A collection of caged compounds for probing roles of local translation in neurobiology

Article abstract of DOI:10.1016/j.bmc.2010.04.005

Spatially localized translation plays a vital role in the normal functioning of neuronal systems and is widely believed to be involved in both learning and memory formation. It is of central interest to understand both the phenomenon and molecular mechani

Full text of DOI:10.1016/j.bmc.2010.04.005

Bioorganic & Medicinal Chemistry 18 (2010) 7746–7752
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A collection of caged compounds for probing roles of local translation
in neurobiology
Oleg Sadovski a, Anna S. I. Jaikaran a, Subhas Samanta a, Marc R. Fabian b, Ryan J. O. Dowling b,
Nahum Sonenberg b, G. Andrew Woolley a,
*
a Department of Chemistry, University of Toronto, 80 St. George ST., Toronto, ON, Canada M5S 3H6
b Department of Biochemistry, McGill University, Montreal, QC, Canada H3G 1Y6
a r t i c l e i n f o
a b s t r a c t
Article history:
Spatially localized translation plays a vital role in the normal functioning of neuronal systems and is
widely believed to be involved in both learning and memory formation. It is of central interest to under-
stand both the phenomenon and molecular mechanisms of local translation using new tools and
approaches. Caged compounds can, in principle, be used as tools to investigate local translation since
optical activation of bioactive molecules can achieve both spatial and temporal resolution on the micron
scale and on the order of seconds or less, respectively. Successful caging of bioactive molecules requires
the identification of key functional groups in appropriate molecules and the introduction of a suitable
caging moiety. Here we present the design, synthesis and testing of a collection of three caged com-
pounds: anisomycin caged with a diethylaminocoumarin moiety and dimethoxynitrobenzyl caged ver-
sions of 4E-BP and rapamycin. Whereas caged anisomycin can be used to control general translation,
caged 4E-BP serves as a probe of cap-dependent translation initiation and caged rapamycin serves a
probe of the role of mTORC1 in translation initiation. In vitro translation assays demonstrate that these
caging strategies, in combination with the aforementioned compounds, are effective for optical control
making it likely that such strategies can successfully employed in the study of local translation in living
systems.
Received 19 January 2010
Revised 25 March 2010
Accepted 1 April 2010
Available online 7 April 2010
Keywords:
Caged
Optical
Photo-control
Protein synthesis
Translation
Rapamycin
4E-BP
Anisomycin
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Biochemical techniques for manipulating translation have been
instrumental in establishing the connection between protein syn-
Spatial and temporal control of protein synthesis (translation)
plays a central role in the neural processes that underlie learning
and memory (for reviews see Ref. 1–7). However, key questions re-
main unresolved, for example, does translation occur only in a
stimulated dendritic spine or does it occur elsewhere, and are pro-
teins targeted to that spine? In addition to establishing functional
correlates of changes in local translation, one often wishes to probe
the molecular mechanisms involved, that is, how does synaptic
activity leading to long term potentiation (LTP) trigger translation
of specific mRNAs in neurons? Is synthesis cap-dependent or is a
cap-independent process involved?8 Does signaling occur through
the mTORC1 pathway (a central regulator of translation) or via
other signaling cascades?9 Answering such questions requires
tools to dissect the regulation of local translation at a single syn-
thesis and development, learning and memory. In general, tools fall
into two broad groups: (i) specific small molecule inhibitors and (ii)
genetic approaches (e.g., overexpression and knock-outs). A variety
of small molecules are known to inhibit eukaryotic translation.
These include emetine, puromycin, anisomycin and cycloheximide.
These inhibitors act primarily at the ribosome, preventing peptide
bond formation, tRNA binding, or protein elongation;10 however,
additional small molecule inhibitors that other steps in protein syn-
thesis, including translation initiation, are currently under develop-
ment.11–13 These small molecules are generally cell permeable but
wash-in and wash-out are not very rapid and, although spatially re-
stricted perfusion has been attempted,14 these drugs are difficult to
localize with confidence within micron sized areas.
Genetic approaches have also been used to alter translational
efficiency by manipulating components of the protein-synthesis
machinery.15,16 While such approaches offer insights into the roles
of specific proteins, as opposed to the all-or-none translational re-
sponse in studies using general protein-synthesis inhibitors, they
usually target global neuronal protein synthesis. With conditional
control (e.g., using the CreER/loxP or DICE-K system) with cell type
specific promoters, some degree of spatiotemporal control can be
apse (length scale of ꢀ1
lm) on the timescale of <1 min. Such tools
could contribute substantially to the fundamental understanding
of a variety of important neuronal processes.
* Corresponding author.
E-mail address: awoolley@chem.utoronto.ca (G.A. Woolley).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.005

O. Sadovski et al. / Bioorg. Med. Chem. 18 (2010) 7746–7752
7747
achieved as has been demonstrated in studies of hippocampal syn-
aptic pathway function.17. Nevertheless, cell-based studies using
these strategies may be difficult to interpret, particularly if the bio-
logical event under study is relatively fast (seconds/minutes) (e.g.,
LTP) or has a complex spatial dependence.
Photo-control, in which light is used to directly control a bio-
chemical process, offers the possibility of rapid, spatially localized,
external control. Caged compounds (compounds in which a light
pulse is used to remove a protecting group and thereby render
the molecule bioactive) already enjoy wide use in neurobiology.18
Caged glutamate, in particular, has been used very successfully in
studies of neural function including LTP.19 In pioneering work,
Dore, Schuman and colleagues designed and synthesized a caged
version of anisomycin, a protein-synthesis inhibitor that functions
at the level of the ribosome.20
We report here the design, synthesis, and in vitro evaluation of
a collection of three new caged compounds that permit the photo-
control of different molecular steps involved in protein synthesis:
(i) a diethylaminocoumarin-caged anisomycin derivative that ex-
tends the work of Goard et al.; (ii) a caged 4E-BP peptide that per-
mits selective control of cap-dependent versus cap-independent
modes of translation initiation and (iii) a caged rapamycin deriva-
tive that enables probing of the role of the mTORC1 pathway in
regulating local translation.
thoxy-2-nitrobenzyl))-OH was carried out as described previ-
ously.22 Dimethoxy-2-nitrobenzyl bromide was prepared from
veratraldehyde as described.23,24 Peptides were prepared using
standard Fmoc-based solid-phase peptide synthesis in a micro-
wave peptide synthesizer (CEM Corp.). After synthesis, peptides
were HPLC purified using a Zorbax SB-C18 column (Chromato-
graphic Specialties Inc.) using a linear gradient of 5–60% acetoni-
trile/water containing 0.1% trifluoroacetic acid in 20 min was
used with a flow rate of 2 mL/min. Peptide composition was con-
firmed by ESI-MS. 4E-BP1a caged: PGGARIIY(dmnb)DRKFLMAib-
AibRNAPVAKAPP; calcd: 3003.8, found: 3002.7. Uncaging was
tested by exposing peptide solutions in water to light from a
365 nm high intensity LED (Opto Technology, Inc., Wheeling, IL,
Model OTLH-0480-UV (365 nm, ꢀ20 mW/cm2) and following con-
version using ESI-MS (uncaged PGGARIIYDRKFLMAibAibRNAPVA
KAPP) calcd: 2808.34, found: 2807.6.
3.1. Caged rapamycin (7-demethoxy-7-dimethoxy-2-nitrobenz-
yloxy-rapamycin)
Rapamycin (LC Laboratories, Woburn, MA) (100 mg,
0.110 mmol) was dissolved in dry dichloromethane (3 mL) at
ꢁ40 °C under argon then 100
lL trifluoroacetic acid was added
and the solution was stirred for 10 min essentially as described.25
Ten equivalents of dimethoxy-2-nitrobenzyl alcohol (prepared
from veratraldehyde as described23,24) were added and the solu-
tion stirred for 1 h at ꢁ40 °C. The mixture was then extracted with
5% aqueous sodium bicarbonate and dried over anhydrous sodium
sulfate. The product was purified by preparative HPLC using a Zor-
bax SB-C18 column 9.4 ꢂ 25 cm (Chromatographic Specialties Inc.)
using isocratic methanol/water 85/15 conditions with 0.1% acetic
acid and a flow rate of 5 mL/min (eluted 10 min) followed by a sec-
ond HPLC step using acetonitrile/water 80/20 Zorbax SB-C18
4.5 ꢂ 25 cm 2 mL/min (eluted at 4.5 min) (yield 11 mg, ꢀ10%). 1H
NMR (CDCl3, 400 MHz) (mixture of trans/cis conformational iso-
mers; data for major isomer) d 7.71 (s, 1H, DMNB Ar H-60), 7.54
(s, 1H, DMNB Ar H-30), 6.39–6.32 (m, 1H), 6.22–6.09 (m, 2H),
6.02–6.00 (m, 1H), 5.60–5.50 (m, 1H), 5.50–5.35 (m, 2H), 5.35–
5.27 (m, 1H), 5.25–5.1 (m, 1H), 4.72 (s, 1H, OH-13), 4.35–4.25
(m, 2H (CH2 on DMNB)), 4.18 (1H, H-28, indicating C7-S epimer),
3.99 (s, 3H, CH3O on DMNB), 3.94 (s, 3H, CH3O on DMNB), 3.90–
3.85 (m, 1H), 3.7 (s, 1H, H-29), 3.7 (m, 1H), 3.41 (s, 3H), 3.34 (s,
3H), 2.94 (m, 1H), 2.64 (d, 2H), 1.75 (s, 3H), 1.40–0.83 (set of broad
unresolved peaks, similar to rapamycin) (note singlet at 3.14 ppm
(C7-methoxy in rapamycin is not present as expected)). ESI-HRMS:
(C59H86N2O17Na)(MNa+), calcd 1117.5819, found 1117.5822. UV–
vis (MeOH) kmax 263, 274, 287, 350 nm. Uncaging was tested by
exposing DMNB-caged rapamycin solutions in water with 50%
methanol for solubility to light from a 365 nm high intensity LED
(Opto Technology, Inc., Wheeling, IL, Model OTLH-0480-UV
(365 nm, ꢀ20 mW/cm2)). Disappearance of caged rapamycin and
appearance of demethyl-rapamcyin (i.e., rapamycin with OH at
C7) were followed by HPLC (Zorbax SB-C18 acetonitrile/water 70/
30 3.5 mL/min, detected at 254 nm) and ESI (demethyl-rapamycin)
ESI-MS: (C50H77NO13Na)(MNa+), calcd: 922.5287, found: 922.5293.
UV–vis (MeOH) kmax 266, 275, 288.
2. Materials and methods
2.1. DEAC-caged anisomycin (N-([7-N,N-diethylaminocoumarin-
4-yl]methyloxycarbonyl)anisomycin)
To a solution of 15 mg (0.06 mmol) of 7-(diethylamino)-4-
(hydroxymethyl)-2H-chromen-2-one (prepared exactly as de-
scribed,21) in dry acetonitrile was added 12 mg (0.06 mmol) 4-
nitrophenyl chloroformate and the solution was stirred for 7 h at
room temperature under argon. Then 10 mg (0.08 mmol) of 4-
dimethylaminopyridine and 10 mg (0.038 mmol) of anisomycin
(AG Scientific) was added and the solution was stirred overnight.
The reaction was quenched with 3–4 vol 15% citric acid, and ex-
tracted with chloroform. The product was purified by HPLC using
a Zorbax SB-C18 column (Chromatographic Specialties Inc.) using
isocratic methanol/water 70/30 conditions with a flow rate of
5 mL/min. The product eluted at 13.4 min. 1H NMR (400 MHz,
CD3OD, 1.3:1 mixture of conformational isomers) d 7.41 (d,
J = 9.0 Hz, 1H), 7.00 and 6.89 (d, J = 8.0 Hz, 2H), 6.72 (d, J = 8.0 Hz,
2H), 6.67–6.60 (m, 2H), 6.46 (s, 1H), 5.98 and 5.92 (s, 2H), 5.26
and 5.13 (AB q, 2H), 4.35–4.28 (m, 1H), 4.02 and 3.90 (br s, 1H),
3.65 and 3.60 (s, 3H), 3.50–3.47 (m, 1H), 3.39 (q, J = 7.0 Hz, 4H),
3.25–3.08 (m, 1H), 2.92–2.87 (m, 1H), 2.70 (t, J = 13.2 Hz, 1H),
1.99 and 1.95 (s, 3H), 1.09 (t, J = 7.0 Hz, 6H); ESI-HRMS: (C29H35
-
N2O8)(MH+), calcd 539.2387, found 539.2396. Uncaging was tested
by exposing DEAC-caged anisomycin solutions in 50 mM HEPES
buffer pH 7.0 with 5% methanol for solubility to light from a
405 nm high intensity LED (Super Bright LEDs Inc. St. Louis Mis-
souri, Model RL5-UV2030 (405 nm; ꢀ20 mW/cm2)). Disappearance
of caged anisomycin and appearance of anisomycin were followed
by HPLC (Zorbax SB-C18 column acetonitrile/water 60/40 (+0.1%
trifluoroacetic acid) for caged anisomycin (eluted at 22 min (detect
at 380 nm); acetonitrile/water 35/65 (+0.1% trifluoroacetic acid) for
anisomycin (eluted at 9.3 min (detect at 225 nm)) and ESI-HRMS:
(C14H20NO4)(MH+), calcd 266.1386, found 266.1393.
3.2. DNA/RNA preparation
The bicistronic plasmid pSP-(CAG)33-FF-HCV-Ren-p(A)51 was a
kind gift from Jerry Pelletier.12,11 The plasmid was linearized by
digestion with BamHI. The transcription reaction contained ribo-
nucleotides from NEB (0.5 mM ATP, 0.5 mM CTP, 0.5 mM UTP,
0.1 mM GTP), 0.5 mM m7G(50)ppp(50)G (Ambion), 100 U RNase
3. 4E-BP peptides
FMOC protected amino acids, Rink amide resin and HBTU were
obtained from Anaspec Inc. Preparation of FMOC-Tyr(O-(4,5-dime-
inhibitor (BioShop), 10 L 10ꢂ transcription buffer, 150 U SP6
l
polymerase (NEB), 3 lg linearized pSP-(CAG)33-FF-HCV-Ren-

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O. Sadovski et al. / Bioorg. Med. Chem. 18 (2010) 7746–7752
p(A)51 and water to 100
l
L. The reaction was incubated at 40 °C for
ribosome.27,28 Atomic resolution structures of the antibiotic in com-
plex with the large ribosomal subunit indicate that bulky substitu-
ents on the amino group of anisomycin will severely inhibit
binding.28 Dore, Schumann and colleagues reported anisomycin
derivatives in which the amino group was caged with either dimeth-
oxynitrobenzyl (DMNB) (2) or bromohydroxycoumarin (BHC) (3)
derivatives. Both compounds were shown to be effective caged
inhibitors of protein synthesis with uncaging at 365 nm (kmax are
350 and 373 nm, respectively). Here we extend these studies by
replacing the BHC group with diethylaminocoumarin moiety (4).
While undergoing analogous photochemistry to BHC, the DEAC
group has longer wavelength absorption (kmax 395 with significant
absorbance to 420 nm, compared to kmax 373 nm for BHC) and a
significantly higher molar extinction coefficient (ꢀ20,000 vs
14,500 Mꢁ1 cmꢁ1). In addition, quantum yields for uncaging as high
as 0.3 have been reported.21,29–31 Because an integrated 405 nm
laser line that can be used for uncaging is becoming a common
feature of a range of optical microscopes used for neuroimaging,32,33
a caged anisomycin derivative that could be activated effectively
with this light source may prove practically useful (Fig. 1).
2 h, following which 6
lL of RNase-free DNase was added and the
mixture incubated a further 15 min at 37 °C. For coupled transcrip-
tion/translation reactions in rabbit reticulocyte lysate control Fire-
fly luciferase DNA was used as supplied by the manufacturer.
3.3. In vitro translation reactions
In vitro translations in rabbit reticulocyte lysate were per-
formed according to manufacturer’s recommendations (Promega).
In vitro translations in Krebs extracts were preformed as described
previously.11,12,26 Translation reactions contained 7
l
L Krebs-2 cell
g 4E-BP peptide and were made up
L with 25 mM Hepes, pH 7.5. Reactions
were incubated at 30 °C for 1 h and terminated with the addition
of 20 L of ice-cold PBS. Three microliters of reaction mix were
added to 25
L luciferase reagent from the Dual-LuciferaseÒ repor-
ter system (Promega) to quantify firefly luciferase translation and
25
L of Stop & GloÒ Reagent to quantify Renilla luciferase.
extract, 50 ng RNA, 0.625–10
to a final volume of 10
l
l
l
l
l
3.4. HeLa cell studies
DEAC-caged coumarin (4) was synthesized via oxidation of
7-(diethylamino)-4-methyl-2H-chromen-2-one with selenium
dioxide as described21 followed by coupling to anisomycin using
p-nitrophenylchloroformate. Uncaging was tested using an HPLC
assay (see Section 2) and using an in vitro translation assay. Trans-
lation was performed using rabbit reticulocyte lysate coupled tran-
scription translation system using firefly luciferase DNA as
template. Translational activity is measured by the amount of
luciferase produced, which can be assayed accurately as relative
light units produced under a standard set of conditions. Caged
anisomycin was either uncaged immediately prior to adding to ly-
sate or uncaged in situ with a 405 nm LED (20 mW/cm2 radiant
flux). Absorbance by the hemoglobin in the lysate resulted in long-
er half lives for uncaging in situ. The transcription/translation reac-
tions were incubated for a further 1 h at 30 °C after uncaging
before assaying for luciferase activity. Very large changes (three or-
ders of magnitude) in translation rates were observed upon uncag-
ing DEAC-anisomycin (Fig. 2).
Cell lines were purchased from the American Type Culture Col-
lection (Manassas, VA). Cells were cultured in DMEM, supplemented
with 10% fetal bovine serum (FBS) and 100 units/mL gentamicin at
37 °C and 5% CO2 in 75-cm2 flasks. Cells were passaged by 0.25%
Trypsin–EDTA when they reached ꢀ80% confluence. Rapamycin
and caged rapamycin stock solution were prepared in MeOH/water
50/50 then diluted in water so that volumes of 20–50 lL of each
sample was added to the cell culture well (final concentration 20–
50 nM). For uncaging experiments samples were irradiated for
1 min using a 365 nm LED (Opto Technology, Inc., Wheeling, IL,
Model OTLH-0480-UV (365 nm, ꢀ20 mW)). Cells were then incu-
bated with drug for 1 h then washed with 1 mL PBS followed by
0.3 mL SDS gel loading buffer. This procedure caused detaching of
the cells as a slurry, which was then boiled for 6 min and frozen.
Samples (30 lL) were then applied to a 10% SDS–polyacrylamide
gel, then transferred to a nitrocellulose membrane (Protran, Per-
kin–Elmer). Membranes were blocked in 5% nonfat milk in TBS con-
taining 0.1% Tween 20 and probed with the appropriate primary and
secondary antibodies (phospho-S6K1 (Thr389), from Cell Signaling,
phospho-S6 (Ser240/244) also from Cell Signaling, and beta-actin,
from Sigma) at 4 °C overnight. Immunoreactive proteins were visu-
alized using enhanced chemiluminescence (Perkin–Elmer).
Effective applied concentrations and light exposure times for use
in living cells will require optimization in specific systems since
maximal localization of the effect will require release of sufficient
anisomycin to cause inhibition but not enough that diffusion leads
to inhibitory concentrations outside the zone of interest. DEAC-
caged anisomycin provides an easy-to-use starting point for probing
the role of local translation in the nervous system.
4. Results and discussion
4.2. Caged 4E-BP peptide for the inhibition of cap-dependent
translation initiation
4.1. DEAC-caged anisomycin
In most cases translation initiation is the rate limiting step in
protein synthesis.34 Translation initiation in eukaryotes can be
Anisomycin (1) is a cell permeable natural product that inhibits
protein synthesis by binding to the peptidyl transferase site of the
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
NH
N
N
N
HO
HO
HO
HO
O
O
O
NO2
HO
Br
O
O
O
O
O
N
O
1
2
3
4
Figure 1. Structures of anisomycin (1), DMNB-caged anisomycin (2), BHC-caged anisomycin (3) and DEAC-caged anisomycin (4).

O. Sadovski et al. / Bioorg. Med. Chem. 18 (2010) 7746–7752
7749
The sequence is shown below, numbered from the consensus Tyr
at position zero. The symbols: b, X, f, u and PO4 represent partially
conserved hydrophobic, any residue, partially conserved phenylal-
anine, conserved hydrophobic and phosphorylation site,
respectively.
dark
50000000
5000000
500000
50000
5000
irradiated
Peptides corresponding to eIF4E binding sequences from 4E-
BP1, 4E-BP2, and eIF4G have been shown to directly interact with
purified recombinant eIF4E in vitro13,45 with affinities ranging from
10 to 100 nM. These peptides have also been reported to inhibit
in vitro translation in cell lysates.46 The effectiveness of 4E-BP1
peptides as translation initiation inhibitors in vivo has also been
tested by microinjection of peptides into sea urchin embryos
where dramatic effects on maturation were observed consistent
with peptide inhibition of cap-dependent translation initiation.47
An effective means for developing photo-control of cap-depen-
dent translation initiation would therefore be to develop a caged
4E-BP analogue. Although small molecule inhibitors of the 4E/4G
interaction are under development,12,13 the affinity and selectivity
of these does not yet match the native peptide sequences. For cell-
based studies of local translation where photochemical tools will
be applied locally over relatively short time scales, there is no
requirement for tools to withstand the gut or the bloodstream, fac-
tors that limit the use of peptides broadly in drug development.
Substitution of the conserved Tyr (Y0) and Leu residues (L5) is
known to severely inhibit binding of 4E-BPs to eIF4E.45 This effect
can be understood in structural terms because X-ray crystal data
show both conserved residues make extensive contacts with the
eIF4E protein.48,49 Figure 3 shows a close up view of the interac-
tions between Tyr0 and eIF4E. Caging this Tyr residue is expected
to lead to severe steric clashes between the peptide and the pro-
tein, so that peptide binding is prevented.
500
0
0.8
8
80
DEAC anisomycin (µM)
Figure 2. Measured luciferase activity (relative light units) (note log scale). The
final concentration of caged anisomycin in the translation assays was 80, 8 and
0.8
l
M. Dark gray bars are caged, light gray bars are uncaged (after exposure to a
M concentration provides three orders of magnitude
405 nm LED for 10 s). The 8
greater inhibition after uncaging.
l
We prepared FMOC-Tyr-DMNB and incorporated it into an ex-
tended 4E-BP peptide analogue (caged 4E-BP, Table 1). Starting
with the 4E-BP1 sequence (for which a Kd of <50 nM has been mea-
sured with purified eIF4E48) we replaced the GluCys dipeptide se-
quence with AibAib and we removed potential phosphorylation
sites (Ser11, Thr14). The AibAib unit promotes helical backbone
conformations50 thereby preorganizing the peptide into the bound
conformation to enhance affinity. Removing phosphorylation sites
eliminates the possibility that inhibitory effects of the 4E-BP ana-
logues will be modified by the activity of kinases or phosphatases.
To test the ability of the caged 4E-BP peptide to photo-control
cap-dependent translation, we prepared bicistronic mRNAs con-
taining a capped transcript for firefly luciferase together with a
Renilla luciferase transcript under control of an IRES sequences12
(Fig. 4). Since firefly and Renilla luciferase enzymes use different
substrates, they can be assayed together. Comparing the ratio of
firefly luciferase to Renilla luciferase activity provides a direct mea-
sure of the relative rates of cap-dependent versus cap-independent
(IRES mediated) translation in vitro.12 We carried out in vitro trans-
lation reactions in Krebs ascites lysates using this bicistronic RNA
in the presence of different amount of 4E-BP peptides. The Krebs
lysate has been found to more faithfully reproduce the cap-depen-
dent translation initiation process as compared to the rabbit retic-
ulocyte system.26
Figure 3. Structure of the 4E-BP1 peptide (cyan) bound to the eIF4E protein (gray
surface). The essential Tyr(0) is labeled and the site of attachment for the caging
group circled. The image is based on the X-ray data of Marcotrigiano et al.48 (PDB
code 1EJ4). Figure prepared using Pymol.
cap-dependent requiring the presence of the m7GpppN cap struc-
ture (where ‘m’ represents a methyl group and ‘N’ is any nucleo-
tide) at the 50 end of the messenger (m)RNA. Conversely, in
specific instances ribosomes can assemble at an IRES (internal ribo-
some entry site) sequence in the mRNA36–38 to initiate cap-inde-
pendent translation. Local control of the mode of translation
initiation has been proposed to play a role in neurohormone secre-
tion,8 amyloid precursor protein production,39 and FGF-2 produc-
tion during brain development.40
The primary mechanism for regulating cap-dependent transla-
tion initiation is via binding of the 4E-BP proteins to the cap-bind-
ing translation initiation factor eIF4E. Three 4E-BPs have been
identified (4E-BP1, 4E-BP2, 4E-BP3) together with the related pro-
teins cup, maskin and neuroguidin.41–44 These proteins appear to
act as competitive inhibitors of eIF4G binding to eIF4E.41,42 All
4E-BPs share with eIF4G a consensus eIF4E binding sequence.
Figure 5 shows results obtained using in vitro translations in
Krebs lysates. Irradiation with 365 nm light for 30 s produced un-
Table 1
Consensus 4E binding sequence and 4E-BP1
Position
Consensus
4E-BP1
Caged-
ꢁ3
R/K
R
ꢁ2
ꢁ1
X
I
0
Y
Y
Y
1
2
X
R
R
3
X
K
K
4
f
F
F
5
L
L
L
6
u
M
M
7
X
E
Aib
8
X
C
Aib
9
X
R
R
10
X
N
11
PO4
S
12
X
P
13
X
V
14
X
T
b
I
X
D
D
P
G
G
A
R
I
I
N
A
P
V
A
K
A
PP
4E-BP

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O. Sadovski et al. / Bioorg. Med. Chem. 18 (2010) 7746–7752
Tyr
Tyr
365 nm light
O
OH
O2N
O
O
pSP-(CAG) -FF-HCV-REN-p(A)
33
51
SP6
Promoter
HCV
IRES
Renilla luciferase
Firefly luciferase
[CAG]33
[A]51
Figure 4. (a) Structure of DMNB-caged Tyr, (b) structure of bicistronic construct for analysis of cap-dependent translation.
3.5
3.0
2.5
2.0
1.5
1.0
Figure 6. Rapamycin/FKBP/mTORC1 ternary complex. The 7-methoxy substituent
of rapamycin (circled) points directly at mTORC1 (ribbon).
2
4
6
2
4
6
2
4
6
0.01
0.1
1
10
µ
g 4EBP peptide
phorylation of S6K1 by PDK1. Active S6K1 can in turn stimulate
the initiation of protein synthesis through activation of S6 ribo-
somal protein (a component of the ribosome) and other compo-
nents of the translational machinery.55 Translation may also be
regulated in other ways including pathways involving ERK and
p38 MAPK signaling.56,57 For instance, ARC synthesis during LTP
consolidation in the dentate gyrus appears to be controlled via
ERK-MNK signaling rather than via mTORC1.9 A tool for the local
control of mTORC1 would enable determination of the role of
mTORC1 in local translation in variety of settings.mTORC1 is
known to be specifically inhibited by the membrane-permeable
natural product rapamycin.58 Rapamycin acts by binding to FKBP
(Kd = 0.5 nM) and this complex binds to and inhibits mTORC1.25
A large medicinal chemistry patent literature describes numerous
chemical modifications of rapamycin and their activities. One face
of the rapamycin molecule interacts with FKBP and another face
(called the effector domain) interacts with mTORC1.58,59 Detailed
structural information is available for complexes of rapamycin
with FKBP and for the ternary complex FKBP–rapamycin–
mTOR.59,60 An examination of the X-ray crystal structure of the
complex of FKBP/rapamycin with mTOR shows the position of
the C7-methoxy group of rapamycin directly exposed to the mTOR
interface (Fig. 6). Thus, introduction of a bulky caging group here is
expected to effectively block mTORC1 activation.
Figure 5. Inhibition of cap-dependent translation as measured by the ratio of firefly
to Renilla luciferase activity in Krebs ascites extracts. Increasing amounts of caged
(d), irradiated (caged, irradiated for 60 s) (s), and native 4E-BP peptides (4) were
added to the reactions.
caged 4E-BP peptide which showed identical activity to pure syn-
thetic 4E-BP peptide (Fig. 5) and more than an order of magnitude
stronger inhibition of cap-dependent translation than the caged
peptide. This result demonstrates that the Tyr0 site is indeed an
effective site for caging. Even more effective caging may be ob-
tained with a bulkier caging group such as a coumarin derivative51
or the nitrodibenzofuran compound recently reported by Momo-
take et al.52
In order to be used for probing the role for cap-dependent ver-
sus cap-independent inhibition of translation in a cellular setting,
caged 4E-BP peptides would have to be microinjected47 or loaded
via cell-uptake sequences. A variety of such sequences can be ap-
pended to the peptide during synthesis. Indeed Herbert et al. have
reported 4E-BP peptides bearing penetrating cell-uptake se-
quences to be active in human lung fibroblast cells in culture.45
4.3. Caged rapamycin for the inhibition of mTORC1
Luengo et al. discovered that the methoxy position at C7 could
be selectively substituted in a mild acid catalyzed process.25 This
remarkable transformation presumably results from the participa-
tion of the adjacent triene unit. Using this acid catalyzed substitu-
tion reaction we introduced a DMNB group at the C7 position of
rapamycin (Fig. 7). We opted to use the DMNB group since (i)
4E-BP activity is post-translationally regulated via phosphoryla-
tion.53 Upstream of 4E-BP is the central regulatory kinase
mTORC1.42 mTORC1 integrates the input from multiple pathways
such as those triggered by insulin and growth factors.54 mTORC1
phosphorylates S6K1 and this stimulates the subsequent phos-

O. Sadovski et al. / Bioorg. Med. Chem. 18 (2010) 7746–7752
7751
O
O
O
OH
OH
OH
O
O
O
O
O
O
N
N
N
O
O
O
OH
OH
OH
O
HO
O
HO
O
HO
O
O
O
O
O
O
O
O
O
O
O
O
(i)
(ii)
O
O
7
+
7
7
O
NO2
rapamycin
O
C7-DMNB-caged
rapamycin
Figure 7. Synthesis of DMNB-caged rapamycin. (i) Trifluoroacetic acid in dichloromethane, (ii) DMNB alcohol.
A
B
C
D
E
F
G
H
A
B
C
D
E
F
G
H
actin
P-S6
P-S6K
actin
Figure 8. Western blots for phospho-S6 (a) and phosphor-S6K (b) (actin serves as an internal control for total protein concentration). A: control, B: vehicle, C: 50 nM
rapamycin, D: 20 nM rapamycin, E: 50 nM caged rapamycin, F: 20 nM caged rapamycin, G: 50 nM uncaged (+UV) rapamycin, H: 20 nM uncaged (+UV).
the dimethoxynitrobenzyl alcohol undergoes the acid catalyzed
substitution reaction easily, (ii) it provides sufficient steric bulk
other rapamycin derivatives.58 We therefore anticipate that they
will be of direct use in experiments aimed at uncovering the role
of mTORC1 in local translation.
for caging and (iii) it undergoes uncaging at
a wavelength
(365 nm) that does not overlap significantly with the triene unit
or rapamycin (near 280 nm). Uncaging is expected to produce 7-
hydroxy rapamycin rather than native rapamycin. This compound
has been reported to be active,61 presumably because it retains the
favorable interactions of rapamycin with its protein partners and
does not introduce any steric hindrance.
Cell-based activity assays of this DMNB-caged rapamycin ana-
logue were carried out using established assays.62,63 HeLa cells
were incubated with either vehicle, native rapamycin, DMNB-
caged rapamycin or UV irradiated DMNB-caged rapamycin. Inhibi-
tion of mTORC1 was assayed by probing for changes in S6 and S6K
phosphorylation levels via Western blot (Fig. 8). Caging was ob-
served to block rapamycin activity very effectively since >10-fold
differences in phospho-S6 were observed (Fig. 8a, lane D vs F).
Importantly, near native levels of activity were restored upon
uncaging indicating that DMNB-caged rapamycin behaves as
designed.
5. Summary
We have reported the successful caging of three compounds,
anisomycin, 4E-BP and rapamycin, with well-defined roles in the
molecular mechanisms of translation. The sites for caging and
the choice of caging group lead in each case to a compound that
shows a substantial (10–1000 times) difference in activity upon
optical activation. These compounds or related derivatives, when
optimized for particular applications may prove useful for probing
the roles and mechanisms of local translation in the nervous sys-
tems of animal models.
Acknowledgements
We would like to thank the Canadian Institutes for Health Re-
search and the NIH for financial support of this work.
An NBDF caged rapamycin could be produced by an analogous
synthetic route. The larger size of the NBDF group may lead to even
great differences between caged and uncaged derivatives. In addi-
tion, the NDBF group is expected to show better two-photon
uncaging properties than the DMNB-caged rapamycin.52 While
coumarin derivatives such as diethylaminocoumarin are appealing
for longer wavelength uncaging, they have been reported to be
ineffective for caging alcohols directly.29 Instead a 7-hydroxy rap-
amycin derivative would have to be caged as a coumarin carbon-
ate. Alternatively, Wosnick et al. have reported that coumarins
may be used to cage thiols directly.64 Thus, a caged C7 mercapto
rapamycin may be envisaged. All these derivatives are expected
to be membrane permeable based on their similarity to numerous
References and notes
1. Bramham, C. R.; Wells, D. G. Nat. Rev. Neurosci. 2007, 8, 776.
2. Pfeiffer, B. E.; Huber, K. M. J. Neurosci. 2006, 26, 7147.
3. Schuman, E. M.; Dynes, J. L.; Steward, O. J. Neurosci. 2006, 26, 7143.
4. Banko, J. L.; Klann, E. Prog. Brain Res. 2008, 169, 59.
5. Klann, E.; Dever, T. E. Nat. Rev. Neurosci. 2004, 5, 931.
6. Govindarajan, A.; Kelleher, R. J.; Tonegawa, S. Nat. Rev. Neurosci. 2006, 7, 575.
7. Bourne, J.; Harris, K. M. Curr. Opin. Neurobiol. 2007, 17, 381.
8. Wayne, N. L.; Lee, W.; Michel, S.; Dyer, J.; Sossin, W. S. Biol. Reprod. 2004, 70,
277.
9. Panja, D.; Dagyte, G.; Bidinosti, M.; Wibrand, K.; Kristiansen, A. M.; Sonenberg,
N.; Bramham, C. R. J. Biol. Chem. 2009, 284, 31498.
10. Janzen, D. M.; Geballe, A. P. Cold Spring Harb. Symp. Quant. Biol. 2001, 66, 459.

7752
O. Sadovski et al. / Bioorg. Med. Chem. 18 (2010) 7746–7752
11. Novac, O.; Guenier, A. S.; Pelletier, J. Nucleic Acids Res. 2004, 32, 902.
12. Cencic, R.; Robert, F.; Pelletier, J. Methods Enzymol. 2007, 431, 269.
13. Moerke, N. J.; Aktas, H.; Chen, H.; Cantel, S.; Reibarkh, M. Y.; Fahmy, A.; Gross, J.
D.; Degterev, A.; Yuan, J.; Chorev, M.; Halperin, J. A.; Wagner, G. Cell 2007, 128,
257.
14. Sutton, M. A.; Ito, H. T.; Cressy, P.; Kempf, C.; Woo, J. C.; Schuman, E. M. Cell
2006, 125, 785.
15. Banko, J. L.; Hou, L.; Poulin, F.; Sonenberg, N.; Klann, E. J. Neurosci. 2006, 26,
2167.
16. Costa-Mattioli, M.; Gobert, D.; Harding, H.; Herdy, B.; Azzi, M.; Bruno, M.;
Bidinosti, M.; Ben Mamou, C.; Marcinkiewicz, E.; Yoshida, M.; Imataka, H.;
Cuello, A. C.; Seidah, N.; Sossin, W.; Lacaille, J. C.; Ron, D.; Nader, K.; Sonenberg,
N. Nature 2005, 436, 1166.
17. Nakashiba, T.; Young, J. Z.; McHugh, T. J.; Buhl, D. L.; Tonegawa, S. Science 2008,
319, 1260.
36. Pelletier, J.; Sonenberg, N. Nature 1988, 334, 320.
37. Jackson, R. J. Biochem. Soc. Trans. 2005, 33, 1231.
38. Filbin, M. E.; Kieft, J. S. Curr. Opin. Struct. Biol. 2009, 19, 267.
39. Beaudoin, M. E.; Poirel, V. J.; Krushel, L. A. Nucleic Acids Res. 2008, 36, 6835.
40. Audigier, S.; Guiramand, J.; Prado-Lourenco, L.; Conte, C.; Gonzalez-Herrera, I.
G.; Cohen-Solal, C.; Recasens, M.; Prats, A. C. RNA 2008, 14, 1852.
41. Lee, V. H.; Healy, T.; Fonseca, B. D.; Hayashi, A.; Proud, C. G. FEBS J. 2008, 275,
2185.
42. Mamane, Y.; Petroulakis, E.; LeBacquer, O.; Sonenberg, N. Oncogene 2006, 25,
6416.
43. Jung, M. Y.; Lorenz, L.; Richter, J. D. Mol. Cell. Biol. 2006, 26, 4277.
44. Macdonald, P. M. Curr. Biol. 2004, 14, R282.
45. Herbert, T. P.; Fahraeus, R.; Prescott, A.; Lane, D. P.; Proud, C. G. Curr. Biol. 2000,
10, 793.
46. Fletcher, C. M.; McGuire, A. M.; Gingras, A. C.; Li, H.; Matsuo, H.; Sonenberg, N.;
Wagner, G. Biochemistry 1998, 37, 9.
18. Ellis-Davies, G. C. Nat. Methods 2007, 4, 619.
19. Tanaka, J.; Horiike, Y.; Matsuzaki, M.; Miyazaki, T.; Ellis-Davies, G. C.; Kasai, H.
Science 2008, 319, 1683.
47. Salaun, P.; Boulben, S.; Mulner-Lorillon, O.; Belle, R.; Sonenberg, N.; Morales, J.;
Cormier, P. J. Cell Sci. 2005, 118, 1385.
20. Goard, M.; Aakalu, G.; Fedoryak, O. D.; Quinonez, C.; St Julien, J.; Poteet, S. J.;
Schuman, E. M.; Dore, T. M. Chem. Biol. 2005, 12, 685.
21. Shembekar, V. R.; Chen, Y.; Carpenter, B. K.; Hess, G. P. Biochemistry 2005, 44,
7107.
22. Wang, Q.; Dai, Z.; Cahill, S. M.; Blumenstein, M.; Lawrence, D. S. J. Am. Chem.
Soc. 2006, 128, 14016.
48. Marcotrigiano, J.; Gingras, A. C.; Sonenberg, N.; Burley, S. K. Mol. Cell 1999, 3,
707.
49. Tomoo, K.; Matsushita, Y.; Fujisaki, H.; Abiko, F.; Shen, X.; Taniguchi, T.;
Miyagawa, H.; Kitamura, K.; Miura, K.; Ishida, T. Biochim. Biophys. Acta 2005,
1753, 191.
50. Mahalakshmi, R.; Balaram, P. Methods Mol. Biol. 2006, 340, 71.
51. Hagen, V.; Dekowski, B.; Kotzur, N.; Lechler, R.; Wiesner, B.; Briand, B.;
Beyermann, M. Chemistry 2008, 14, 1621.
52. Momotake, A.; Lindegger, N.; Niggli, E.; Barsotti, R. J.; Ellis-Davies, G. C. Nat.
Methods 2006, 3, 35.
53. Gingras, A. C.; Raught, B.; Gygi, S. P.; Niedzwiecka, A.; Miron, M.; Burley, S. K.;
Polakiewicz, R. D.; Wyslouch-Cieszynska, A.; Aebersold, R.; Sonenberg, N. Genes
Dev. 2001, 15, 2852.
54. Hay, N.; Sonenberg, N. Genes Dev. 2004, 18, 1926.
55. Kapp, L. D.; Lorsch, J. R. Annu. Rev. Biochem. 2004, 73, 657.
56. Roux, P. P.; Shahbazian, D.; Vu, H.; Holz, M. K.; Cohen, M. S.; Taunton, J.;
Sonenberg, N.; Blenis, J. J. Biol. Chem. 2007, 282, 14056.
57. Proud, C. G. Biochem. J. 2007, 403, 217.
58. Hartford, C. M.; Ratain, M. J. Clin. Pharmacol. Ther. 2007, 82, 381.
59. Choi, J.; Chen, J.; Schreiber, S. L.; Clardy, J. Science 1996, 273, 239.
60. Van Duyne, G. D.; Standaert, R. F.; Karplus, P. A.; Schreiber, S. L.; Clardy, J. J. Mol.
Biol. 1993, 229, 105.
23. Fletscher, C. A. Org. Synth. 1953, 33, 65.
24. Wilcox, M.; Viola, R. W.; Johnson, K. W.; Billington, A. P.; Carpenter, B. K.;
Mccray, J. A.; Guzikowski, A. P.; Hess, G. P. J. Org. Chem. 1990, 55, 1585.
25. Luengo, J. I.; Yamashita, D. S.; Dunnington, D.; Beck, A. K.; Rozamus, L. W.; Yen,
H. K.; Bossard, M. J.; Levy, M. A.; Hand, A.; Newman-Tarr, T., et al Chem. Biol.
1995, 2, 471.
26. Svitkin, Y. V.; Sonenberg, N. Methods Enzymol. 2007, 429, 53.
27. Hansen, J. L.; Moore, P. B.; Steitz, T. A. J. Mol. Biol. 2003, 330, 1061.
28. Blaha, G.; Gurel, G.; Schroeder, S. J.; Moore, P. B.; Steitz, T. A. J. Mol. Biol. 2008,
379, 505.
29. Furuta, T. In Dynamic Studies in Biology: Phototriggers, Photoswitches and Caged
Biomolecules; Goeldner, M., Givens, R., Eds.; Wiley-VCH: Weinheim, 2005; p 29.
30. Shembekar, V. R.; Chen, Y.; Carpenter, B. K.; Hess, G. P. Biochemistry 2007, 46,
5479.
31. Hagen, V.; Frings, S.; Wiesner, B.; Helm, S.; Kaupp, U. B.; Bendig, J. Chembiochem
2003, 4, 434.
32. Lutz, C.; Otis, T. S.; DeSars, V.; Charpak, S.; DiGregorio, D. A.; Emiliani, V. Nat.
Methods 2008, 5, 821.
33. Olympus Website, 2009.
34. Gandin, V.; Miluzio, A.; Barbieri, A. M.; Beugnet, A.; Kiyokawa, H.; Marchisio, P.
C.; Biffo, S. Nature 2008, 455, 684.
61. Guadliana, M. A.; Truesdell, S. U.S.A. Patent, WO 9606847 1996, 21.
62. Gingras, A. C.; Raught, B.; Sonenberg, N. Prog. Mol. Subcell. Biol. 2001, 27, 143.
63. Raught, B.; Gingras, A. C.; Sonenberg, N. Proc. Natl. Acad. Sci. U.S.A. 2001, 98,
7037.
64. Wosnick, J. H.; Shoichet, M. S. Chem. Mater. 2008, 20, 55.