Treatment
with benztropine in models of high FKBP51 ex-
pression rescued FKBP51 suppression of GRE acti-
vation and GR nuclear translocation, likely by in-
hibiting the FKBP51-GR interaction. Using recom-
binant protein studies, we found that benztropine
can bind directly to FKBP51 and that disruption of
the FKBP51/GR/Hsp90 heterocomplex was in-
creased in the presence of hormone. Together,
these findings, in addition to identifying benztro-
pine as a FKBP51 inhibitor, highlight the feasibil-
ity of high-throughput screening for modulators
of steroid hormone receptor activity.
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The identification of FKBP51-specific inhibitors
is of great interest to both provide tools to further
study the effects of FKBP51 inhibition and as po-
tential therapeutics for FKBP51-related disorders.
However, the direct targeting of FKBP51 has been
challenging due to the high sequence similarity
among the FKBP family members. Target specific-
ity is critical for inhibitors of FKBP51, since other
isomerases, like FKBP52, have different, but es-
sential, functions. Mice lacking FKBP51 showed an
anti-depressive behavior without cognitive im-
pairment, while mice lacking FKBP52 experienced
detrimental effects on developmental, stress sen-
Since benztropine is BBB permeable, it could
serve as a solid platform for a medicinal chemis-
try campaign in the hopes of refining its specific-
ity,
improving
its
potency
for
the
FKBP51/GR/Hsp90 interaction while abrogating
the negative effects. In fact, a number of N-sub-
stituted benztropine analogues have been devel-
oped, which retain high affinity for dopamine
sitivity, and neuroendocrine processes5, 41
.
Recently, some compounds have been developed
that are selective for FKBP51 over other family
members, including FKBP5249. These molecules
have been shown to reduce anxiety-like behavior
in mice50, indicating that targeting FKBP51 may be
a viable approach to treat anxiety disorders and
other stress-related diseases. These molecules
were designed to target the hydrophobic FK506-
binding pocket of FKBP51, which disrupts PPIase
activity. However, PPIase activity has been shown
to be dispensable for GR activity10, 51, so the link
between the physiological effects and mechanism
of action of these compounds are still uncertain.
Therefore, it is unclear if these compounds will
achieve the desired outcome on GR activity and
stress regulation even though they are highly se-
lective for this domain of FKBP51. Perhaps com-
pounds such as benztropine that target FKBP51-
independently of regulating PPIase activity and
that appears to have increased activity in the
presence of hormone will have better clinical util-
ity.
transporters with minimal off-target effects66, 67
.
An even greater number of benztropine deriva-
tives have been developed with the goal of discov-
ering novel monoamine transporter inhibitors68.
Similar future studies may be used to improve
specificity against the FKBP51/GR/Hsp90 com-
plex.
In conclusion, our cell-based screen identified a
very promising molecule that can selectively in-
hibit FKBP51-mediated GR suppression. Attenua-
tion of FKBP51 activity, or expression, is desirable
for neuropsychiatric disorders, Alzheimer’s dis-
ease, and cancer. Moreover, the unique design of
our screen could be used to identify inhibitors of
other steroid hormone receptors, other co-chap-
erones, or components tertiary to steroid signal-
ing pathways. Therefore, these studies have broad
implications and utility in the development of
novel therapeutics targeting not only FKBP51
complexes, but also other dynamic complexes in
the cell.
Benztropine, labeled as Cogentin, is a blood-
brain barrier permeable compound that has been
widely used to reduce extrapyramidal side effects
of antipsychotic treatments and as a second line
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Materials and Methods: Plasmids: FKBP51 and
RFP-GR constructs were generated in our labora-
tory and inserted into pCMV6 vectors. The mouse
mammary tumor virus (MMTV) glucocorticoid re-
sponse element (GRE) luciferase reporter con-
struct was a gift from Dr. Marc Cox (University of
Texas, El Paso).
previously described34, 71. A Zeiss LSM 880 Axi-
oObserver laser scanning confocal microscope
was used for representative images. A 63×/1.40
PLAN APO oil objective was 1-μm Z-stacked im-
ages with Argon (for FKBP51-positive signal in
green), and Red HeNe (for GR-positive signal in
red).
Cell Culture: HeLa, M17, and HEK293T cells
were maintained in Dulbecco’s Modified Eagle
Medium (DMEM) with 10% fetal bovine serum
(FBS) as previously described69, 70, for all hormone
treatments charcoal-stripped FBS was used in-
stead for at least 16 hours before treatment
started. For the high-throughput screen, HeLa
cells were transfected in suspension with 200 ng
of the GRE luciferase reporter and FKBP51 or 6/TR
(6 repeats of the tetracycline repressor sequence)
as a control using 3 µl/µg of Mirus TransIT2020
transfection reagent (Mirus Bio, Madison, WI) in
serum-free media and plated in 96-well plates.
Cells were treated 24 hours later with hydrocorti-
sone (50 nm; Sigma-Aldrich) and compounds from
the LOPAC 1280 drug library (Sigma-Aldich, St.
Louis, MO) in triplicate wells at 10 µM. Cells were
harvested 16 hours later in Cell Culture Lysis Re-
agent (Promega, Madison, WI) and transferred to
opaque, white polystyrene 96-well plates. Lucif-
erase assays were performed with the Luciferase
Assay System (Promega) according to manufac-
turer’s protocols and luminescence was immedi-
ately read on a multimodal plate reader (Biotek
Synergy H1, Biotek, Winooski, VT).
For RFP-GR experiments, HEK293T cells were
plated on poly-L-lysine coated coverslips and
transfected with 1 µg of RFP-GR and 1 µg of FKBP51
or 6/TR using 2.5 µl/µg of Lipofectamine 2000
(Life Technologies) in Opti-Mem. After 4 hours,
media was replaced with DMEM supplemented
with 10% charcoal stripped FBS, Glutamax, and
Pen/Strep. Cells were treated 24 hours later with
the synthetic glucocorticoid, dexamethasone (100
nm), and benztropine (10 µM) for 90 minutes and
then fixed in 4% PFA for 30 minutes. Cells were
permeabilized with 0.1% Triton-X-100, stained
for FKBP51, and DAPI was applied for 5 minutes to
stain nuclei. All conditions were run in duplicate
and confocal microscopy images were captured by
a blinded experimenter.
Expression, Purification, and Labeling of Recom-
binant Proteins: The GR-LBD protein, a stabilized
variant of the GR ligand binding domain was pu-
rified as previously reported45. FKBP51, FKBP52,
and Hsp90α proteins were generated as de-
scribed72. Briefly, full-length wild-type human
FKBP51, FKBP52, or Hsp90α were cloned into a
pET28 vector, expressed in E. coli, and purified
via Ni-NTA-agarose chromatography followed by
size exclusion chromatography. Protein purity
was verified by Coomassie blue SDS-PAGE. Puri-
fied proteins were labeled for flow cytometric
protein-protein interaction evaluation as previ-
ously described73, 74. Recombinant GR-LBD was in-
cubated with biotin for 1 hour followed by washes
to remove excess biotin. The biotin-labeled pro-
tein was then immobilized with streptavidin
coated polystyrene particles (Spherotech, Lake
Forest, IL) for 1 hour followed by washes to re-
move any unbound protein. For the experiment
using biotinylated benztropine, we used the Dyna-
beads M-280 Streptavidin particles (Thermo
Fisher Scientific, #11205D) that have a smaller di-
ameter and a strong binding affinity for biotin al-
lowing better drug-protein interaction. FKBP51,
FKBP52 and BSA were chemically labeled with
Alexafluor 488 carboxylic acid succinimidyl ester
(Life Technologies) following the manufacturer’s
protocol.
Biotinylated benztropine Synthetic Procedures:
All materials were obtained from commercial sup-
pliers and used without further purification. Dry
THF was obtained via distillation from sodium
benzophenone ketyl. Preparative chromatography
was carried out using Sorbtech silica gel (60 Å po-
rosity, 40-63 µm particle size) in fritted MPLC car-
tridges and eluted with Thomson Instrument
SINGLE StEP pumps. Thin layer chromatography
analyses were conducted with 200 µm pre-coated
Sorbtech fluorescent TLC plates. Plates were vis-
ualized by UV. LC/MS and LRMS data was ob-
tained using an Agilent 1100 HPLC/MSD system
equipped with a diode array detector running an
acetonitrile/water gradient and 0.1% formic acid.
High resolution mass spectral data were obtained
using an Agilent 6540 QTOF mass spectrometer.
Nuclear magnetic resonance spectrometry was
run on a Varian Inova 500 MHz spectrometer, and
chemical shifts are listed in ppm correlated to the
solvent used as an internal standard. Benztropine
(2)39. Tropine (1, 1.052 g, 7.45 mmol) and benzhy-
dryl chloride (1.4 mL, 7.9 mmol) were added to a
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Microscopy: Slides were imaged on an Olympus
FV1200 confocal microscope as previously de-
scribed for analysis71. Fifteen confocal z-stack im-
ages were captured per treatment at 60x magnifi-
cation. To examine the extent of GR nuclear colo-
calization, ImageJ software (National Institutes of
Health) was utilized. Briefly, DAPI-positive nuclei
were masked and the percent of total GR-positive
signal to DAPI-overlapping were calculated as
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25 mL round bottomed flask containing a stir bar was quenched with brine, and the mixture was ex-
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8
to give a tan solution. The mixture was heated
with stirring to 160 °C for 1 hour, then allowed to
cool to room temperature. The residue was dis-
solved in dichloromethane and then evaporated to
obtain a brown oil. The oil was triturated in ether
to yield a tan solid as the HCl salt of the crude
product. Saturated sodium carbonate was added
to this suspension, then the aqueous layer was ex-
tracted with ethyl acetate. The combined organic
layers were dried with sodium sulfate and evapo-
rated under reduced pressure to yield 2 (2.12 g,
tracted with ethyl acetate. The combined organic
extracts were dried with brine, then concentrated
on a rotary evaporator to give a brown oil. Purifi-
cation by flash column chromatography provided
unreacted 3 (364.3 mg) as well as pure 4 (65.7 mg,
7%). 1H NMR (500 MHz, CDCl3) δ 7.36 – 7.26 (m,
8 H), 7.25 – 7.07 (m, 2 H), 5.40 (s, 1 H), 3.59 (br
s, 1 H), 3.35 (br s, 2 H), 3.26 – 3.07 (m, 2 H), 2.28
– 2.15 (m, 3 H), 2.03 – 1.86 (m, 6 H) ppm. 13C NMR
(126 MHz, CDCl3) δ 143.0, 128.4, 127.2, 126.8,
80.7, 71.6, 69.3, 58.3, 41.6, 37.6, 36.5, 25.7 ppm.
LRMS m/z: [M+H]+ = 332. HRMS m/z: [M+H]+
Calcd for C23H26NO 332.2014; Found 332,2012. Fi-
nal Compound (5). A solution of 4 (49.0 mg, 148
µmol) and azide PEG3 biotin conjugate (85 mg,
190 µmol, used as obtained from Sigma Aldrich) in
water (500 µL) and t-butanol (500 µL) was stirred
in a 10 mL round bottomed flask. Sodium ascor-
bate (6.90 mg, 35 µmol) and copper (II) sulfate
pentahydrate (4.20 mg, 17 µmol) was added (caus-
ing the mixture to turn blue), and the reaction was
stirred at room temperature overnight. The mix-
ture was concentrated on a rotary evaporator,
then taken up in water and extracted with di-
chloromethane. The combined organic extracts
were dried over sodium sulfate, then concen-
trated on a rotary evaporator. The residue was pu-
rified by flash column chromatography to give 5
(92 mg, 80%) as a white solid. 1H NMR (500 MHz,
CD3OD) δ 8.08 (s, 1 H), 7.37 (d, J = 7.3 Hz, 4 H),
7.33 – 7.27 (m, 4 H), 7.26 – 7.19 (m, 2 H), 5.53 (s,
1 H), 5.04 – 4.90 (m, 1 H), 4.60 (t, J = 5.0 Hz, 2
H), 4.47 (dd, J = 7.8 Hz, 4.8 Hz, 1 H), 4.28 (dd, J =
7.8 Hz, 4.5 Hz, 1 H), 3.99 (s, 2 H), 3.90 (t, J = 5.0
Hz, 2 H), 3.67 – 3.55 (m, 10 H), 3.51 (t, J = 5.5 Hz,
2 H), 3.36 – 3.33 (m, 3 H), 3.22 – 3.14 (m, 1 H),
2.90 (dd, J = 12.7 Hz, 5.0 Hz, 1 H), 2.68 (d, J=12.8
Hz, 1 H), 2.45 – 2.35 (m, 2 H), 2.19 (br t, J=7.3 Hz,
4 H), 2.14 – 2.00 (m, 4 H), 1.76 – 1.53 (m, 5 H),
1.48 – 1.37 (m, 2 H) ppm. 13C NMR (126 MHz,
CDCl3, mixture of amide rotamers) δ 175.0, 164.6,
142.6, 140.9, 128.0, 127.0, 126.5, 125.5, 80.8,
70.10, 70.08, 69.98, 69.8, 69.1, 68.0, 61.9, 60.2,
59.5, 55.6, 50.1, 48.4, 45.6, 39.6, 38.9, 35.3, 34.5,
31.8, 28.3, 28.1, 25.4, 24.7, 20.6 ppm. LRMS m/z:
[M+H]+ = 776. HRMS m/z: [M+H]+ Calcd for
C41H58N7O6S 776.4169; Found 776.4180.
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93%) as a light brown oil. H NMR (500 MHz,
CDCl3) δ 7.36 – 7.27 (m, 8 H), 7.24 – 7.16 (m, 2 H),
5.40 (s, 1 H), 3.74 – 3.53 (m, 1 H), 3.17 (br s, 2 H),
2.32 (s, 3 H), 2.21 (br d, J = 7.5 Hz, 2 H), 2.07 –
1.86 (m, 6 H) ppm. LRMS m/z: [M+H]+ = 308. Me-
thyl
(3-endo)-3-benzhydryl-8-azabicy-
clo[3.2.1]octane-8-carboxylate (3). A solution of 2
(4.07 g, 13.2 mmol) in 1,2-dichloroethane (65 mL)
was stirred in a 250 mL round bottomed flask, and
sodium carbonate (5.64 g, 53.2 mmol) and 1-chlo-
roethyl chloroformate (5.78 mL, 53.0 mmol) was
added. The mixture was heated with stirring to 85
°C for 3 h, then cooled and filtered. The filtrate
was concentrated on a rotary evaporator, then
dissolved in methanol and stirred at room temper-
ature overnight. This mixture was concentrated
on a rotary evaporator to give an off-white solid
that was dissolved in dichloromethane and then
washed with 5.5 M ammonium hydroxide and wa-
ter. The organic layer was then dried with magne-
sium sulfate and concentrated on a rotary evapo-
rator to give 3 as a brown oil (4.34 g, 93%) that
1
was used without further purification. H NMR
(500 MHz, CDCl3) δ 7.37 – 7.27 (m, 8 H), 7.25 –
7.17 (m, 2 H), 5.42 (s, 1 H), 4.24 (br s, 2 H), 3.70
– 3.65 (m, 4 H), 2.23 (q, J = 6.3 Hz, 2 H), 2.07 –
1.86 (m, 6 H) ppm. LRMS m/z: [M+H]+ = 352. (3-
endo)-3-Benzhydryl-8-propargyl-8-azabicy-
clo[3.2.1]octane (4). A stirred mixture of 3 (1.334
g, 3.79 mmol) in 2 M potassium hydroxide (37 mL,
74 mmol) and ethanol (35 mL) in a 100 mL round
bottomed flask was heated to 100 °C for 14 h, then
concentrated on a rotary evaporator and ex-
tracted with ether. The combined organic extracts
were washed with water and concentrated on a
rotary evaporator to give the crude secondary
amine (1.22 g), which was used without further
purification. This residue was taken up in dry THF
(5 mL) and added dropwise to a suspension of
60% sodium hydride (183 mg, 4.57 mmol) in dry
THF (5 mL) at 0 °C under argon. Upon completion
of the addition, the mixture was allowed to warm
to room temperature for 10 minutes, and then
propargyl bromide (500 µL, 4.64 mmol) was
added via syringe and the reaction was allowed to
stir at room temperature overnight. The reaction
Flow Cytometry Protein Interaction Assay: The
assay was conducted as previously described73, 74
.
For the drug binding and complex interaction ex-
periment, biotinylated GR-LBD and Alexafluor
488 conjugated FKBP51 were incubated together,
with Hsp90α to facilitate the interaction, in
equimolar ratios for 30 minutes with varying con-
centrations of drug. For the benztropine specific-
ity assay, Dynabeads Streptavidin particles were
washed three times with BSA/PBS (0.2 mg/mL).
The streptavidin particles were then incubated for
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30 minutes with recombinant fluorescently la- minute incubation on ice. Supernatant was col-
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beled FKBP51, FKBP52, or BSA in the presence of
absence of a biotinylated-benztropine. All condi-
tions were run in triplicate on separate days.
Binding was evaluated using an Accuri C6 flow cy-
tometer (BD Biosciences, San Jose, CA) to quantify
median bead-associated fluorescence74. Back-
ground scatter and fluorescence were subtracted
from the signal.
Co-Immunoprecipitation: Co-immunoprecipita-
tion (co-IP) was performed as previously de-
scribed48. Briefly, HeLa cells were transfected
with 3 µg FKBP51 and treated overnight with hy-
drocortisone (50 nm) and benztropine (10 µM).
Cells were harvested in M-PER buffer and lysates
were incubated overnight in rabbit anti-GR anti-
body (Cell Signaling) at 4° C. Magnetic protein A
beads (Life Technologies) were incubated with
samples for 4 hours at 4° C, followed by five
washes and a denaturing elution. The resulting
precipitates were subjected to SDS-PAGE.
lected and centrifuged again at 107,000 x g for 30
minutes to remove the membrane fraction. The
resulting supernatant contained the cytosolic
fraction. Pellets from the initial spin were resus-
pended in 1 mL TSE buffer (10 mM Tris, 300 mM
sucrose, 1 mM EDTA, 0.1% NP-40, 1 mM PMSF,
and protease/phosphatase inhibitors) and centri-
fuged at 4,000 x g for 5 minutes. The supernatant
was discarded, and pellets were washed with 0.5
mL TSE buffer until the supernatant was clear.
The pellet was resuspended with 100 µL TSE
buffer and stored as the nuclear fraction.
Animal studies: All animal studies were ap-
proved by the University of South Florida Institu-
tional Animal Care and Use Committee and car-
ried out in accordance with the National Institutes
of Health (NIH) guidelines for the care and use of
laboratory animals. Mice were group housed un-
der a 12-hour light-dark cycle (lights on at 06:00)
and permitted ad libitum access to food and water.
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SDS/PAGE: Western blot experiments were per-
formed as previously described75. Membranes
were incubated overnight with primary antibod-
ies directed against rabbit GR (1:1000; Cell Sig-
naling), mouse GAPDH (1:5000; Meridian Life Sci-
ences, Mempthis, TN), rabbit Histone H3 (1:1000;
Abcam, Cambridge, MA) or goat Lamin B (1:500;
Santa Cruz, Dallas, TX). Chemiluminescence was
detected with an ImageQuant LAS 4000 imaging
system (GE Life Sciences, Pittsburgh, PA) and
band densitometry was analyzed using Scion Im-
age. All samples were run in duplicate and nor-
malized to GAPDH for cytosolic fractions or His-
tone H3 or Lamin B for nuclear fractions prior to
analysis.
Ex Vivo Slice Culture and Subcellular Fractiona-
tion: Ex vivo slices were processed as previously
described76. Briefly, mice were decapitated and
whole brains were rapidly dissected. Horizontal
400 µm slices between 2.5 and 4.0 mm ventral to
the surface of the skull were prepared on a vi-
bratome and acclimated in artificial cerebrospinal
fluid (ACSF). Slices from 9-month-old wild-type
mice were treated with benztropine (Sigma-Al-
drich) or dimethyl sulfoxide (DMSO) and/or the
synthetic CORT, dexamethasone, or DMSO for 4
hours prior to harvest. To examine GR transloca-
tion, slices underwent subcellular fractionation as
described previously for isolation of specific tis-
sue fractions77. Briefly, slices were homogenized
in a buffer containing 10 mM HEPES, 10 mM NaCl,
1 mM KH2PO4, 5 mM NaHCO3, 5 mM ethylenedia-
minetetraacetic acid (EDTA), 1 mM CaCl2, 0.5 mM
MgCl2, 250 mM sucrose, 1 mM phenylmethyl-
sulfonyl fluoride (PMSF), and protease/phospha-
tase inhibitors (Sigma-Aldrich) and centrifuged at
1000 x g for 10 minutes at 4° C following a 20-
Primary Neuronal Cultures: Primary cortical
neurons were isolated from E16.5 wild-type
mouse brains using previous established proto-
cols78. Briefly, brains were extracted, meninges
were removed, and cortices were placed in ice-
cold isotonic buffer (137 mM NaCl, 5 mM KCl, 0.2
mM NaH2PO4, 0.2 mM KH2PO4, 5.5 mM glucose,
and 6 mM sucrose, pH 7.4). Following washes,
cortices were minced, digested in trypsin, tritu-
rated, and resuspended in DMEM supplemented
with 10% FBS, penicillin/streptomycin and Am-
photericin B. The DMEM was exchanged 24 hours
later for Neurobasal medium supplemented with
Glutamax and B27 supplement (Life Technolo-
gies). Primary neurons were transduced at DIV4
with 2 µL/well of eGFP or FKBP51 AAV in PBS at
1013 viral particles per microliter. At DIV14, neu-
rons were treated with dexamethasone (0.5 µM)
and/or benztropine (10 µM) for 3 hours prior to
harvesting using subcellular fractionation to iso-
late cytosolic and nuclear fractions as described
above.
Statistical Analyses: Statistical significance for
each analysis was determined with Student’s t-
tests or one- or two-way analysis of variance
(ANOVA) with Tukey or Bonferroni post-tests to
compare groups. All figures and statistics were
generated using GraphPad Prism software; each
graph represents the mean ± the standard error
of the mean (SEM). For analysis of the compound
library screen, luciferase values were normalized
on each plate to two separate values: hormone-in-
duced GR activity and FKBP51+hormone GR activ-
ity. Using SPPS statistical software, standardized
residuals were calculated from the normalized
FKBP51-hormone values with hormone alone val-
ues used as a covariate. Z scores were generated.
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2. Baughman, G., Wiederrecht, G. J., Chang, F.,
Significance was determined by SPPS with
p<0.0001 used as criterion.
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Martin, M. M., and Bourgeois, S. (1997) Tissue
distribution and abundance of human FKBP51, and
FK506-binding protein that can mediate calcineurin
inhibition, Biochem. Biophys. Res. Commun. 232,
437-443.
Supporting Information
The Supporting Information is available free of
charge on the ACS Publication Website. The Sup-
porting Information file for the current manu-
script contains additional details on methods, as
well as Supplementary Figures 1 and 2, Supple-
mentary Table 1 and their captions.
3. Touma, C., Gassen, N. C., Herrmann, L., Cheung-
Flynn, J., Bull, D. R., Ionescu, I. A., Heinzmann, J. M.,
Knapman, A., Siebertz, A., Depping, A. M., Hartmann,
J., Hausch, F., Schmidt, M. V., Holsboer, F., Ising, M.,
Cox, M. B., Schmidt, U., and Rein, T. (2011) FK506
Binding Protein 5 Shapes Stress Responsiveness:
Modulation of Neuroendocrine Reactivity and Coping
Behavior, Biol. Psychiatry.
4. Scharf, S. H., Liebl, C., Binder, E. B., Schmidt, M.
V., and Muller, M. B. (2011) Expression and
regulation of the Fkbp5 gene in the adult mouse
brain, PLoS One 6, e16883.
5. O'Leary, J. C., 3rd, Dharia, S., Blair, L. J., Brady, S.,
Johnson, A. G., Peters, M., Cheung-Flynn, J., Cox, M.
B., de Erausquin, G., Weeber, E. J., Jinwal, U. K., and
Dickey, C. A. (2011) A new anti-depressive strategy
for the elderly: ablation of FKBP5/FKBP51, PLoS One
6, e24840.
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AUTHOR INFORMATION
Corresponding Author
*Corresponding Author: Laura J. Blair, PhD. Univer-
sity of South Florida Department of Molecular Medi-
cine, USF Health Byrd Institute, 4001 E. Fletcher
Ave.
MDC
36
Tampa,
FL
33613,
USA
lblair@health.usf.edu Tel.: +1-813-396-0639.
ORCiD
Laura J. Blair: 0000-0002-4981-5564
6. Wochnik, G. M., Ruegg, J., Abel, G. A., Schmidt, U.,
Holsboer, F., and Rein, T. (2005) FK506-binding
proteins 51 and 52 differentially regulate dynein
interaction and nuclear translocation of the
glucocorticoid receptor in mammalian cells, The
Journal of biological chemistry 280, 4609-4616.
7. Riggs, D. L., Roberts, P. J., Chirillo, S. C., Cheung-
Flynn, J., Prapapanich, V., Ratajczak, T., Gaber, R.,
Picard, D., and Smith, D. F. (2003) The Hsp90-
Notes
CAD and JJS are co-inventors for the following pa-
tent: “Inhibitors of the FKBP51 Protein from a High-
Throughput Drug Screen and Methods of Use” US Pa-
tent# US9399039 B1. The other authors have no con-
flicts of interest to disclose.
ACKNOWLEDGMENT
This work was originally conceived and designed by
C. Dickey. We would like to dedicate this work to him
for his inspirational brilliance, creativity, and deter-
mination. This work was funded by NIMH R01
MH103848, NINDS R01 NS073899, and Alzheimer’s
binding
peptidylprolyl
isomerase
FKBP52
potentiates glucocorticoid signaling in vivo, EMBO J
22, 1158-1167.
8. Denny, W. B., Valentine, D. L., Reynolds, P. D.,
Smith, D. F., and Scammell, J. G. (2000) Squirrel
monkey immunophilin FKBP51 is a potent inhibitor
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Association
MCDN
15
370051.
ABBREVIATIONS
9. Davies, T. H., Ning, Y. M., and Sanchez, E. R.
(2005) Differential control of glucocorticoid
Acetylcholine, Ach; Adeno-associated virus, AAV; Ar-
tificial cerebrospinal fluid, ACSF; Cornus Ammonis 1,
CA1; Corticosterone/hydrocortisone/glucocorticoid,
receptor
hormone-binding
function
by
tetratricopeptide repeat (TPR) proteins and the
immunosuppressive ligand FK506, Biochemistry 44,
2030-2038.
CORT;
dopamine
transporters,
protein
DATs;
4;
FKBP4/FKBP52,
FK506-binding
FKBP5/FKBP51, FK506-binding protein 5; Flow cy-
tometry protein interaction assay, FCPIA; Glucocor-
ticoid receptor, GR; Glucocorticoid Response Ele-
ment, GRE; 90kDa Heat Shock Protein, Hsp90; Major
Depressive Disorder, MDD; National Institute of
Health, NIH; Paraventricular Nucleus, PVN; Pep-
tidyl-prolyl isomerase, PPIase; Post-traumatic stress
disorder, PTSD; single-nucleotide polymorphisms,
SNPs.
10. Wochnik, G. M., Ruegg, J., Abel, G. A., Schmidt,
U., Holsboer, F., and Rein, T. (2005) FK506-binding
proteins 51 and 52 differentially regulate dynein
interaction and nuclear translocation of the
glucocorticoid receptor in mammalian cells, J. Biol.
Chem. 280, 4609-4616.
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Galigniana, M. D., and Erlejman, A. G. (2016)
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The
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