Boronic acid-appended molecular glues for ATP-responsive activity modulation of enzymes

Article abstract of DOI:10.1021/jacs.6b02664

Water-soluble linear polymers Gu_mBA_n (m/ n = 18/6, 12/12, and 6/18) with multiple guanidinium ion (Gu⁺) and boronic acid (BA) pendants in their side chains were synthesized as ATP-responsive modulators for enzyme activity.

Full text of DOI:10.1021/jacs.6b02664

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Communication
Boronic Acid–Appended Molecular Glues for ATP-
Responsive Activity Modulation of Enzymes
Kou Okuro, Mizuki Sasaki, and Takuzo Aida
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b02664 • Publication Date (Web): 18 Apr 2016
Downloaded from http://pubs.acs.org on April 19, 2016
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Boronic Acid–Appended Molecular Glues for ATP-Responsive Activi-
ty Modulation of Enzymes
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Kou Okuro,*,† Mizuki Sasaki,† and Takuzo Aida*,†,‡
† Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo
113-8656, Japan
‡ RIKEN Center for Emergent Matter Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
Supporting Information Placeholder
tiple guanidinium ion (Gu+) and boronic acid (BA) pen-
ABSTRACT: Water-soluble linear polymers GumBAn (m/n =
dants in their side chains (Figure 1a). GumBAn polymers can
temporarily suppress the activity of an enzyme by hybridiza-
tion (Figure 2). However, upon addition of ATP, the en-
zyme is liberated and can retrieve its intrinsic activity. Most
importantly, this event occurs in a much lower range of
[ATP] (1–10 µM) than previous examples including our
chaperon nanotubes.5–7
18/6, 12/12, and 6/18) with multiple guanidinium ion
(Gu+) and boronic acid (BA) pendants in their side chains
were synthesized as ATP-responsive modulators for enzyme
activity. GumBAn polymers strongly bind to the phosphate
–
ion (PO4 ) and 1,2-diol units of ATP via the Gu+ and BA
pendants, respectively. As only the Gu+ pendants can be
used for proteins, GumBAn is able to modulate the activity of
enzymes in response to ATP. As a proof-of-concept study,
we demonstrated that trypsin (Trp) can be deactivated by
hybridization with GumBAn. However, upon addition of
ATP, Trp was liberated to retrieve its hydrolytic activity due
to a higher preference of GumBAn toward ATP than Trp.
This event occurred in a much lower range of [ATP] than
reported examples. Under cellular conditions, the hydrolytic
activity of Trp was likewise modulated.
Previously, we developed dendritic8a–8e and linear8f–8i mo-
Chemicals that act site-selectively in tumor tissue are at-
tractive for tumor chemotherapy and drug delivery. Tumor
targeting is mostly based on a difference in pH between the
tumor (pH 5.9–7.6) and normal tissue (pH 7.3–8.0).1,2
Adenosine triphosphate (ATP; Figure 1b) represents a
promising tumor indicator, as the concentration of extracel-
lular ATP in tumor tissue ([ATP] >100 µM)3 is more than
four orders of magnitude higher than that in normal tissue
([ATP] = 1–10 nM).4 Previously, a few examples of ATP-
responsive drug delivery carriers using DNA aptamers5 and
phenylboronic acid6 have been reported. However, these
systems were designed to distinguish extracellular and intra-
cellular matrices, and operate at [ATP] > 1 mM. Recently,
we reported an ATP-responsive drug carrier, consisting of
tubularly connected chaperon units, which can operate at
[ATP] = 10–50 µM, albeit that the system requires esterase
to cleave off a drug linker for the release of encapsulated
drugs.7 Here we report a series of water-soluble polymers,
GumBAn, as ATP-responsive molecular glues that carry mul-
Figure 1. (a) Molecular glues GumBAn (m/n = 18/6, 12/12, and
6/18) with randomly incorporated guanidinium ion (Gu+) and
boronic acid (BA) moieties in their side chains, as well as of
GumOHn (m/n = 12/12) without BA units; (b) adenosine tri-
phosphate (ATP); (c) schematic illustration of (left) a salt-
–
bridge between phosphate ion (PO4 ) and Gu+, and (right) co-
valent bonds between 1,2-diol and BA moieties.
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Figure 2. Schematic illustration of the modulation of the enzy-
matic activity of trypsin (Trp) by GumBAn and ATP. GumBAn
adheres to the surface of Trp to form agglomerates and thus
suppresses Trp enzymatic activity (dormant state). ATP com-
petitively binds to GumBAn, which allows its dissociation from
Trp, thus restoring the enzymatic activity.
Figure 3. Fluorescence spectra (λext = 560 nm) of sulforhoda-
lecular glues containing multiple Gu+ units.9 These molecu-
lar glues strongly adhere to proteins,8a,8c,8e,8g nucleic acids,8h,8i
phospholipid membranes,8d and clay nanosheets8b,8f via mul-
tiple salt bridges between Gu+ and oxyanionic groups9 locat-
ed on the targets. GumBAn (Figure 1a) were designed as a
new class of molecular glues containing not only Gu+ but
also BA units that can covalently bind to 1,2-diols (Figure
mine B-labeled Trp (RhdTrp, 0.5 µM) at 25 °C in Tris-HCl buffer
(20 mM, pH 7.8) upon successive titration with (a) Gu12BA12
(0–750 nM) and (b) ATP (0–10 µM). (c) Dissociation profiles
of Gu12B12 (red; 750 nM), Gu18BA6 (blue; 800 nM), Gu6BA18
(green; 4.5 µM), and Gu12OH12 (orange; 700 nM) from RhdTrp
(0.5 µM) at 25 °C in Tris-HCl buffer. Fractions of bound Trp
were calculated from the fluorescence intensity at 578 nm. (d)
DLS histograms of Trp (0.5 µM) in the absence (dark blue) and
presence (red) of Gu12BA12 (700 nM) at 25 °C in Tris-HCl buff-
er. (e) Association constants of Gu12B12, Gu18BA6, Gu6BA18, and
Gu12OH12 toward Trp (Ktrp) and ATP (KATP) determined ac-
cording to the reported methods.15,16
–
1c).10 The cooperative effect of multiple Gu+/PO4 salt
bridges and covalently bound BA/1,2-diol moieties (Figure
1c) should result in a high affinity of GumBAn toward ATP
(Figure 1b), which may be able to liberate proteins from
their GumBAn conjugates (Figure 2). As a proof-of-concept
study, we chose a hydrolytic enzyme trypsin (Trp) as a target
protein. Trp is known to inhibit the proliferation and metas-
tasis of cancer cells,11 and thus may possibly exhibit tumor-
suppressing activity. However, Trp is not tissue selective and
thus also deteriorates normal tissue.11 Here we highlight that
the hydrolytic activity of Trp, which is intrinsically inde-
pendent of ATP, can be modulated via conjugation with
GumBAn, and that this conjugation is essentially reversible
upon addition of ATP (Figure 2).
by quantification of the thiol termini (Table S1).13 Subse-
quently, we labeled Trp with a fluorescence probe sulforho-
damine B (RhdTrp, 0.5 µM) and conducted titration experi-
ments with Gu12BA12 (0–750 nM) in Tris-HCl buffer (20
mM, pH 7.8) at 25 °C, as the adhesion of Gu12BA12 to RhdTrp
quenches the fluorescence emission at 578 nm (λext = 560
nm; Figure 3a).14 According to an established method,15 we
fitted the fluorescence intensity changes to a 1:1 binding
model, and obtained an association constant (KTrp) of 1.3 ×
107 M–1 for the binding between Gu12BA12 and Trp (Figure
3e). A similar fluorescence-quenching profile was observed
for RhdTrp with Gu18BA6 (KTrp = 2.4 × 107 M–1; Figures 3e
and S12a),13 as well as Gu12OH12 (KTrp = 2.5 × 107 M–1; Fig-
ures 3e and S12e).13 In contrast, Gu6BA18 barely quenched
the fluorescence (Figure S12c),13 and a much smaller KTrp
value (3.1 × 105 M–1; Figure 3e) relative to that of Gu12BA12
was observed, indicating that the binding to Trp occurs pre-
GumBAn polymers (Figure 1a) were synthesized using the
“thiol–yne” reaction12 between alkyne-appended monomers
containing Gu+ and BA units, and a triethylene glycol with
thiol termini. With different molar ratios of two alkyne-
appended monomers, Gu18BA6, Gu12BA12, and Gu6BA18
were synthesized. The oxidation of Gu12BA12 with H2O2
furnished Gu12OH12 as a reference, which contains hydroxyl
(OH) groups instead of BA units (Figure 1a). The average
molecular weights of GumBAn and GumOHn were evaluated
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sponding KATP. The relative affinity toward ATP and Trp
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(KATP/KTrp; Figure 3e) can be tuned via the Gu+/BA ratio.
Among the examined GumBAn polymers, Gu6BA18 exhibited
the highest KATP/KTrp ratio (11; Figure 3e) due to its small
KTrp value. Although Gu12BA12 exhibited KATP/KTrp < 1, it
was used for the following studies on account of its high KATP
and KTrp values (Figure 3e). In principle, BA/1,2-diol bind-
ing is pH-sensitive.10 However, judging from the binding
behavior to Alizarin Red S, a fluorescent diol (Figure S12),13
the BA units of Gu12BA12 could certainly bind to the 1,2-diol
unit of ATP also at a typical pH (6.8) of tumor tissue.
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We then examined the ATP-responsive enzymatic activity
of Trp.
Upon mixing N-α-benzoyl-DL-arginine 4-
nitroanilide (BAPNA, 1 mM) with Trp (0.5 µM) at 37 °C in
Tris-HCl buffer (50 mM, pH 7.8) containing CaCl2 (10
mM), the characteristic absorption at 405 nm increased
(Figure 4b, dark blue), indicating that BAPNA was hydro-
lyzed to p-nitroaniline (Figure 4a).17 Conversely, BAPNA
was hydrolyzed only very sluggishly (Figure 4b, red) when
Gu12BA12 (10 µM) was used under otherwise identical condi-
tions. This result might be ascribed to the agglomeration of
Trp (Figure 3d), which would lower the accessibility of the
active site of Trp. However, the hydrolysis, which was sup-
pressed by Gu12BA12, could be accelerated by addition of
ATP (1 and 100 µM; Figure 4b, purple and pink, respective-
ly). When GumBAn was absent, the hydrolytic activity of Trp
remained intact to the addition of ATP (Figure S17).13 The
hydrolytic activity of Trp in the presence of Gu12BA12 and
ATP was evaluated by using pseudo-first order reaction ki-
netics, and normalized relative to that of untreated Trp. A
sigmoidal correlation was observed between the %-activity of
Trp and [ATP] (Figure 4c), which resulted in an abrupt in-
crease in hydrolytic activity of Trp upon increasing [ATP]
from 1 µM to 10 µM. Considering the concentrations of
ATP in tumor and normal tissues (vide supra), this threshold
is favorable for tumor targeting.
Figure 4. (a) Schematic representation of Trp-catalyzed hy-
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drolysis of BAPNA to p-nitroaniline. (b) Absorption changes at
405 nm of Trp (0.5 µM, dark blue), as well as of a mixture of
Trp, Gu12BA12 (10 µM), and ATP (0, 1, and 100 µM; red, pur-
ple, and pink, respectively) in Tris-HCl buffer (50 mM, pH 7.8)
containing BAPNA (1 mM) and CaCl2 (10 mM) at 37 °C. (c)
Normalized enzymatic activity of Trp (0.5 µM) in the presence
of Gu12BA12 (10 µM) and ATP (0.01–100 µM).
dominantly via Gu+. Dynamic light scattering (DLS) meas-
urements revealed that Gu12BA12 (700 nM) adheres to Trp
(0.5 µM; Figure 3d, dark blue) to yield agglomerates with an
average diameter of ~70 nm (Figure 3d, red).8g
Subsequently, GumBAn that adheres to Trp can be de-
tached by addition of ATP. For example, when ATP (0–10
µM) was added to a conjugate of RhdTrp (0.5 µM) and
Gu12BA12 (750 nM; Figure 3a), the fluorescence emission at
578 nm increased as expected (Figure 3b). The fraction of
Gu12BA12-bound RhdTrp decreased nonlinearly as a function
of [ATP] (Figure 3c, red), as evaluated from the change of
the fluorescence profile using the extinction coefficient of
Gu12BA12 for RhdTrp.15 For example, when 10 µM of ATP
was added, the %-fraction of Gu12BA12-bound RhdTrp
dropped from 80% to 20% (Figure 3c, red). An association
constant for Gu12BA12 toward ATP (KATP) of 3.8 × 106 M–1
(Figure 3e) was obtained by fitting the dissociation profile
(Figure 3c, red) to the competitive binding model.16 Like-
wise, KATP values for Gu6BA18 (3.5 × 106 M–1; Figure 3c,
green) and Gu18BA6 (2.7 × 106 M–1; Figure 3c, blue) were
determined (Figure 3e). Although the KTrp of Gu12OH12 is
comparable to that of Gu12BA12, ATP was virtually unable to
liberate Trp from Gu12OH12 (Figure 3c, orange). For
Gu12OH12, a much smaller KATP value (5.6 × 104 M–1; Figure
3e) relative to that of Gu12BA12 was observed, which high-
lights the crucial role of the BA units in the ATP-responsive
nature of GumBAn. The binding of GumBAn to ATP strongly
Then, we attempted to extend this strategy to cellular sys-
tems. Trp can be used to detach cells from a culture sub-
strate by degrading cell surface components involved in the
adhesion to the substrate. Therefore, we immersed human
hepatocellular carcinoma Hep3B cells, fluorescently labelled
with CellBrite Orange,13 in a Hank’s balanced salt solution
(HBSS, pH 7.8) containing Trp (5 µM), before we subjected
the sample to confocal laser scanning microscopy (λext = 552
nm). As shown in Figure 5a, the cells were detached from
the substrate. Accordingly, the cell/substrate contact area,
calculated from the fluorescence micrographs by using the
ImageJ software, decreased over a period of 60 min (Figure
5d, dark blue).18 In sharp contrast, Hep3B cells barely de-
tached (Figures 5b and 5d, red) when they were treated with
a mixture of Trp (5 µM) and Gu12BA12 (10 µM) under oth-
erwise identical conditions, thus indicating the suppression
of enzymatic activity of Trp. Subsequently, we added 100
µM of ATP to the system, whereupon the cells started to
depends on multivalent Gu+/PO4 salt-bridge interactions.
–
–
In fact, toward AMP, an ATP analogue with only one PO4
unit, the association constant for Gu12BA12 (KAMP: 8.7 × 102
M–1; Figure S14)13 was 4,400-fold smaller than the corre-
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AUTHOR INFORMATION
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Corresponding Authors
okuro@macro.t.u-tokyo.ac.jp
tokyo.ac.jp (T.A.)
(K.O.);
aida@macro.t.u-
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Notes
The authors declare no competing financial interests.
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ACKNOWLEDGMENT
This work was partially supported by the Grant-in-Aid for
Young Scientists (B) (26810046) to K.O.
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REFERENCES
(1) Gullotti, E.; Yeo, Y. Mol. Pharmaceutics 2009, 6, 1041.
(2) Wike-Hooley, J. L.; Haveman, J.; Reinhold, H. S. Radiother. Oncol.
1984, 2, 343.
(3) Pellegatti, P.; Raffaghello, L.; Bianchi, G.; Piccardi, F.; Pistoia, V.; Di
Virgilio, F. PLoS One 2008, 3, e2599.
(4) Trabanelli, S.; Očadlíková, D.; Gulinelli, S.; Curti, A.; Salvestrini, V.;
Vieira, R. de P.; Idzko, M.; Di Virgilio, F.; Ferrari, D.; Lemoli, R. M. J. Im-
munol. 2012, 189, 1303.
(5) (a) Zhu, C.-L.; Lu, C.-H.; Song, X.-Y.; Yang, H.-H.; Wang, X.-R. J.
Am. Chem. Soc. 2011, 133, 1278. (b) He, X.; Zhao, Y.; He, D.; Wang, K.;
Xu, F.; Tang, J. Langmuir 2012, 28, 12909. (c) Mo, R.; Jiand, T.; Di Santo,
R.; Tai, W.; Gu, Z.; Nat. Commun. 2014, 5, 3364.
Figure 5. Confocal laser scanning micrographs (λext = 552 nm;
0–60 min) of Hep3B cells (5.0 × 103 cells/chamber) fluores-
cently labelled with CellBrite Orange in HBSS (pH 7.8) con-
taining Trp (5 µM) at 25 °C. Micrographs recorded in the ab-
sence (a) and presence of (b) Gu12BA12 (10 µM) or (c) both
Gu12BA12 (10 µM) and ATP (100 µM). Scale bars = 100 µm.
(d) Normalized contact areas between Hep3B cells and the
substrate in HBSS containing Trp (5 µM) at 25 °C in the ab-
sence (dark blue) and presence of Gu12BA12 (10 µM, red), as
well as in the presence of both Gu12BA12 and ATP (1 or 100 µM,
purple and pink, respectively); calculated from the micrographs
using the ImageJ software.
(6) Naito, M.; Ishii, T.; Matsumoto, A.; Miyata, K.; Miyahara, Y.;
Kataoka, K. Angew. Chem., Int. Ed. 2012, 51, 10751.
(7) Biswas, S.; Kinbara, K.; Niwa, T.; Taguchi, H.; Ishii, N.; Watanabe,
S.; Miyata, K.; Kataoka, K.; Aida, T. Nat. Chem. 2014, 5, 613.
(8) (a) Okuro, K.; Kinbara, K.; Tsumoto, K.; Ishii, N.; Aida, T. J. Am.
Chem. Soc. 2009, 131, 1626. (b) Wang, Q.; Mynar, J. L.; Yoshida, M.; Lee,
E.; Lee, M.; Okuro, K.; Kinbara, K.; Aida, T. Nature 2010, 463, 339. (c)
Okuro, K.; Kinbara, K.; Takeda, K.; Inoue, Y.; Ishijima, A.; Aida, T. Angew.
Chem., Int. Ed. 2010, 49, 3030. (d) Suzuki, Y.; Okuro, K.; Takeuchi, T.;
Aida, T. J. Am. Chem. Soc. 2012, 134, 15273. (e) Uchida, N.; Okuro, K.;
Niitani, Y.; Ling, X.; Ariga, T.; Tomishige, M.; Aida, T. J. Am. Chem. Soc.
2013, 135, 4684. (f) Tamesue, S.; Ohtani, M.; Yamada, K.; Ishida, Y.;
Spruell, J. M.; Lynd, N. A.; Hawker, C. J.; Aida, T. J. Am. Chem. Soc. 2013,
135, 15650. (g) Mogaki, R.; Okuro, K.; Aida, T. Chem. Sci. 2015, 6, 2802.
(h) Hashim, P. K.; Okuro, K.; Sasaki, S.; Hoashi, Y.; Aida, T. J. Am. Chem.
Soc. 2015, 137, 15608. (i) Hatano, J.; Okuro, K.; Aida, T. Angew. Chem., Int.
Ed. 2016, 55, 193.
(9) (a) Sakai, N.; Matile, S. J. Am. Chem. Soc. 2003, 125, 14348. (b)
Hennig, A.; Gabriel, G. J.; Tew, G. N.; Matile, S. J. Am. Chem. Soc. 2008,
130, 10338. (c) Shukla, D.; Schneider, C. P.; Trout, B. L. J. Am. Chem. Soc.
2011, 133, 18713. (d) Yonamine, Y.; Yoshimatsu, K.; Lee, S.-H.; Hoshino,
Y.; Okahata, Y.; Shea, K. J. ACS Appl. Mater. Interfaces 2013, 5, 374. (e)
Gasparini, G.; Bang, E.-K.; Molinard, G.; Tulumello, D. V.; Ward, S.; Kelley,
S. O.; Roux, A.; Sakai, N.; Matile, S. J. Am. Chem. Soc. 2014, 136, 6069.
(10) (a) Barker, S. A.; Chopra, A. K.; Hatt, B. W.; Somers, P. J. Carbo-
hydr. Res. 1973, 26, 33. (b) Springsteen, G.; Wang, B. Tetrahedron 2002,
58, 5291.
(11) Novak, J. F.; Trnka, F. Anticancer Res. 2005, 25, 1157.
(12) (a) Hoogenboom, R. Angew. Chem., Int. Ed. 2010, 49, 3415. (b)
Lowe, A. B.; Hoyle, C. E.; Bowman, C. N. J. Mater. Chem. 2010, 20, 4745.
(13) See Supporting Information.
(14) Zhang, X.-F.; Zhang, Y.; Liu, L. J. Lumin. 2014, 145, 448.
(15) Yi, L.; Morgan, J. T.; Ragsdale, S. W. J. Biol. Chem. 2010, 285,
20117.
(16) Qi, X.; Loiseau, F.; Chan, W. L.; Yan, Y.; Wei, Z.; Milroy, L. G.;
Myers, R. M.; Ley, S. V.; Read, R. J.; Carrell, R. W.; Zhou, A. J. Biol. Chem.
2011, 286, 16163.
(17) Fritz, H.; Hartwich, G.; Werle, E. Hoppe-Seyler’s Z. Physiol. Chem.
1966, 345, 150.
detach efficiently, even in the presence of Gu12BA12 (Figures
5c and 5d, pink). It is noteworthy that 1 µM of ATP hardly
affected the cell detachment rate (Figure 5d, purple). These
results suggest that Gu12BA12 remains bound to Trp in an
ordinary cellular environment, but liberates Trp in ATP-rich
areas such as tumor tissue.
In conclusion, we developed novel molecular glues, Gum-
BAn, carrying multiple guanidinium ion (Gu+) and boronic
acid (BA) pendants, as ATP-responsive modulators for the
activities of enzymes. GumBAn binds tightly to ATP as well as
to proteins. The enzymatic activity of trypsin (Trp) was
effectively modulated in vitro and in cellular systems using
GumBAn and ATP. As Trp/GumBAn conjugates may poten-
tially be sensitive to ATP-rich (>100 µM) tumor tissue, in-
vivo pharmacological studies may furnish interesting results.
ASSOCIATED CONTENT
Supporting Information. Synthesis of GumBAn and GumOHn;
analytical data: NMR, MALDI-TOF mass spectrometry, fluo-
rescence spectroscopy, and related experimental procedures.
(18) Sen, S.; Kumar, S. Cell. Mol. Bioeng. 2009, 2, 218.
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