Development of a Photoactivatable Protein Phosphatase-1-Disrupting Peptide

Article abstract of DOI:10.1021/acs.joc.9b02548

We describe here the development of a photoreleasable version of a protein phosphatase-1 (PP1)-disrupting peptide (PDP-Nal) that triggers protein phosphatase-1 activity. PDP-Nal is a 23 mer that binds to PP1 through several interactions. It was photocaged

Full text of DOI:10.1021/acs.joc.9b02548

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Note
Development of a photo-activatable Protein Phosphatase-1 Disrupting Peptide
Malgorzata Trebacz, Yansong Wang, Leslie Makotta, Lars Henschke, and Maja Köhn
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.9b02548 • Publication Date (Web): 16 Dec 2019
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JOC Note
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Development of a photo-activatable Protein Phosphatase-1
Disrupting Peptide
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Malgorzata Trebacz†,‡,§,⊥, Yansong Wang§,⊥, Leslie Makotta§, Lars Henschke§,
Maja Köhn†,‡,§,
*
†Faculty of Biology, Institute of Biology III, University of Freiburg, Schänzlestraße 18, 79104,
Germany
‡Signalling Research Centres BIOSS and CIBSS, University of Freiburg, Freiburg, Germany.
§European Molecular Biology Laboratory, Genome Biology Unit, Meyerhofstraße 1, 69117
Heidelberg, Germany.
⊥These authors contributed equally.
*correspondence to: maja.koehn@bioss.uni-freiburg.de
ABSTRACT
We describe here the development of a photo-releasable version of a PP1-disrupting peptide
(PDP-Nal) that triggers protein phosphatase-1 (PP1) activity. PDP-Nal is a 23mer that binds to
PP1 through several interactions. It was photo-caged on a tyrosine residue, which required the
exchange of phenylalanine in PDP-Nal to tyrosine in order to disrupt the most important binding
interface. This PDP-caged can be light-controlled in live cells.
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Protein phosphatase-1 (PP1) is a ubiquitously expressed phospho-serine/threonine-specific
phosphatase that is involved in numerous signaling processes like cell division or insulin
secretion, making it a potential therapeutic target in diseases such as cancer or diabetes.1,2 PP1
counteracts more than 100 kinases through regulation by more than 200 regulatory interactors of
protein phosphatase one (RIPPOs), with which PP1 forms holoenzymes3,4. The most common
motif, which is involved in binding of RIPPOs, is the so-called RVxF-type motif (single amino
acid abbreviation code, x = any amino acid except proline). PP1 disrupting peptides (PDPs) are
currently the only selective modulators targeting the PP1 catalytic subunit, and they contain the
sequence RVTF as the RVxF-type motif.5 They disrupt a subset of PP1 holoenzymes, which
results in rapid dephosphorylation of nearby substrates. PDPs were designed based on the
sequence of the RIPPO nuclear inhibitor of PP1 (NIPP1)5. They are proteolytically stable in
cellular assays5,6 due to the incorporation of an unnatural amino acid in the sequence and because
of amidation and acetylation at the C- and N-terminus, respectively5,6. The polybasic sequence at
the N-terminus of PDPs was optimized to render these peptides cell-permeable5. Optimization of
the sequence has led to the creation of PDP-Nal (for the sequence see Table 1), which exhibits
improved stability and potency6. PDPs have been used to study PP1 substrates in the mitogen-
activated protein kinase (MAPK) pathway6, PP1’s role in calcium release7, and for activation of
PP1 to treat arrythmia and heart failure8,9. Since this rather general release of PP1 activity is
expected to impact several substrates and pathways, in order to study specific roles of PP1 there
is a need for a more controlled modulation of its activity. Photo-cleavable groups (cages) can be
used to block the interaction of a ligand to a protein. Irradiation with light releases the cage,
activating the ligand that can then bind to a protein and modulate its activity. Thus, photocaging
allows for temporal control by light irradiation of the ligand binding to a protein10-12. Nitrobenzyl
derivatives are the most commonly applied photolabile protecting group. They are simple to
synthesize, relatively stable, and easy to mimic with a benzyl group to screen for the optimal
position of a cage in a peptide. They can be cleaved with UV light at 365 nm. Coumarin
derivatives allow irradiation at longer wavelengths. Coumarins are also relatively simple to
synthesize and release the substrate rapidly after visible light and two-photon irradiation.
Moreover, their fluorescent properties can be used for tracking molecules inside the cells. Yet,
handling them under ambient light can cause partial uncaging. Both coumarin-based and
nitrobenzyl-based caging groups have been applied in cells11-13. Cyanine-based caging groups
exhibit a similar fluorescent character and are uncaged with near-infrared light. However, the
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complexity and size of the molecules can be a disadvantage11,13. Applications of the caging
approach for peptides have been described previously. For example, nitrobenzyl-derived cages
have been used to study kinase activity14,15, have been applied in hydrogels for caging RGDS
peptide in order to create a non-adhesive surface with patches containing the uncaged RGDS
peptide where cells can bind16, and for making a cell penetrating peptide light sensitive in order
to enhance drug delivery to tumor cells17. Here, we report the development of a caged PDP for
photo-control of PP1 activity. Due to the fact that most studies with caged peptides were so far
done with the nitrobenzyl (Nb) cage or a derivative14-17, and because the Nb group is relatively
stable under ambient light allowing simple handling, we focused here on the Nb group as cage.
PDPs are 23mer peptides that have several interaction points with PP15, which could be
problematic when designing a caged molecule. A single cage would only interrupt one area of
interaction, and more cages might be required to efficiently disrupt the interaction between these
long peptides and PP1. However, exchange of the RVTF sequence in the PDPs to RATA (this
version is called “PDPm”) caused the PDPs to not bind PP1 anymore5, which provided the
possibility that introducing one cage within this motif could be sufficient to disrupt the
interaction PP1 with the PDP. Previously, serine was caged with a 4,5-dimethoxy-2-nitrobenzyl
group (DMNb) and applied successfully in photo-release studies with peptides14,15. Therefore,
Fmoc-Ser(DMNb)-OH was synthesized according to literature procedures15 and incorporated into
PDP-Nal, replacing the threonine of the RVTF sequence (PDP-RVS(DMNb)F). Then, the efficacy
of PDP-RVS(DMNb)F to disrupt the PP1:Inhibitor 2 (I2) holoenzyme was tested, which would
lead to free PP1 catalytic subunit that dephosphorylates an unnatural substrate (6,8-difluoro-4-
methylumbelliferyl phosphate, DiFMUP) measured by a fluorescence change5,6. However, this
modification did not significantly lower the potency of the PDP (data not shown), which was
likely due to the fact that the caged serine was placed at the flexible position of the RVxF-type
motif.
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Due to the known importance of valine and phenylalanine of the RVTF sequence for binding of
PDPs to PP1 as shown by the inactive RATA version5, we next interrogated if caging of F and/or
V would result in loss of potency. As caging of valine and phenylalanine would only be possible
upon introduction of an alcohol, they were replaced by the structurally similar threonine and
tyrosine. Because testing of different positions for the cage was required, for synthetic ease we
used commercially available benzylated tyrosine and threonine (Table 1), mimicking the Nb
group. When monitoring the potency of PDP-Nal and PDP-RVTY peptide only a slight change
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was observed, with the EC50 changing from 144 nM to 194 nM, respectively. However, replacing
valine with threonine in the PDP-RVTY peptide to PDP-RTTY led to an about 40-times higher
EC50, which excluded this change from further use. The benzylated PDP-RVTY(Bn) peptide was
more than 8-times less potent that the non-benzylated PDP-RVTY (Table 1, Figure 1). This was
judged to be a large enough difference to develop the caged PDP-RVTY(Nb) peptide.
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Table 1. Activity of (nitro-) benzylated PDP-Nal derived peptides. Amino acids crucial for the binding to
the pocket of PP1 carried a benzyl group (Bn) on the hydroxyl group on the side chain of the amino acid
in order to mimic the Nb cage. EC50 values were determined by a dephosphorylation assay using
recombinant PP1 and I2 with DiFMUP as substrate. The RVxF motif is marked in bold, and changes are
marked in red. Error for PDP-Nal, PDP-RVTY, PDP-RVTY(Bn): standard error of the mean of two
independent triplicate measurements (n = 6); error for PDP-RTTY: standard deviation from the curve taken
from one triplicate measurement (n = 3). PDP-caged was measured in two independent triplicate
measurements, PDP-RT(Bn)TY(Bn) in one triplicate measurement.
Peptide
Sequence
EC50 [µM]
0.144 ± 0.040
0.194 ± 0.005
1.63 ± 0.04
> 1.00
PDP-Nal
RRKRPKRKRKNARVTFNalEAAEII
RRKRPKRKRKNARVTYNalEAAEII
RRKRPKRKRKNARVTY(Bn)NalEAAEII
RRKRPKRKRKNARVTY(Nb)NalEAAEII
RRKRPKRKRKNARTTYNalEAAEII
RRKRPKRKRKNART(Bn)TY(Bn)NalEAAEII
PDP-RVTY
PDP-RVTY(Bn)
PDP-caged
PDP-RTTY
7.59 ± 0.89
> 2.30
PDP-RT(Bn)TY(Bn)
150
100
50
PDP-RVTY
PDP-RVTY(Bn)
PDP-Nal
0
-8
-7
-6
-5
-4
-3
log(conc)
-50
Figure 1. Difference of the kinetics of PDP-Nal (red triangles), PDP-RVTY (black dots) and PDP-
RVTY(Bn) (blue squares). The area between the curves illustrates the activity difference between the
peptides. The dephosphorylation assay was carried out with recombinant PP1 and I2 using DiFMUP as
substrate. The error is the standard deviation of two independent triplicate measurements (n = 6).
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First, the building block Fmoc-Tyr(Nb)-OH 1 (Scheme 1) needed to be synthesized. The
synthesis of 1 had been described previously18-20. We adapted the synthesis procedure starting
with Tyr-OtBu 2 for synthetic simplicity and reported good yields20, but optimized conditions for
scaling up to several grams (Scheme 1). Firstly, the free amine was protected using Boc-
anhydride. For the nitrobenzylation step, compound 3 was reacted with nitrobenzyl bromide and
Cs2CO3 in acetonitrile (ACN), which gave compound 4 in an excellent yield of 96% at the gram
scale. Deprotection of the tBu-ester and the Boc-group was achieved with neat TFA, and after
subsequent protection of the amine using Fmoc-OSu Fmoc-Tyr(Nb)-OH 1 was obtained in
almost quantitative yield (Scheme 1). This building block was then incorporated into the PDP
using standard Fmoc-solid phase synthesis, resulting in the peptide PDP-RVTY(Nb), which we
called PDP-caged (see the sequence in Table 1).
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Scheme 1. Synthetis of Fmoc-Tyr(Nb)-OH 1. ACN: acetonitrile; Boc-ON: 2-(tert-
butoxycarbonyloxyimino)-2-phenylacetonitrile; d: day(s); DMF: N,N-dimethyl formamide; Fmoc-OSu: 9-
fluorenylmethyl-succinimidyl carbonate; o.n.: overnight; RT: room temperature; TEA: triethylamine; tBu:
tert-buryl; TFA: trifluoroacetic acid; THF: tetrahydrofuran.
When testing PDP-caged in the activity assay we noted large error bars at higher concentrations
and varying results (data not shown). It is possible that this was due to uncaging during the light
exposure in the plate reader. Therefore, we were not able to determine an exact EC50.
Nevertheless, we assumed that this peptide would behave similarly to the PDP-RVTY(Bn)
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peptide, and would disrupt the PP1:I2 interaction with a lesser potency than PDP-RVTY, and
therefore carried on with PDP-caged.
Next, we characterized the stability of PDP-caged. After 24 hours of exposure under ambient
light as well as in the dark, we did not notice any degradation (Supporting Figure S1). This
confirms that the nitrobenzyl group is cleaved only with UV light, which makes it easier to
handle PDP-caged under ambient light. We then tested if PDP-caged could be uncaged in vitro.
To this end, the peptide was exposed to light at 365 nm for a certain amount of time, and the
product formation to PDP-RVTY was monitored using HPLC. After 3 minutes the product had
formed to 94%, and after 5 minutes the caged peptide was visible only in a negligible amount
(Figure 2A). Finally, we asked whether a difference in activity of PDP-caged and PDP-RVTY
could be detected in a cell assay, and whether the activity of PDP-caged could be released
through uncaging. To address this, we adapted the previously established dephosphorylation of
the phosphorylated threonine 3 on histone H3 (H3pT3) upon PDP treatment5,6 to uncaging
conditions. H3pT3 is a substrate of PP1 that is phosphorylated at the beginning of mitosis. The
PP1:Repoman holoenzyme dephosphorylates H3pT3 at the end of mitosis, enabling mitotic
exit21,22. PDP3 and PDP-Nal treatment both led to the dephosphorylation of H3pT3 during
mitotic arrest5,6. Here, U2OS cells were synchronized in mitotic arrest and then treated with PDP-
RVTY (positive control), PDPm-Nal (negative control containing the RATA sequence instead of
RVTF)6, and PDP-caged, or left untreated (Figure 2B). Treatment occurred with 40 µM final
concentration of the peptides for 15 minutes, then the cells were washed and afterwards irradiated
for 5 minutes with UV light (λ=365 nm). Following another 10 minutes of incubation, the cells
were fixed and stained with antibodies against total histone H3 and H3pT3. For the experimental
read-out we used the so-called in-cell Western method, which is similar to
immunocytochemistry. The analysis showed that phosphorylation of H3pT3 was enhanced in
arrested cells. As expected, treatment with PDP-RVTY led to a reduction of the phosphorylation
level, whereas the addition of the negative control PDPm-Nal did not reduce the phosphorylation
on H3pT3. Importantly, PDP-caged treatment only reduced the H3pT3 phosphorylation level
after irradiation with UV light to a similar level as the positive control PDP-RVTY did. All other
conditions were inert to UV light treatment, showing that the UV light-dependent activation is
specific to PDP-caged. Thus, PDP-RVTY shows a similar activity like PDP-Nal6 in this cellular
assay, and the activity of PDP-caged can be controlled using UV light irradiation in live cells.
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Figure 2. Uncaging experiments of PDP-caged in vitro and in cells. A) In vitro photo-cleavage of PDP-
caged. HPLC traces for the peptide exposed to λ = 365 nm light for 1, 3 or 5 minutes are shown. B)
Dephosphorylation of H3pT3 after treatment with 40 µM PDPs, with or without UV irradiation (+ UV/ -
UV). U2OS cells were synchronized in mitotic arrest. In-cell Western analysis was used to detect
phosphorylation levels. An inactive mutant of PDP-Nal containing a RATA sequence instead of RVTF
was used as a negative control (PDPm-Nal). Red: total histone 3; green: H3pT3. The respective signal
intensity of H3pT3/total histone 3 was compared to “mitotic arrest, no peptide”. For quantification three
independent experiments were used (see Supporting Figure S2 for the other two replicates). Results were
analyzed with GraphPad Prism 6. Statistical analysis is based on 2way ANOVA and Bonferroni multiple
comparison test. (nsP > 0.05, ****P < 0.0001; n = 3).
In summary, we developed here PDP-caged as a light-controllable modulator of PP1 activity in
live cells. While PDP-caged contains 23 amino acids, of which several interact with PP15,
targeting phenylalanine of the RVxF-type binding motif for replacement with a caged tyrosine
was sufficient to disrupt the interaction between the PDP and PP1. PDP-caged allows releasing
PP1 activity in a timely precise manner through UV light irradiation at specific time points. A
limitation of the Nb group when used in biological systems is that UV light irradiation is
cytotoxic and has a limited penetration ability through tissue11,12. In the proof-of-concept settings
used here, the wavelength (365 nm) is close to visible light and the irradiation time is short.
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Therefore, it is not expected that the cytotoxicity would cause problems in these settings, as seen
in the controls. For more complex applications however, red-shifted derivatives of Nb or
Coumarin and possibly two-photon excitation might have to be applied11,12. We expect that this
compound will prove to be a useful tool in time-sensitive studies of PP1 activity in cellular
processes, such as in different stages of mitosis23,24 and in regulation of calcium homeostasis in
the heart25.
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EXPERIMENTAL SECTION
General Information. Tyr(OtBu) was bought from Bachem, Switzerland. DIPEA (N,N-
diisopropylethylamine), Fmoc-Thr(Bn)-OH and Fmoc-Tyr(Bn)-OH were purchased from ABCR.
HOBt (N-hydroxybenzotriazole) was bought from MOLEKULA. Triisopropylsilane and cesium
carbonate were from Alfa Aesar. Dithiothreitol (DTT), imidazole, MnCl2, bovine serum albumin
(BSA), pyridine, di-tertbutyl dicarbonate (Boc2O), triethylamine (TEA), 2-nitrobenzyl bromide,
sodium hydroxide, acetic anhydride (Ac2O) and d-chloroform were from Sigma. Dioxane, DCM
(dichloromethane), Cy (cyclohexane), EA (ethyl acetate), methanol and sodium sulphate, TFA
(trifluoroacetic acid), piperdine and ACN (acetonitrile) were from Roth. Other Fmoc-amino
acids, 9-fluorenylmethyl-succinimidyl carbonate (Fmoc-OSu) and HBTU (O-(benzotriazol-1-yl)-
N,N,N’,N’-tetramethyluronium hexafluorophosphate) were from Novabiochem. Acetic anhydride
and DMF (N,N-dimethylformamide) were from Merck Millipore. DiFMUP (6,8-difluoro-4-
methylumbelliferyl phosphate) was purchased from Life Technologies.
1H and 13C Nuclear Magnetic Resonance: All spectra were recorded on a 400 MHz Bruker
Avance DPX and a 500 MHz Bruker DRX. LC-MS analysis and HPLC purification: LC-MS
analysis and HPLC peptide purifications were carried out on a Shimadzu High Performance
Liquid Chromatograph coupled with a Mass Spectrometer using a UV-Vis Photodiode array
detector SPD-M20A Prominence unless otherwise noted. ESI-MS of PDP-caged was performed
on an Agilent Technologies 1260 Infinity I/II coupled to 6120 Quadrupole LC/MS. The solvent
used for all analytical and semi-preparative runs was a mixture of H2O containing 0.05% TFA
and ACN containing 0.05% TFA. RP-HPLC analytical runs were realized with a Macherey Nagel
C18 EC 250/4.0 NUCLEODUR 100-5 C18 ec column and a pump rate of 1.5 mL/min. RP-HPLC
semi-preparative separations were performed with a Macherey Nagel C18 VP 250/10
NUCLEODUR 110-5 C18 ec column in gradients ranging between 31 and 43% of ACN over 30
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minutes using a flow rate of 5 mL/min. Semi-preparative HPLC of PDP-RVTY was run on an
Agilent Technologies 1260 I/II Infinity system using gradient of ACN 10 – 70% over 30 minutes.
Flash chromatography: Flash chromatography purifications were performed using silica gel 60
(Roth), grain size 0.04-0.063 mm, 230-400 mesh ASTM. MALDI-TOF: The mass of all peptides
was recorded on MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization coupled with Time
Of Flight) with the MALDI micro MX mass spectrometer (Waters, Manchester, UK) equipped
with a reflection analyzer used in positive ion mode with activated delayed mode. The matrix was
prepared by weighing out 10 to 20 mg of α-cyano-4-hydroxycinnamic acid (CHCA) in an
Eppendorf then adding 1.0 mL of a 50:50 H2O/ACN with 0.1% TFA final concentration.
Peptide Synthesis. All peptides were synthesized using solid phase peptide synthesis on the
automated liquid handling system Syro I from Multisyntech using the Fmoc protection strategy5,6.
Peptide elongation was carried out on the Rink Amide Resin, using HBTU/HOBT double
coupling (5 eq. amino acid, 5 eq. HBTU, 2 eq. HOBt and 10 eq. DIPEA in DMF) for 40 minutes
with 15 seconds vortexing every 4 minutes. The Fmoc deprotection was carried out using 20%
piperidine. The capping step was done twice after every double coupling with 10 %
Ac2O/pyridine mixture for 5 minutes with 10 seconds vortexing every minute. The cleavage
cocktail contained 95% TFA, 2.5% TIS and 2.5% water. After cleavage the peptides were
precipitated in ice-cold diethylether, HPLC purified and analysed by MALDI (Supporting Table
S1). The synthesis of PDP-Nal was described before6.
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Preparation of (2S)-2-tert-butyl carbamate-3-(4-hydroxyphenyl)propanoic acid tert-butyl
ester: Boc-Tyr-OtBu 3. Tyr-OtBu 2 (2.00 g, 8.42 mmol), Boc2O (1.84 g, 8.42 mmol) and TEA
(114 µl, 0,84 mmol) were stirred in 100 ml THF at RT overnight. THF was removed in vacuo.
The mixture dissolved in EA, washed three times with water, and dried over sodium sulphate. It
was purified using the gradient 20%-40% EA in Cy. Yield: 2.84 g (8.42 mmol), 100%, white
solid. 1H NMR (400 MHz, DMSO-d6) δ 9.21 (s, 1H), δ 7.09 (d, J = 7.92 Hz, 1H), δ 7.00 (d, J =
8.36 Hz, 2H), δ 6.65 (d, J = 8.32 Hz, 2H), δ 3.91 (dt, J = 6.22, 8.38 Hz, 1H), δ 2.83 – 2.66 (m,
2H), δ 1.36 – 1.32 (m, 18H). These data are in agreement with reference 20.
Preparation of (2S)-2-tert-butyl carbamate-3-{4-[2-nitrophenyl)methoxy]phenyl} propanoic
acid tert-butyl ester: Boc-Tyr(Nb)-OtBu 4. Boc-Tyr-OtBu 3 (2.76 g, 8.19 mmol), 2-nitrobenzyl
bromide (1.77 g, 8.19 mmol) and Cs2CO3 (2.93 g, 3.00 mmol) were dissolved in ACN (30 ml).
The reaction was stirred for three days at room temperature. The reaction mixture was filtered,
the solvent was reduced in vacuo, and the compound was purified using flash chromatography
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(2% EA in Cy to 10% EA in Cy). Yield: 3.70 g (7.83 mmol), 96%, orange oil. Rf = 0.45 (Cy/EA
= 12/1); 1H NMR (CDCl3, 400 MHz) δ 8.16 (d, J = 8.16 Hz, 1H), δ 7.87 (d, J = 7.80 Hz, 1H), δ
7.66 (t, J = 7.56 Hz, 1H), δ 7.47 (t, J = 7.76 Hz, 1H), δ 7.10 (d, J = 8.48 Hz, 2H), δ 6.90 (d, J =
8.52 Hz, 2H), δ 5.45 (s, 2H) δ 5.00 (d, J = 8.00 Hz, 1H), δ 4.44 – 4.37 (m, 1H), δ 3.02 – 2.96 (m,
2H), δ 1.41 (s, 9H), δ 1.40 (s, 9H). 13C{1H} NMR (125 MHz) δ 171.0, δ 157.0, δ 155.1, δ 146.9,
δ 134.0, δ 130.7, δ 129.4, δ 128.5, δ 128.3, δ 125.0, δ 114.8, δ 82.0, δ 79.6, δ 66.8, δ 54.9, δ 37.6,
δ 28.3, δ 28.0. MS m/z: [M + Na]+ Calcd for C25H32N2O7 495.21; Found [M + Na]+ 495.20. These
data are in agreement with reference 20.
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Preparation of (2S)-2-amino-3-{4-[2-nitrophenyl)methoxy]phenyl}propanoic acid: Tyr(Nb)-
OH 5. Boc-Tyr(Nb)-OtBu 4 (3.70 g, 7.83 mmol) was dissolved in TFA (75 ml). The reaction was
stirred for 1 h at room temperature and was monitored by thin layer chromatography (10% EA in
Cy). TFA was co-evaporated with toluene in vacuo and the compound was dried under high
vacuum. Yield: 2.35 g (7.43 mmol), 95%, orange-brown solid. The product was used further
without purification and analytical data collection 20.
Preparation
of
(2S)-2-fluorenylmethyloxycarbamate-3-{4-[2-nitrophenyl)methoxy]
phenyl}propanoic acid: Fmoc-Tyr(Nb)-OH 1. Tyr(Nb)-OH 5 (7.43 mmol), Fmoc-OSu (3.01 g,
8.92 mmol) and Et3N (2.55 ml, 18.6 mmol) were dissolved in ACN:H2O (7:4, 33 ml), stirred
overnight at RT, dried in vacuo and purified using flash chromatography (DCM only, then
DCM/MeOH = 12/1 and 9/1). Yield: 3.98 g (7.39 mmol), 97%, orange-brown solid. Rf = 0.47; 1H
NMR (CDCl3, 400 MHz) δ 8.16 (d, J = 8.1 Hz, 1H), δ 7.86 (d, J = 7.8 Hz, 1H), δ 7.75 (d, J = 7.5
Hz, 2H), δ 7.65 (t, J = 7.54 Hz, 1H), δ 7.56 – 7.52 (m, 2H), δ 7.47 (t, J = 7.8 Hz, 1H), δ 7.39 (t, J
= 7.4 Hz, 2H), δ 7.30 (t, J = 7.4 Hz, 2H), δ 7.07 (d, J = 8.2 Hz, 2H), δ 6.90 (d, J = 8.3 Hz, 2H), δ
5.43 (s, 2H), δ 5.22 (d, J = 7.9 Hz, 1H), δ 4.68 – 4.63 (m, 1H), δ 4.46 (dd, J = 10.4, 7.0 Hz, 1H), δ
4.36 (dd, J = 10.3, 7.2 Hz, 1H), δ 4.19 (t, J = 6.8 Hz, 1H), δ 3.16 (dd, J = 14.3, 4.9 Hz, 1H), δ
13
3.07 (dd, J = 13.9, 5.8 Hz, 1H). C{1H} NMR (100 MHz) δ 175.5, δ 157.4, δ 155.8, δ 146.9, δ
143.7, δ 143.7, δ 141.3, δ 134.0, δ 133.8, δ 130.6, δ 128.5, δ 128.3, δ128.3, δ 127.8, δ 127.1, δ
125.0, δ 120.0, δ 115.1, δ77.2 δ 67.0, δ 66.8, δ 54.6, δ 47.2, δ 36.9. MS m/z: [M + Na]+ Calcd for
C31H26N2O7 561.16, found [M + Na]+ 561.15. These data are in agreement with reference 20.
Protein expression and purification. Recombinant PP1 and I2 were obtained as described in
Wang et al., 20196.
Activity assay6. Recombinant PP1 protein (25 pM final assaying concentration), I2 phosphatase
inhibitor (1 nM final assaying concentration), and PDP at various concentrations were incubated
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in assay buffer (25 mM imidazole, 50 mM NaCl, BSA 0.1 mg/ml, 1 mM DTT, 0.3 mM MnCl2,
pH 7.4) for approximately 20 min at 25°C followed by addition DiFMUP to reach a final
concentration of 135 µM (Km value). The dephosphorylation of DiFMUP to the fluorescent
product was monitored for 40 min on a TECAN Infinite M1000 PRO fluorescence microplate
reader with excitation at 358 nm and emission at 452 nm. All experiments were performed two
times in triplicates, with the exception of peptides PDP-RTTY and PDP-RT(Bn)TY(Bn), that were
measured in one triplicate. After baseline normalization (assay in the absence of PDP), the
reaction rates were plotted versus the log of the inhibitor concentrations and the EC50 values were
obtained by fitting the curves using the one-site competition model of GraphPad Prism
(GraphPad Software, Version 6).
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In vitro uncaging reaction and PDP-caged stability. 100 µM PDP (10% DMSO in H2O) was
exposed to UV light using the UVP Crosslinker 365 nm (CL-1000L λ=365 nm) with the light
dose 120 mJ per cm2 or ambient light either for 3 min or for 24 hours. t = 0 control sample was
kept in the dark for the time of the experiment. Samples were acquired on the Agilent HPLC
system using 10-90% gradient of ACN in H2O. Data were analysed using the OpenLAB CDS
Software (Agilent).
Cell Line6. U2OS human female osteosarcoma cells were cultured at 37°C in a humidified
incubator under an atmosphere of 5% CO2. The cells were grown in Dulbecco’s modified Eagle’s
medium (DMEM) containing 1 g/L glucose, 10% (v/v) fetal bovine serum (FBS), 1% (v/v)
penicillin/streptomycin and 2 mM L-glutamine.
In-cell Western assay6. Garnier bio-one CELLSTAR 12-well plates were coated with 0.1 mg/ml
poly-D-lysine for 5 minutes, washed with PBS and led dry for 2 hours. U2OS cells were plated in
1:100 dilution one day before starting the experiment. Culture media containing 2 mM thymidine
was added and after 24 h exchanged to the media without thymidine for 2 hours. Cells were
treated with nocodazole for 15 h in the concentration of 100 ng/ml to allow the mitotic arrest. All
peptides were diluted in the imaging buffer (115 mM NaCl, 1.2 mM CaCl2, 1.2 mM MgCl2, 1.2
mM K2HPO4, 20 mM HEPES and 2 g/l D-glucose) containing 100 ng/ml nocodazole to reach the
final concentration of 40 µM and were added to the wells for 15 min. Cells were then washed
with PBS and supplemented with fresh imaging buffer containing nocodazole. 5 min uncaging
was conducted using the UVP Crosslinker 365 nm (CL-1000L λ=365 nm) with a light dose of
120 mJ per cm2. Plates were placed in the incubator for another 10 min to allow PP1 activation.
Cells were fixed with ice cold MeOH for 10 min, washed with PBS and blocked for 1.5 h with
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Odyssey Blocking Buffer (PBS) (LI-COR). Primary antibodies against phosphorylated histone 3
on threonine 3 (H3pT3) (Millipore, cat. no. 07-424) and total histone 3 (Active Motif, cat no.
39763) in dilution 1:500 were added and incubated for 2 h. Wells were washed with In-Cell
Western Washing Buffer (0.5% Triton-X in PBS) 3 times for 5 min. Secondary antibodies
staining was performed using IRDye680RD Goat anti-Mouse IgG (H+L) (LI-COR, cat. no. 925-
68070) and IRDye800CW Goat anti-Rabbit IgG (H+L) (LI-COR, cat. no. 925-32211) in the
dilution of 1:1000 and incubated for 1 h. Wells were washed with the in-cell Western Washing
Buffer (0.5% Triton-X in PBS) 3 times for 5 min. PBS was added and plates were acquired on
the LI-COR Odyssey CLX Imager. Data analysis was carried out using the software Image
Studio Lite (LI-COR).
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI:
XXX
Supporting Figure S1, S2, Supporting Table S1, NMR spectra, analytical data of compounds and
peptides (pdf).
NOTES
The authors declare to have no competing financial interest.
ACKNOWLEDGEMENTS
M.K. acknowledges financial support from the European Research Council through ERC starting
grant #336567 and from the German Science Foundation (DFG) through the German Excellence
Strategy (BIOSS – EXC-294 and CIBSS – EXC-2189 – Project ID 390939984). Gefördert durch
die Deutsche Forschungsgemeinschaft (DFG) im Rahmen der Exzellenzstrategie des Bundes und
der Länder – EXC 2189 – Projekt-nummer 390939984.
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Table of Contents entry
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