Stolonidiol: Synthesis, Target Identification, and Mechanism for Choline Acetyltransferase Activation

Article abstract of DOI:10.1021/jacs.7b01083

Stolonidiol, a marine natural product, has been reported to potentiate the activity of choline acetyltransferase (ChAT), the enzyme that produces the neurotransmitter acetylcholine. Here we report the total synthesis of stolonidiol starting from (R)-(+)-limonene. To identify the mechanism by which ChAT activity is increased, we sought to identify the biological target of stolonidiol. We show that stolonidiol binds to the phorbol ester binding site of protein kinase C (PKC), induces translocation of PKC to the cell membrane, and activates kinase activity. Furthermore, we confirmed the increase in ChAT activity observed upon treatment of cells with stolonidiol and show that this effect is mediated by PKC. Collectively, our data strongly suggest that PKC activation by stolonidiol is responsible for the resulting potentiation of ChAT activity.

Full text of DOI:10.1021/jacs.7b01083

Article
pubs.acs.org/JACS
Stolonidiol: Synthesis, Target Identification, and Mechanism for
Choline Acetyltransferase Activation
Jeremy W. Mason,† Cullen L. Schmid,‡ Laura M. Bohn,‡ and William R. Roush*,†
‡
†Department of Chemistry and Department of Molecular Therapeutics, The Scripps Research Institute, 130 Scripps Way, Jupiter,
Florida 33458, United States
S
* Supporting Information
ABSTRACT: Stolonidiol, a marine natural product, has been
reported to potentiate the activity of choline acetyltransferase
(ChAT), the enzyme that produces the neurotransmitter
acetylcholine. Here we report the total synthesis of stolonidiol
starting from (R)-(+)-limonene. To identify the mechanism by
which ChAT activity is increased, we sought to identify the
biological target of stolonidiol. We show that stolonidiol binds to the phorbol ester binding site of protein kinase C (PKC),
induces translocation of PKC to the cell membrane, and activates kinase activity. Furthermore, we confirmed the increase in
ChAT activity observed upon treatment of cells with stolonidiol and show that this effect is mediated by PKC. Collectively, our
data strongly suggest that PKC activation by stolonidiol is responsible for the resulting potentiation of ChAT activity.
INTRODUCTION
■
Alzheimer’s disease (AD) is the leading cause of dementia and
currently afflicts 5.4 million Americans.1 Alarmingly, current
projections indicate that the prevalence of this disease will
nearly triple over the next 35 years.2 Although the mechanistic
details remain obscure, cholinergic dysfunction appears early in
the course of AD and leads to severe reductions in cholinergic
signaling.3 To counteract these losses, acetylcholinesterase
(AChE) inhibitors are used to prevent the breakdown of
acetylcholine and thus enhance cholinergic neurotransmission.
Indeed, three of the four FDA approved treatments for AD are
AChE inhibitors, which have been the first-line therapies for
over two decades.4
As an alternative to preventing the breakdown of acetylcho-
line, a complementary approach for enhancing cholinergic
Figure 1. Cholinergic neurons are especially sensitive to AD
signaling could be the activation of acetylcholine synthesis
pathology. Current therapeutics target acetylcholinesterase (AChE),
preventing the breakdown of neurotransmitter and thereby increasing
(Figure 1). For example, if a small molecule could be identified
that enhanced choline acetyltransferase (ChAT) activity, the
enzyme responsible for acetylcholine synthesis, enhancements
in cholinergic signaling might be realized. Dramatic reductions
in ChAT activity have been observed in AD patients,5 and
combining therapies that prevent neurotransmitter breakdown
and amplify neurotransmitter synthesis may prove beneficial.
Stolonidiol (1), a natural product isolated from a Japanese
soft coral,6 has been reported to enhance ChAT activity.7 Given
the potential therapeutic benefits of enhancing ChAT activity,
we became interested in identifying the mechanism by which 1
is able to produce this effect. We first developed a synthetic
route to access the natural product and subsequently surveyed
candidate cellular targets that might account for the reported
increase in ChAT activity. We find that activation of protein
kinase C (PKC) is the likely mechanism for the observed
increase in ChAT activity.
cholinergic signaling. Strategies aimed at increasing the production of
acetylcholine via the activation of choline acetyltransferase (ChAT)
may provide a synergistic approach to potentiating cholinergic
neurotransmission.
There is currently a single total synthesis reported for 1 that
required 36 steps.8 The published synthesis employed a
Horner−Wadsworth−Emmons (HWE) macrocyclization to
form the 11-membered ring, which was followed by selective
epoxidations to complete the natural product (Scheme 1). We
hoped to develop a more expedient route to 1, but chose to
adopt the HWE ring closing strategy and targeted 3 as our key
intermediate. Proceeding retrosynthetically from 3, we felt that
Received: January 31, 2017
© XXXX American Chemical Society
A
DOI: 10.1021/jacs.7b01083
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Article
column chromatography. To overcome this lack of selectivity,
we screened a variety of dihydroxylation conditions, including
several asymmetric methods.13−15 Unfortunately, we were
unable to identify conditions that provided useful levels of
selectivity in this system.
Scheme 1. Retrosynthesis of Stolonidiol (1)
As an alternative, we developed a procedure for converting
the undesired diol diastereomer into the desired one (Scheme
3). First, selective tert-butyl dimethylsilyl ether (TBS)
Scheme 3. Conversion of Dihydroxylation Products into
Desired Isomer 6 and Initial Ireland−Claisen
Rearrangement
the pendant disubstituted alkene might be disconnected via
alkyl-Suzuki coupling of an intermediate derived from 4. We
believed that the all-carbon quaternary stereocenter of 4 could
be installed through a Claisen rearrangement via the cyclic
chairlike transition state depicted in 5. Finally, we anticipated
that allylic alcohol 6 would be accessible from readily available
(R)-(+)-limonene (7).
SYNTHESIS OF STOLONIDIOL
■
Our synthesis began with hydration of the exocyclic alkene of 7
using a procedure previously reported for the kilogram scale
production of fragrance chemical α-terpineol (8) (Scheme 2).9
Scheme 2. Conversion of Limonene into Diene 12
protection of the primary hydroxyls provided a mixture of 13
and 6, which were separable by chromatography. Identification
of the hydroxyl configuration was established using Mosher
ester analysis16 of each isomer (see Supporting Information
Figure S1). Unfortunately, 13 could not be converted directly
to 6, presumably due to steric hindrance around the secondary
hydroxyl. We eventually found that deprotection of the TBS
group of 13, followed by Mitsunobu inversion of the free diol17
and reprotection of the TBS group provided a three-step
sequence that afforded 6 in 58% yield from 13.
With 6 in hand, we began exploring the use of an Ireland−
Claisen rearrangement18 to generate the required quaternary
stereocenter. Acetylation of 6 provided 14, which was
converted into the silyl enol ether derivative by treatment
with LDA in THF−HMPA and TBS−Cl at −78 °C. This
solution was then slowly warmed to 80 °C (for 16 h) to
promote the Claisen rearrangement. Using this procedure, we
were able to isolate the carboxylic acid corresponding to 15 in
74% yield as the only isomer detected in the reaction mixture.
Esterification of the carboxylic acid with trimethylsilyl diazo-
methane then provided 15 in 91% yield.
While the Ireland−Claisen procedure provided efficient
access to intermediate 15, we wondered if this three-step
sequence (acetylation, rearrangement, and esterification) could
be reduced to a single-step by employing a Johnson−Claisen
protocol.19 We were initially leery of the high temperature and
acidic conditions required for this rearrangement, but to our
delight, we found that treatment of 6 with triethylorthoacetate
and catalytic pivalic acid at 145 °C provided 4 in 74% yield on
gram scale (Scheme 4). Ester 4 was further processed to give
alkyl-iodide 16 in 91% yield over two steps.
Methoxymethyl ether (MOM) protection of the resulting
tertiary hydroxyl produced 9, and then the remaining
endocyclic olefin was cleaved by ozonolysis. Next, we sought
to cyclize keto-aldehyde 10 to cyclopentenone 11 using an
enamine-based cyclization approach. While analogous cycliza-
tions have been reported for substrates lacking the tertiary
hydroxyl,10−12 this additional functionality blocked the
production of 11 using piperidine or pyrrolidine catalysis.
Screening a variety of amine and acid catalysts led to the
identification of dibenzylamine and acetic acid as the optimal
catalyst combination (Supporting Information Table S1).
Wittig olefination of aldehyde 11 then provided diene 12 in
90% yield.
At this point, we hoped to perform a regio- and
diastereoselective dihydroxylation of 12. We anticipated that
if the diene system adopted a planar s-trans conformation, then
dihydroxylation from the top face of the exocyclic olefin would
be blocked by the MOM-protected isopropoxy substituent,
which would direct dihydroxylation to the bottom face of this
olefin. Surprisingly, dihydroxylation of 12 produced a 1:1
mixture of diol diastereomers which were inseparable by
Coupling of 16 with vinyl iodide 17 using the alkyl-Suzuki
conditions developed by Furstner and colleagues20 provided
̈
the desired product, which was directly deprotected to give
B
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Scheme 4. Johnson−Claisen Rearrangement and Conversion
to Coupling Precursor 16
known intermediate 3 and complete a formal total synthesis
(Scheme 5). With intermediate 3 in hand, we were able to
Figure 2. (A) Crystal structure of a phorbol ester bound to PKC
(PDB: 1PTR). (B) Possible binding model of 1 docked in the phorbol
ester binding site of PKC using AutoDock Vina.26
Scheme 5. Completion of the Synthesis of 1
Figure 3. PKCα radioligand displacement assay using [3H]-PDBu.
Maximum displacement represents 10 μM cold PDBu and %
displacement curves are shown for cold PDBu (IC50 = 39 nM) and
1 (IC50 = 3.1 μM). Data points were run in triplicate ( SEM).
Prior to activation, PKC translocates to the cell membrane
where further structural rearrangements result in kinase
activation.28 As such, translocation to the cell membrane
provides a well-established marker of PKC activation. To
investigate whether binding of 1 to PKC leads to kinase
activation, we next performed a translocation assay using a
green-fluorescent protein (GFP)-tagged PKC (Figure 4).29
Following transfection of a PKC-GFP plasmid into HEK293
cells, the labeled protein was found distributed throughout the
complete the synthesis of 1 using the previously reported
route,8 with one notable modification. In our hands, the
reported one-step conversion of 3 to 18 provided inconsistent
results (see Supporting Information). We developed a two-step
procedure that begins with allylic chlorination of 3 using the
procedure of Meyers,21 followed by phosphonate displacement
of an in situ formed allylic iodide providing intermediate 18 in
57% yield over the two steps. Full experimental details for the
conversion of 3 to 1 are provided in the Supporting
Information.
BIOLOGICAL TARGET IDENTIFICATION
■
After evaluating the various pathways that have been reported
in the literature to enhance ChAT activity, we identified PKC
as a promising potential target. Most notably, the Rylett
laboratory has reported a series of studies that identified ChAT
as a substrate for PKC-mediated phosphorylation, which was
further shown to modulate ChAT activity.22−24 We performed
docking studies that suggested that the phorbol ester binding
site of PKC25 may accommodate stolonidiol, adding plausibility
to our hypothesis that PKC could be a biological target of 1
(Figure 2).
To test this hypothesis, we began by performing a
radioligand displacement assay with PKCα and [3H]-phorbol
dibutyrate (PDBu) (Figure 3).27 Addition of increasing
concentrations of unlabeled PDBu or 1 both displaced the
radioligand and gave IC50 values of 39 nM and 3.1 μM,
respectively, suggesting that 1 does in fact interact with the
phorbol ester binding site of PKC, although less potently then
PDBu.
Figure 4. GFP-labeled PKCγ was transfected in HEK293 cells, and
cells were treated with PMA or stolonidiol at the indicated
concentrations. Vehicle treated cells show PKC-GFP distributed
throughout the cytosol, whereas treatment of cells with 1 μM PMA
and 10 μM 1 produces pronounced translocation to the plasma
membrane. Treatment with 0.1 μM PMA and 5 μM 1 induced partial
translocation; 1 μM 1 did not induce translocation.
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cytosol and addition of vehicle produced no change in PKC
localization (Figure 4). However, when cells were treated with
1 μM phorbol 12-myristate-13-acetate (PMA), or 10 μM 1, we
observed robust translocation of the labeled protein to the cell
membrane. Treating cells with 0.1 μM PMA or 5 μM 1 induced
a partial response, whereas 1 μM 1 did not induce
translocation. Therefore, treatment with 1 is able to induce
PKC translocation to the cell membrane, indicative of kinase
activation.
activity over control treated cells (Figure 6). We next examined
ChAT activity following treatment with 1 and found that 1 and
Upon activation, PKC phosphorylates a consensus sequence
that is found on multiple cellular proteins. To further examine
the ability of 1 to induce activation of PKC, a commercially
available monoclonal antibody mixture (MultiMab) that
specifically recognizes PKC consensus sequences containing a
phosphorylated serine was used to assess kinase activity. We
treated HEK293 cells with 1 μM PMA, 1 μM 1, or 10 μM 1
and performed a Western blot analysis of cell lysates (Figure 5).
Figure 6. HEK293 cells stably expressing 69 kDa ChAT were treated
as indicated for 16 h, and ChAT activity (relative to control) was
measured in crude cell lysates. Data are expressed as mean SEM of
five experiments. Statistical significance (Student’s t-test) versus
control: * p < 0.05; *** p < 0.0005; n.s. = not significant.
10 μM treatments produced 48% and 97% increases in ChAT
activity, respectively. To confirm that this increase in ChAT
activity was mediated by PKC, we cotreated cells with 10 μM 1
and 2 μM GF. Cotreatment with the PKC inhibitor reversed
the observed increase in ChAT activity, suggesting that the
increased activity is mediated by PKC.
Reduced PKC activity has been observed in AD patients, and
PKC activity is known to play a crucial role in memory
formation.32,33 The culmination of these effects has led to the
hypothesis that PKC activators may provide symptomatic
benefits in AD. Indeed, the potent PKC activator bryostatin 1 is
currently being investigated as a potential AD therapeutic.33−36
We show here that an additional benefit of PKC activation may
arise from subsequent ChAT activation, adding further promise
to this approach.
Figure 5. HEK293 cells were treated for 20 min with DMSO
(control), 1 μM PMA, 1 μM 1, or 10 μM 1, and cell lysates were
probed with antibody specific for substrates of PKC that have been
phosphorylated at a serine residue within the PKC consensus
sequence. Cotreatment of cells with 10 μM 1 and 2 μM
GF109203X (GF) reverses the increase in substrate phosphorylation,
whereas treatment with 2 μM GF alone does not affect substrate
phosphorylation.
CONCLUSIONS
■
In summary, we describe the use of the natural product
stolonidiol to probe pathways responsible for activating
neurotransmitter biosynthesis. Similar mechanistic investiga-
tions of small molecules that enhance neurotransmission may
provide a general approach for identifying therapeutic targets
for complex neurodegenerative diseases.
In comparison to control (DMSO) treated cells, those treated
with PMA or with 1 showed a strong increase in substrate
phosphorylation, further supporting the notion that 1 is able to
activate PKC in cells. Cotreatment of cells with 10 μM 1 and 2
μM GF109203X (GF), a bis-indolylmaleimide inhibitor of
PKC,30 reversed the observed increase in substrate phosphor-
ylation indicating that PKC activation is mediating phosphor-
ylation.
EXPERIMENTAL SECTION
For experimental details, see Supporting Information.
■
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.7b01083.
■
S
Finally, having generated strong evidence in support of the
ability of 1 to activate PKC, we wanted to confirm that
treatment of cells with 1 resulted in increased ChAT activity.
We first generated stably transfected ChAT expressing HEK293
cells and confirmed ChAT expression via immunofluorescence
(Supporting Information Figure S2). We then treated these
cells with 1 μM PMA and measured ChAT activity using the
method of Fonnum,31 after 16 h of treatment. In agreement
with previous reports,23 we observed a 61% increase in ChAT
Experimental details and procedures (PDF)
Spectral data (PDF)
AUTHOR INFORMATION
Corresponding Author
*roush@scripps.edu
ORCID
■
William R. Roush: 0000-0001-9785-5897
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Article
(32) Favit, A.; Grimaldi, M.; Nelson, T. J.; Alkon, D. L. Proc. Natl.
Acad. Sci. U. S. A. 1998, 95, 5562.
(33) Alkon, D. L.; Epstein, H.; Kuzirian, A.; Bennett, M. C.; Nelson,
T. J. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 16432.
(34) ClinicalTrials.gov NCT02431468.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
́
(35) Etcheberrigaray, R.; Tan, M.; Dewachter, I.; Kuiperi, C.; Van der
This work was supported by National Sciences and Engineering
Research Council of Canada (NSERC) and The Scripps
Research Institute. We thank Dr. Jane Rylett for the ChAT
expression plasmid and Dr. Tobias Meyer for the PKC-GFP
plasmid (Addgene plasmid no. 21204).
Auwera, I.; Wera, S.; Qiao, L.; Bank, B.; Nelson, T. J.; Kozikowski, A.
P.; Van Leuven, F.; Alkon, D. L. Proc. Natl. Acad. Sci. U. S. A. 2004,
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(36) Alkon, D. L.; Sun, M.-K.; Nelson, T. J. Trends Pharmacol. Sci.
2007, 28, 51.
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