19F NMR monitoring of the eukaryotic 20S proteasome chymotrypsin-like activity: An investigative tool for studying allosteric regulation

Article abstract of DOI:10.1039/c4ob00962b

The proteasome displays three distinct proteolytic activities. Currently, proteasome inhibitors are evaluated using specific fluorescent substrates for each of the individual active sites. However, the photophysical properties of the commonly used fluorophores are similar and thus, the simultaneous monitoring of the three proteolytic activities is not possible. We have developed a bimodal fluorescent fluorinated substrate as a novel tool to study the chymotrypsin-like (ChT-L) proteolytic activity and its regulation by inhibitors and by substrates of trypsin-like (T-L) and caspase-like sites (PA). We demonstrate that this substrate is reliable to evaluate the ChT-L inhibitory activity of new molecules either by fluorescence or ¹⁹F NMR spectroscopy. We have found that the ChT-L activity is dramatically reduced in the presence of T-L and PA substrates. This work provides a proof of concept that the fluorinated substrate enables investigation of the allosteric regulation of the ChT-L activity. This journal is

Full text of DOI:10.1039/c4ob00962b

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Biomolecular Chemistry
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19F NMR monitoring of the eukaryotic 20S
proteasome chymotrypsin-like activity: an
investigative tool for studying allosteric
regulation†‡
Cite this: Org. Biomol. Chem., 2014,
12, 4576
Received 9th April 2014,
Accepted 13th May 2014
DOI: 10.1039/c4ob00962b
M. Keita,a J. Kaffy,*a C. Troufflard,b E. Morvan,b B. Croussea and S. Ongeri*a
www.rsc.org/obc
The proteasome displays three distinct proteolytic activities. and caspase-like or post-acid (PA) activities corresponding
Currently, proteasome inhibitors are evaluated using specific fluor- to the peptide cleavage on the carboxyl side of hydrophobic,
escent substrates for each of the individual active sites. However, basic and acidic amino acid residues, respectively.2 Fluo-
the photophysical properties of the commonly used fluorophores rescent substrates for each of the individual active sites have
are similar and thus, the simultaneous monitoring of the three been designed and are commonly used to evaluate the inhibi-
proteolytic activities is not possible. We have developed a bimodal tory activity and the specificity of the proteasome inhibitors
fluorescent fluorinated substrate as a novel tool to study the towards the three active sites. These substrates are peptides
chymotrypsin-like (ChT-L) proteolytic activity and its regulation by with a fluorophore capped at the C-terminus which gives a
inhibitors and by substrates of trypsin-like (T-L) and caspase-like fluorescent response upon hydrolysis. However, commonly
sites (PA). We demonstrate that this substrate is reliable to evaluate used fluorophores, 7-amino-4-methyl coumarin (AMC) or
the ChT-L inhibitory activity of new molecules either by fluo- β-naphthylamide (β-NA), possess very similar photophysical
rescence or 19F NMR spectroscopy. We have found that the ChT-L properties (the wavelengths of excitation and emission are 360
activity is dramatically reduced in the presence of T-L and PA sub- and 460 nm for AMC and 340 and 405 nm for β-NA). AMC is
strates. This work provides a proof of concept that the fluorinated usually preferred because β-NA is much less fluorescent than
substrate enables investigation of the allosteric regulation of the AMC and it is carcinogenic.3b Thus, the use of these fluoro-
ChT-L activity.
phores is a major lock for the simultaneous monitoring of the
three proteolytic activities.3,4 The current knowledge of the
catalytic mechanism of proteasome is also essentially based
on these fluorescent substrates. However, some previous
studies suggest that an active site can allosterically activate or
inhibit the others.3–6 In particular, the ChT-L activity can be
modulated in the presence of T-L or PA substrates (both sub-
strates might induce a change in the activity and the confor-
mation of 20S proteasome resulting from the binding of T-L
and PA substrates at their specific site or at another site).4,5,7
The importance and significance of allostery in molecular
therapies in general and in particular in the proteasome field
must be more carefully examined and validated. However, in
the absence of investigative tools, the study of allostery
remains challenging.8 A better understanding of proteasome
regulation is expected to enable the development of more
selective therapeutic agents. In particular, since inhibition of
ChT-L activity has been the focus of antineoplastic agents,
there is a crucial need for a simple and reliable method to
study ChT-L proteolytic activity and to evaluate potential
ChT-L proteasome inhibitors in the presence of one or both
substrates with T-L and PA activities. The finding of
fluorescent probes with a distinct and univocal wavelength has
been scarcely described and is challenging.4,5
Introduction
Recognized as the central enzyme of non-lysosomal protein
degradation, the proteasome represents a main therapeutic
target as it regulates a vast array of pathways during cell life
and death, including cell-cycle progression. Consequently,
selective inhibitors of the proteasome are promising drug can-
didates for treating diseases such as cancer. Bortezomib and
Carfilzomib are clinically used for the treatment of incurable
multiple myeloma (both compounds) and mantle lymphoma
(Bortezomib).1 Three distinct proteasome proteolytic activities
are described as chymotrypsin-like (ChT-L), trypsin-like (T-L)
aMolécules Fluorées et Chimie Médicinale, BioCIS UMR-CNRS 8076, LabEx LERMIT,
Université Paris-Sud, Faculté de Pharmacie, 5 rue Jean-Baptiste Clément, 92296
Châtenay-Malabry Cedex, France. E-mail: sandrine.ongeri@u-psud.fr,
julia.kaffy@u-psud.fr
bNMR Service, BioCIS UMR-CNRS 8076, Université Paris-Sud, Faculté de Pharmacie,
5 rue Jean-Baptiste Clément, 92296 Châtenay-Malabry Cedex, France
†Dedicated to Dr Danièle Bonnet-Delpon.
‡Electronic supplementary information (ESI) available: Inhibitors synthesis and
experimental details. See DOI: 10.1039/c4ob00962b
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For this purpose, we developed a new fluorine-containing
substrate with ChT-L proteasome activity to evaluate univocally
ChT-L proteasome inhibitors by 19F NMR in the presence of
PA or T-L substrates. The powerful and attractive role of the
fluorine atom as a tag lies in the high sensitivity of 19F NMR
spectroscopy, the 100% natural abundance of the isotope 19F
and the lack of background signal (unlike 1H and 13C
nuclei).9,10 The FABS (Fluorine Atoms for Biochemical Screen-
ing) method uses 19F NMR spectroscopy to detect the initial
and enzymatically modified substrates. It has been used to
screen inhibitors of a large panel of enzymes and to explore
protein functions.9,10 Moreover, 19F NMR-based detection
methods are known to be less subject to artefacts than
fluorescence spectroscopy.9,10
Scheme 1 Synthesis of Suc-LLVY-AFC. (a) Boc-Tyr-OH, POCl3, −15 °C,
15 min, 46%; (b) 4 M HCl, dioxane, rt, 2 h, quantitative; (c) Boc-Val-OH,
HOBt, EDCI, DIPEA, DMF, rt, 16 h, 64%; (d) Boc-Leu-OH, HOBt, EDCI,
DIPEA, DMF, rt, 16 h, 46%; (e) Boc-Leu-OH, HOBt, EDCI, DIPEA, DMF, rt,
16 h, 60%; (f) succinic anhydride, DIPEA, DMF, rt, 16 h, 56%.
Results and discussion
Design of the new fluorine-containing substrate with ChT-L
proteasome activity
Requirements for our new substrate were as follows: a CF3
group was introduced in order to enhance the NMR signal
intensity (as demonstrated by Dalvit et al. with the 3-FABS
method9,11); the CF3 group was placed on a fluorescent core in
order to offer a multimodal probe; the hydrolysis of the sub-
strate must liberate the fluorinated fluorescent moiety with a
19F chemical shift different from that of the substrate; a quan-
titative monitoring of the hydrolysis is essential to determine
molecule inhibitory activities (IC50).
In order to fulfill all these criteria, the fluorophore AMC of
Suc-LLVY-AMC, which is the commonly used specific substrate
of the ChT-L site, was replaced by the fluorophore containing
a CF3-probe, 7-amino-4-trifluoromethylcoumarin (AFC, see
Fig. 1). The use of such a multimodal probe has been very
seldom reported; however, multimodal probes enable comp-
lementary experiments and can be further used in cellular
systems and living bodies to perform fluorescence measure-
ment or 19F magnetic resonance imaging (MRI).12–14 In par-
ticular, to our knowledge, AFC has only been used as a probe
to detect an enzymatic process by fluorescence and 19F NMR
but not to quantify the enzymatic reaction and consequently
evaluate the inhibitory activity of compounds on the enzyme
activity.12,13
Fig. 2 19F NMR spectrum across 9h27 depicting the production of AFC
(free amine) from Suc-LLVY-AFC (substrate, 30 µM) in the presence of
rabbit 20S proteasome (6 nM).
Enzymatic activity of the rabbit 20S proteasome on
Suc-LLVY-AFC
The enzymatic activity of the rabbit 20S proteasome on Suc-
LLVY-AFC was controlled by the concomitant decrease of the
19F NMR substrate signal and increase of the 19F NMR AFC
signal (Fig. 2). Notably, although the fluoro group is far from
the cleavage site, the released amine AFC displays a distinct
chemical shift to Suc-LLVY-AFC (−64.09 and −64.34 ppm
respectively). The Suc-LLVY-AFC signal disappeared totally in
favour of the AFC signal in less than 10 hours (Fig. 2). The
found Km value of Suc-LLVY-AFC (20 μM, determined by
fluorescence, see ESI‡) was similar to that of Suc-LLVY-AMC
(30 μM15).
The substrate Suc-LLVY-AFC was easily synthesized in nine
steps from the commercially available AFC by a series of de-
protection and coupling reactions classically used in peptide
chemistry (Scheme 1 and ESI‡).
Inhibition of the ChT-L activity of rabbit 20S proteasome by
hydrazino acid-based pseudopeptides
The Suc-LLVY-AFC substrate was then compared with the non-
fluorinated substrate Suc-LLVY-AMC for its ability to be used
to determine the inhibitory activity of molecules on the rabbit
Fig. 1 Structure of the substrate Suc-LLVY-AFC and representation of
the enzymatic reaction releasing the bimodal probe AFC.
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spectroscopy using the classical Suc-LLVY-AMC substrate
(1.1 0.1 μM).15
After having proved that Suc-LLVY-AFC is a good reference
substrate, we took advantage of this novel easy method to
evaluate new ChT-L proteasome non-covalent inhibitors.
While most of the described proteasome inhibitors interact co-
valently with the active site of the enzyme, non-covalent
bonding inhibitors are being studied to a lesser extent. They
might be of particular interest because they are devoid of reac-
tive electrophilic function prone to nucleophilic attack and,
thus, they might induce lower side effects and improved bio-
availability.17,18 Three new α- and β-hydrazino acid-based
pseudopeptides 2, 3, 4 were prepared (Fig. 3b). In compound
2, a tryptophan residue replaced the lysine residue previously
described in molecule 1.15 In the N-terminal part of pseudo-
peptides 3 and 4, 3-phenoxyphenylmethyl and 3,4-dimethoxy-
phenylmethyl replaced respectively the 3-phenoxyphenylacetyl
and 3,4-dimethoxyphenylacetyl groups in previously described
inhibitors.15
Fig. 3 (a) Schematic representation of hydrazino acid-based pseudo-
peptides. (b) Structures of molecules 1–4.
The synthesis of compounds 2–4 was undertaken by a
series of classical deprotection and coupling reactions (see
ESI‡).
The inhibition of the ChT-L activity of the rabbit 20S protea-
some by molecules 2–4 was investigated by fluorescence
spectroscopy and by 3-FABS using the substrate Suc-LLVY-AFC.
Again, results obtained with the two methods were very similar
(Table 1). Molecules 2–4 inhibited the ChT-L activity with IC50
values up to the micromolar range. The inhibitory potency of 2
was not affected by the replacement of the lysine residue by
tryptophane. Similarly for compounds 3 and 4, increasing the
flexibility of the N-terminal part of pseudopeptides by introdu-
cing 3-phenoxyphenylmethyl and 3,4-dimethoxyphenylmethyl,
afforded similar and good inhibitory activities (the 3-phenoxy-
phenylacetyl and 3,4-dimethoxyphenylacetyl analogous com-
pounds exhibited IC50 values of 1.4 and 8.8 µM15).
Fig. 4 Inhibition of the ChT-L activity of rabbit 20S proteasome by
compound 1 at pH 7.5 and 37 °C, using the substrate Suc-LLVY-AFC.
(a) Followed by fluorescence spectroscopy, λex = 360 nm, λem = 480 nm;
(b) followed by 3-FABS.
Table 1 Chymotrypsin-like (ChT-L) proteasome inhibition potency
of molecules. IC50 (µM) at pH 7.5 and 37 °C, using the substrate
Suc-LLVY-AFC
a
b
Compound
IC50
IC50
Monitoring of the ChT-L activity in the presence of PA and T-L
substrates
1
2
3
4
5.3 2.2
3.9 0.6
1.7 0.3
9.4 1.4
9.7 3.2
8.5 1.1
3.1 0.3
10.5 2.7
We then employed Suc-LLVY-AFC (40 μM) to simultaneously
monitor the ChT-L activity in the presence of fluorescent non-
fluorinated substrates commonly used for PA (Z-LLE-AMC,
100 μM) and T-L (Boc-LRR-AMC, 50 μM) activities. A concen-
tration of about twice the Km of Z-LLE-AMC was used.
However, due to a low solubility of Boc-LRR-AMC a lower con-
centration was used (50 μM). The presence of both substrates
causes no background 19F NMR signal. The ChT-L activity
decreased dramatically upon exposure to both PA and T-L sub-
strates (Fig. 5 and 6). We observed 69 and 75% of inhibition of
the ChT-L activity by Z-LLE-AMC (100 μM) and Boc-LRR-AMC
(50 μM) respectively (Fig. 5 and 6). The hypothesis is that the
binding of these substrates to their respective specific active
sites might induce a change in the conformation of 20S protea-
some conformation and therefore induce a change in the
ChT-L proteolytic activity. These results confirmed the pre-
viously reported negative effect of PA substrate on ChT-L
activity.4,5,7 However, the allosteric effect of T-L substrate on
a Determined by fluorescence spectroscopy. b Determined by 3-FABS.
20S proteasome ChT-L activity. For that purpose, we employed
the α-hydrazino acid-based molecule 1, previously reported by
our group.15 Molecule 1 is one element of a library of new
pseudopeptides where a natural α-amino acid is replaced by an
α- or a β-hydrazino acid (Fig. 3).15,16
The inhibition of the ChT-L activity of rabbit 20S protea-
some by compound 1 was followed by fluorescence spec-
troscopy and by 3-FABS using the substrate Suc-LLVY-AFC
(Fig. 4). The IC50 of 1 determined by 3-FABS was similar to that
determined by fluorescence spectroscopy (9.7 3.2 µM and
5.3 2.2 µM respectively, Table 1). These two values of IC50
were similar to the value obtained previously by fluorescence
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50% of the ChT-L activity, while in the simultaneous presence
of PA substrate (100 μM, Z-LLE-AMC) or of T-L substrate
(50 μM, Boc-LRR-AMC), the ChT-L activity was further
decreased (68 and 78% of inhibition respectively, Fig. 5 and 6).
Surprisingly, in both cases, no synergic effect could be
observed and the inhibition of the ChT-L activity was not
increased in the case of the simultaneous presence of the
inhibitor 3 and the PA or T-L substrates compared to the case
where the PA or T-L substrates were added in the absence of 3.
The results seem to indicate that the allosteric effect is
superior to the inhibitory effect of 3. The superiority of the
allosteric effect to the inhibitor activity should be further clari-
fied. This may lead to a difference between the activity of some
inhibitors determined in vitro with one specific ChT-L sub-
strate, with thereafter the activity observed in cells in which
natural PA and T-L substrates are present.
Fig. 5 Monitoring of the ChT-L activity as assessed with Suc-LLVY-AFC
■
alone (40 μM, ), in the presence of the PA substrate (100 μM,
●
▲
Z-LLE-AMC, ), in the presence of inhibitor 3 (3 μM, ), or in the simul-
taneous presence of the PA substrate (100 μM, Z-LLE-AMC) and inhibitor
3 (3 μM, X).
Conclusions
In conclusion, we have found a novel bimodal substrate to
monitor the ChT-L activity of the 20S proteasome. We replaced
the fluorophore AMC of the commonly used ChT-L substrate
Suc-LLVY-AMC by the fluorophore containing the CF3-probe,
7-amino-4-trifluoromethylcoumarin (AFC). We confirmed that
this new substrate Suc-LLVY-AFC is reliable to evaluate the
ChT-L inhibitory activity of molecules either by fluorescence or
by the 3-FABS method by 19F NMR spectroscopy giving results
similar to those obtained solely by fluorescence with the com-
monly used substrate Suc-LLVY-AMC. We lifted a major lock
existing with the actual fluorescent substrates that totally
prevent the simultaneous monitoring of the three proteolytic
activities, and demonstrated that Suc-LLVY-AFC is a relevant
tool to simultaneously and quantitatively monitor the ChT-L
activity in the presence of commonly used fluorescent PA and
T-L substrates. Our results confirmed the influence of these PA
and T-L substrates on the ChT-L activity. Indeed, we observed
a dramatic decrease of the ChT-L activity upon exposure to
both PA and T-L substrates, in the absence and presence of an
inhibitor. Such a new fluorinated reference substrate will be
helpful to further study the catalytic mechanism of protea-
some and opens the prospect of deeper investigation of the
allosteric regulation of ChT-L activity. This 19F NMR approach
is very simple due to the easy access to the fluorinated sub-
strate and to the high sensitivity of the fluorine chemical
shifts to local environment changes. It can be conducted with
routine NMR equipment. This approach can also be extended
to studies in living cells.
Fig. 6 Monitoring of the ChT-L activity as assessed with Suc-LLVY-AFC
■
alone (40 μM, ), in the presence the T-L substrate (50 μM, Boc-
●
▲
LRR-AMC, ), in the presence of inhibitor 3 (3 μM, ), or in the simul-
taneous presence of the TL substrate (50 μM, Boc-LRR-AMC) and inhibi-
tor 3 (3 μM, X).
the ChT-L activity has been less studied in the literature and
results have been divergent. Indeed, an increased ChT-L
activity4 or its inhibition5 in the presence of T-L substrates
have been reported. Kisselev and Goldberg indicated that com-
mercially available substrates for assaying T-L activity such as
Boc-LRR-AMC might lack specificity at low concentrations
because the Km of the 26S proteasome for these substrates is
high (>0.5 mM).3 Thus, the decrease of the ChT-L activity by
this T-L substrate might not be due to an allosteric effect but
rather to its lack of specificity.
Monitoring of the ChT-L activity inhibition by pseudopeptides
in the presence of PA and T-L substrates
Experimental
Proteasome activity assays (IC50 determination by
To our knowledge, the evaluation of ChT-L inhibitors in the
presence of both ChT-L and PA or T-L substrates has not been
previously reported. Thus, we evaluated the inhibition of the
fluorescence)
ChT-L activity by the pseudopeptide 3 in the presence of a PA ChT-L activity of purified 20S rabbit proteasome (Boston-
and a T-L substrate. At 3 μM, compound 3 inhibited about BioChem) was determined by monitoring the hydrolysis of the
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Suc-Leu-Leu-Val-Tyr-AFC substrate. Proteasome (0.3 nM final
concentration) was incubated in 96-well plates (200 µL final
Acknowledgements
volume) in the absence (control) or presence of various Financial support from the Ministère de la Recherche et des
concentrations of inhibitors (0.1–100 µM, stock solutions in Technologies (MRT) for MK is acknowledged. Guillaume
DMSO) in the following buffer: 20 mM Tris-HCl, 1 mM DTT, Bernadat and Andrew Pearson (language service of the UFR
0.02% (w/v) SDS, and 10% glycerol, pH 7.4. After 15 min of Pharmacy, UPS) are thanked for helping with kinetics data
incubation at 37 °C, the substrate was added (2 µL of a 5 mM processing automation and for correcting the English text
DMSO solution, 50 µM final concentration) and the rate of respectively.
hydrolysis was monitored with a Fluostar Optima (BMG
Labtech) microtiter plate reader by recording the fluorescence
of the hydrolyzed AFC group (excitation filter: 360 nm; emis-
sion filter: 480 nm). The final DMSO concentration was kept
Notes and references
constant at 2% (v/v) and each inhibitor was tested 3 times in
triplicate. The initial linear portion of the curves (60–300 min)
gave access to Vo and Vi values: the slopes of the reaction pro-
gress curves respectively in the absence or presence of inhibi-
tors (Vo was considered to be 100% of the proteasome activity,
while Vi < 100% were considered as inhibition events). These
data were used to calculate the IC50 values (inhibitor concen-
tration causing 50% decrease in proteasome activity) by fitting
the experimental data to the equation: % inhibition = 100
(1 − Vi/Vo). Dose–response curves were fitted to the data point
by nonlinear regression with a 4-parameter log-logistic model
as implemented in the drc package (v2.3-7)19 within the R soft-
ware environment (v3.0.1).20 IC50 and the corresponding confi-
dence intervals were determined with the same software using
the delta method.
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frequency of 376 MHz with a 5 mm inverse dual probe head
(1H–19F). The 19F-NMR spectra were recorded using a pulse
sequence of proton decoupling with a spectral width of 12 019
Hz, an acquisition time of 1 s and a relaxation delay of 4 s. The
spectra were analyzed with TOPSPIN 2.1 (Bruker). The enzy-
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