Enzyme-Instructed Assembly and Disassembly Processes for Targeting Downregulation in Cancer Cells

Article abstract of DOI:10.1021/jacs.7b00070

Cancer cells differ from normal cells in both gain of functions (i.e., upregulation) and loss of functions (i.e., downregulation). While it is common to suppress gain of function for chemotherapy, it remains challenging to target downregulation in cancer cells. Here we show the combination of enzyme-instructed assembly and disassembly to target downregulation in cancer cells by designing peptidic precursors as the substrates of both carboxylesterases (CESs) and alkaline phosphatases (ALPs). The precursors turn into self-assembling molecules to form nanofibrils upon dephosphorylation by ALP, but CES-catalyzed cleavage of the ester bond on the molecules results in disassembly of the nanofibrils. The precursors selectively inhibit the cancer cells that downregulate CES (e.g., OVSAHO) but are innocuous to a hepatocyte that overexpresses CES (HepG2), while the two cell lines exhibit comparable ALP activities. This work illustrates a potential approach for the development of chemotherapy via targeting downregulation (or loss of functions) in cancer cells.

Full text of DOI:10.1021/jacs.7b00070

Subscriber access provided by University of Newcastle, Australia
Communication
Enzyme-Instructed Assembly and Disassembly
Processes for Targeting Down-Regulation in Cancer Cells
Zhaoqianqi Feng, Huaimin Wang, Rong Zhou, Jie Li, and Bing Xu
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b00070 • Publication Date (Web): 03 Mar 2017
Downloaded from http://pubs.acs.org on March 3, 2017
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of the American Chemical Society is published by the American Chemical
Society. 1155 Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 5
Journal of the American Chemical Society
1
2
3
4
5
6
Enzyme-Instructed Assembly and Disassembly Processes for
Targeting Down-Regulation in Cancer Cells
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Zhaoqianqi Feng, Huaimin Wang, Rong Zhou, Jie Li and Bing Xu*
Department of Chemistry, Brandeis University, 415 South Street, Waltham, Massachusetts 02454, United States.
Supporting Information Placeholder
1
9
a multiple-step process to inhibit cancer cells, EISA promis-
es a unprecedented way for targeting down-regulation for
cancer therapy, which remains a challenge in translational
medicine. Scheme 1 shows the concept: A pair of cell lines
both express alkaline phosphatase (ALP) in comparable lev-
els, but one (e.g., OVSAHO) down-regulates CES, and the
other (e.g., HepG2) upregulates carboxylesterase (CES). Up-
on the action of ALP, precursors turn into self-assembling
molecules to form assemblies, but the assemblies disassoci-
ate upon the action of CES. Because the assemblies are cyto-
toxic and the un-assembled products are innocuous to cells,
the precursors would only inhibit the cells expressing ALP
and down-regulating CES. Thus, the overall result is to target
the down-regulation of enzyme (e.g., CES) in cancer cells.
ABSTRACT: Cancer cells differ from normal cells in both
gain-of-functions (i.e., upregulation) and loss-of-functions
(
i.e., down-regulation). While it is common to suppress gain-
of-function for chemotherapy, it remains challenging to tar-
get down-regulation in cancer cells. Here we show the com-
bination of enzyme-instructed assembly and disassembly to
target down-regulation in cancer cells by designing peptidic
precursors as the substrates of both carboxylesterases (CES)
and alkaline phosphatases (ALP). The precursors turn into
self-assembling molecules to form nanofibrils upon
dephosphorylation by ALP, but CES-catalyzed cleavage of
ester bond on the molecules results in the disassembly of
nanofibrils. The precursors selectively inhibit the cancer cells
(e.g., OVSAHO) that down-regulates CES, but are innocuous
to a hepatocyte (HepG2) that overexpresses CES, while both
the cell lines exhibit comparable ALP activities. This work
illustrates a potential approach for the development of
chemotherapy via targeting down-regulation (or loss-of-
functions) in cancer cells.
Scheme 1. Structures of the precursor and its hydrolysis
products and the concept of targeting the cells that down-
regulate CES, while expressing ALP.
While the self-assembly of small molecules is a well-studied
1
2
phenomenon in organic solvents or on surfaces, the for-
mation of such structures in biological systems has only re-
3
,4,5
cently been described.
At the intersection of supramo-
lecular chemistry and cell biology, supramolecular assem-
6
blies have shown great promises for cell cultures, modulat-
7
8
ing immune responses, delivering drugs, inhibiting drug-
9
10
resistant pathogens, and inhibiting cancer cells. We are
particularly interested in the use of assemblies of molecules
1
1
for cancer therapy because a serendipitous discovery of the
inverse comorbidity between cancer and neurodegenerative
diseases implicates molecular nanofibrils formed by self-
1
2
assembly inhibiting cancer cells, either in animal model or
1
3
in a human trial. This notion, indeed, is supported by the
1
4
development of enzyme-instructed self-assembly (EISA),
which selectively generates nanoscale assemblies of small
Based on the above concept, we design an EISA precursor
-OMe-OP, which contains both CES cleavage site (i.e., car-
1
5,15,16
molecules (e.g., small peptide derivatives
drate derivatives ) in-situ on cancer cells for inhibiting the
or carbohy-
boxylmethylester) and ALP cleavage site (i.e., phosphotyro-
sine). Such a design allows ALP to convert 1-OMe-OP to 1-
OMe-OH, CES to turn 1-OMe-OP into 1-OH-OP, and the
actions of ALP and CES to generate 1-OH-OH. Critical mi-
celle concentration (CMC) measurement and static light
scattering (SLS) reveal that 1-OMe-OH favors self-assembly.
Transmission electron microscopy (TEM) confirms that 1-
4
cancer cells.
EISA, as a process, differs fundamentally from the well-
17
established prodrug approach because, in EISA, only the
assemblies, not the un-assembled products of enzymatic
15,18
conversion, are inhibitory to cancer cells.
Besides acting as
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 2 of 5
OMe-OH, generated by dephosphorylation of 1-OMe-OP,
tion of ALP to that solution increases the ratio to 98.9, but
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
forms nanofibrils, and CES catalyzes the dissociation of the
nanofibrils by converting 1-OMe-OH to 1-OH-OH. Cell via-
bility test indicates that 1-OMe-OP potently inhibits the
cancer cells (e.g., OVSAHO) that down-regulate CES, but is
innocuous to the cells (e.g., HepG2) that up-regulate CES,
while those two cell lines exhibit comparable phosphatase
activities. Control experiments (the addition of esterase in-
the addition of CES decreases the ratio to 0.02. Moreover,
incubating 1-OMe-OP with ALP and CES together results in
1-OH-OH to exhibit a signal intensity ratio of 0.7, two order
of magnitude lower than that of 1-OMe-OH. Agreeing with
the CMC measurement, these results indicate that CES in-
structs the dissociation of the assemblies formed by ALP-
instructed self-assembly of 1-OMe-OH. In addition, the TEM
images (Figure 1B) show that 1-OMe-OP hardly forms any
nanostructures at the concentration of 100 μM, while the
addition of ALP results in nanofibrils with a diameter of 8±2
nm. Being co-incubated with CES and ALP together, 1-OMe-
OP turns to 1-OH-OH, which forms small particles with a
diameter of 7±2 nm. These TEM images confirm that ALP
instructs the assembly of 1-OMe-OH while CES catalyzes the
dissociation of the assemblies.
20
hibitors ) confirm that the action and the expression level of
CES are critical for selectively inhibiting the cancer cells. A
dicarboxylmethylester analog of 1-OMe-OP validates the
generality of the concept. This work, for the first time,
demonstrates the use of molecular assemblies to target the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
21
loss-of-function (i.e., an “untargetable” feature ) in cancer
cells. Thus, it opens a new way for developing anticancer
therapeutics based on the process of self-assembly and the
down-regulation of enzymes.
Figure 2. (A) Relative activities (comparing to culture medi-
um) of esterase secreted from the cells. (B) Percentage of the
molecular species after incubating 1-OMe-OP (500 μM) with
HepG2 or OVSAHO cells for 24 h.
To demonstrate the concept of targeting down-regulation
in cellular milieu, we choose OVSAHO—an ovarian cancer
cell line and HepG2—as a model cell of hepatocyte. Accord-
ing to the CCLE database, the mRNA expression of CES1 of
HepG2 is nearly three times higher than that of OVSAHO
cells, while these two cell lines express comparable levels of
tissue non-specific alkaline phosphatase (ALPL) (Figure S14).
Since the hydrolysis of the methylester bond in 1-OMe-OP
or 1-OMe-OH is able to occur in pericellular space, we
measure the activity of secreted esterases (Equation S1) of
HepG2 or OVSAHO cells in its conditioned medium. As
shown in Figure 2A, the relative activity (0.4) of the secreted
esterases of HepG2 cells is almost 40 times higher than that
of OVSAHO cells (0.01) at 8 hours, indicating that HepG2
cells secrete more esterases than OVSAHO cells do. In the
conditioned medium of HepG2 at 24 h, the relative activity of
the secreted esterases (0.7) becomes about five times of that
of OVSAHO (0.14), suggesting that HepG2 cells constantly
secrete more esterases than OVSAHO cells do.
Figure 1. (A) CMCs—determination with rhodamine 6G for
1-OH-OP, 1-OMe-OP, 1-OH-OH, and 1-OMe-OH. (B) TEM
of the nanostructures formed by 1-OMe-OP (100 μM) before
and after the addition of ALP or both ALP and CES. In PBS
(
pH 7.4) and scale bar = 100 nm.
The key feature of the design is that ALP-generated 1-
OMe-OH to form assemblies and the assemblies disassociate
upon CES catalytically converting 1-OMe-OH to 1-OH-OH.
We synthesize the precursor 1-OMe-OP and the relevant
products (1-OMe-OH, 1-OH-OH and 1-OH-OP) from its
hydrolysis catalyzed by ALP or CES or both (Scheme 1). We
first assess their self-assembling abilities by measure their
CMCs. As shown in Figure 1A, the CMCs follow the order of
We quantify the relevant conversion after incubating 1-
OMe-OP with HepG2 or OVSAHO cells for 24 h (Figure 2B).
LC-MS analysis indicates that only about 50% precursors (1-
OMe-OP) remains in both cells (i.e., 44.3 % for HepG2 and
4
4.2 % for OVSAHO), indicating that HepG2 and OVSAHO,
1-OMe-OH < 1-OMe-OP < 1-OH-OH < 1-OH-OP. This result
in fact, exhibit comparable phosphatase activities. However,
the CES from HepG2 hydrolyzes 14% of carboxylmethylester,
which is four times higher than the CES from OVSAHO does
indicates that the presence of phosphate group decreases
self-assembling ability of the Nap-capped tripeptide (Nap-
ffy), while attaching methyl group to the C-terminal of Nap-
ffy increases the self-assembling ability by about an order of
magnitude. We use SLS to measure the signals change upon
treating 1-OMe-OP by ALP (Figure S13). The signal intensity
ratio of the solution of 1-OMe-OP (20 µM) is 0.3. The addi-
(
i.e., hydrolyzing 3.2 %). Although the difference of 1-OMe-
OH in HepG2 and OVSAHO is only about 8%, the molar
ratios of 1-OMe-OH/1-OH-OH in the cultures of HepG2 and
OVSAHO are 3.3 and 30, respectively. Thus, we speculate
ACS Paragon Plus Environment

Page 3 of 5
Journal of the American Chemical Society
that 1-OH-OH likely promotes the disassembly of 1-OMe-
OH. Congo red, a dye for self-assembled nanofibrils, helps
or loperamide only slightly decreases the IC50 value of 1-
OMe-OP against OVSAHO (Figure 3B, Figure S17). These
results confirm that 1-OMe-OP is able to target the down-
regulation of CES in OVSAHO cells.
1
5
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
directly visualize the formation of nanofibrils in pericellular
space of OVSAHO and HepG2 cells (Figure S15). Moreover,
the pericellular fluorescence deceases upon washing, agree-
ing with that the nanofibrils form on the cell surface. The
confocal images also reveal that more nanofibrils formed on
OVSAHO cells than on HepG2 cells, agreeing with cell viabil-
ity results. These results, agreeing with enzyme expression
levels and the relative activities of the secreted esterases of
the cells, further support the design (Scheme 1) for targeting
cells that down-regulate CES.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. (A) Molecular structure and enzymatic conversion
of precursor 2-(OMe) -OP; (B) IC values (at 72 h) of 2-
2
50
(OMe) -OP against HepG2 or OVSAHO cells.
2
To verify the generality of the concept in Scheme 1, we
develop 2-(OMe) -OP (Figure 4A), a dicarboxylmethylester
2
analog of 1-OMe-OP, as another precursor (Scheme S2). Up-
on the action of ALP, 2-(OMe) -OP turns into 2-(OMe) -
2
2
OH, which self-assembles in water to form nanotubes with
the diameter of 14±2 nm (Figures S19 and S20). Similar to 1-
OMe-OH, 2-(OMe) -OH becomes 2-(OH) -OH upon the
2
2
action of CES. The CMCs (Figure S21) follow the order of 2-
(OMe) -OH (2.66 μM) < 2-(OMe) -OP (30.4 μM) < 2-(OH) -
2
2
2
OH (112 μM) < 2-(OH) -OP(500 μM). Cell assays confirms
2
that 2-(OMe) -OP selectively inhibit OVSAHO over HepG2,
2
exhibiting IC50 of 5 µM against OVSAHO cells and IC50 over
200 µM towards HepG2 cells (Figure 4B). Notably, the IC50 of
2-(OMe) -OP is 4.4 µg/mL, comparable to cisplatin (5.5
2
25
µg/mL ) against OVSAHO in cell assay. Besides supporting
that the molecular design of the substrates of ALP and CES
(
Scheme 1) is a general strategy, this result, together with the
Figure 3. (A) Cell viabilities for HepG2 and OVSAHO cells
treated with 1-OMe-OP; (B) IC50 values (at 72 h) of 1-OMe-
OP against HepG2 or OVSAHO cells without/with addition
of the inhibitors of esterases: BNPP (non-specific),
loperamide (CES2) and troglitazone (CES1).
results of 1-OMe-OP, further validate the approach of target-
ing the down-regulation of CES in cancer cells by enzyme-
instructed assembly and disassembly processes.
In conclusion, this work demonstrates that the combina-
tion of enzyme-instructed assembly and disassembly is able
to target down-regulations (or loss of function) in cancer
cells. The result reported here would be particularly benefi-
cial for treating the metastatic cancers, where the cancerous
cells exist alongside healthy cells (e.g., metastatic ovarian
While 1-OMe-OP potently inhibits OVSAHO cells at 50
μM (Figure 3A), it is almost innocuous to HepG2 cells. The
IC50 value of 1-OMe-OP against HepG2 cells (338 μM) is
about fifteen times higher than that of OVSAHO cells (22
μM), confirming that 1-OMe-OP selectively target OVSAHO
cells. Besides difference in their self-assembling ability, the
carboxylic species (e.g., 1-OH-OH) likely adheres less to cell
membrane than the methylester one (e.g., 1-OMe-OH) does,
thus exhibits less cytotoxicity. To prove further that CES
hydrolysis contributes to the low cytotoxicity of 1-OMe-OP
against HepG2, we co-incubate CES inhibitors and 1-OMe-
OP with HepG2 (Figure 3B). The addition of troglitazone (a
26
cancer into liver ). Notably, the IC50 values of the precursors
against OVSAHO cells follow the lowest CMCs of the corre-
sponding hydrolysis products (Figure S22), indicating that
the CMC values may help predict the effective concentra-
tions of the precursors in cell assays. Interestingly, although
the mRNA expression of ALPL in OVSAHO is slightly higher
than that in HepG2, the two cell lines exhibit comparable
phosphatase activities towards the precursors, which under-
scores the need to validate the enzyme activities experimen-
tally for precisely targeting of cancer cell. Although this work
uses ALP and CES, this principle demonstrated here should
20
22
CES1 inhibitor ) or loperamide (a CES2 inhibitor ) reduces
the IC50 of 1-OMe-OP against HepG2 from 338 μM to 133 μM
and 117 μM, respectively. BNPP (an inhibitor of both CES1
23
27
28
and CES2 ) lowers the IC of 1-OMe-OP against HepG2
be applicable to any other enzymes or cellular difference,
5
0
almost an order of magnitude (from 338 μM to 43 μM).
Agreeing with that HepG2 cells express both CES1 and
especially the difference in lost-of-functions, for spatiotem-
poral control of molecular assemblies that control cell fate.
24
CES2, the inhibition of CES reduces the hydrolysis of 1-
OMe-OH, thus boosting the cytotoxicity of 1-OMe-OP to-
wards HepG2. In contrast, the addition of troglitazone hardly
shows any effect on the cell viability of OVSAHO, and BNPP
ASSOCIATED CONTENT
Supporting Information
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 4 of 5
Synthetic procedures, characterizations, and cell viability.
This material is available free of charge via the Internet at
http://pubs.acs.org.
(16) Wang, H.; Feng, Z.; Wu, D.; Fritzsching, K. J.; Rigney, M.;
Zhou, J.; Jiang, Y.; Schmidt-Rohr, K.; Xu, B. J. Am. Chem. Soc. 2016,
38, 10758; Feng, Z.; Wang, H.; Du, X.; Shi, J.; Li, J.; Xu, B. Chem.
Commun. 2016, 52, 6332; Li, J.; Kuang, Y.; Shi, J. F.; Zhou, J.; Medina,
J. E.; Zhou, R.; Yuan, D.; Yang, C. H.; Wang, H. M.; Yang, Z. M.; Liu, J.
F.; Dinulescu, D. M.; Xu, B. Angew. Chem., Int. Ed. 2015, 54, 13307;
Wang, H. M.; Feng, Z. Q. Q.; Wang, Y. Z.; Zhou, R.; Yang, Z. M.; Xu,
B. J. Am. Chem. Soc. 2016, 138, 16046.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
AUTHOR INFORMATION
Corresponding Author
bxu@brandeis.edu
Notes
The authors declare no competing financial interests.
(
17) Rautio, J.; Kumpulainen, H.; Heimbach, T.; Oliyai, R.; Oh, D.;
Jaervinen, T.; Savolainen, J. Nat. Rev. Drug Discovery 2008, 7, 255;
Brunton, L.; Hilal-Dandan, R.; Goodman, L. S. Goodman and Gilman
manual of pharmacology and therapeutics; Mcgraw Hill Professional:
New York, 2013.
(18) Shi, J.; Du, X.; Yuan, D.; Zhou, J.; Zhou, N.; Huang, Y.; Xu, B.
Biomacromolecules 2014, 15, 3559.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ACKNOWLEDGMENT
This work was partially supported by NIH (R01CA142746)
and NSF (MRSEC-1420382).
(19) Zhou, J.; Xu, B. Bioconjugate Chem. 2015, 26, 987.
(
20) Fu, J.; Sadgrove, M.; Marson, L.; Jay, M. Drug Metab. Dispos.
REFERENCES
2016, 44, 1313.
(
21) Bourzac, K. Nature 2014, 509, S69.
(
1) Terech, P.; Weiss, R. G. Chem. Rev. 1997, 97, 3133; Ikeda, A.;
(22) Quinney, S. K.; Sanghani, S. P.; Davis, W. I.; Hurley, T. D.;
Sun, Z.; Murry, D. J.; Bosron, W. F. J. Pharmacol. Exp. Ther. 2005, 313,
Shinkai, S. Chem. Rev. 1997, 97, 1713; Sangeetha, N. M.; Maitra, U.
Chem. Soc. Rev. 2005, 34, 821; Lehn, J.-M. Proc. Natl. Acad. Sci. U. S.
A. 2002, 99, 4763; Whitesides, G. M.; Mathias, J. P.; Seto, C. T.
Science 1991, 254, 1312.
(2) Love, J. C.; Estroff, L. A.; Kriebel, J. K.; Nuzzo, R. G.;
Whitesides, G. M. Chem. Rev. 2005, 105, 1103.
1
011.
23) Ohura, K.; Sakamoto, H.; Ninomiya, S.-i.; Imai, T. Drug
Metab. Dispos. 2010, 38, 323.
24) Ross, M. K.; Borazjani, A.; Wang, R.; Crow, J. A.; Xie, S. Arch.
Biochem. Biophys. 2012, 522, 44.
25) Elias, K. M.; Emori, M. M.; Papp, E.; MacDuffie, E.; Konecny,
G. E.; Velculescu, V. E.; Drapkin, R. Gynecol. Oncol. 2015, 139, 97.
(
(
(
3) Yang, Z.; Xu, K.; Guo, Z.; Guo, Z.; Xu, B. Adv. Mater. 2007, 19,
(
3
152; Zorn, J. A.; Wille, H.; Wolan, D. W.; Wells, J. A. J. Am. Chem.
Soc. 2011, 133, 19630; Kato, M.; Han, T. N. W.; Xie, S. H.; Shi, K.; Du,
X. L.; Wu, L. C.; Mirzaei, H.; Goldsmith, E. J.; Longgood, J.; Pei, J. M.;
Grishin, N. V.; Frantz, D. E.; Schneider, J. W.; Chen, S.; Li, L.; Sawaya,
M. R.; Eisenberg, D.; Tycko, R.; McKnight, S. L. Cell 2012, 149, 753.
(
(
26) Langyel, E. Am. J. Pathol. 2010, 177, 1053.
27) Dragulescu-Andrasi, A.; Kothapalli, S. R.; Tikhomirov, G. A.;
Rao, J. H.; Gambhir, S. S. J. Am. Chem. Soc. 2013, 135, 11015; Jiang, T.;
Olson, E. S.; Nguyen, Q. T.; Roy, M.; Jennings, P. A.; Tsien, R. Y. Proc.
Natl. Acad. Sci. U. S. A. 2004, 101, 17867.
(
4) Pires, R. A.; Abul-Haija, Y. M.; Costa, D. S.; Novoa-Carballal,
R.; Reis, R. L.; Ulijn, R. V.; Pashkuleva, I. J. Am. Chem. Soc. 2015, 137,
76.
5) Tanaka, A.; Fukuoka, Y.; Morimoto, Y.; Honjo, T.; Koda, D.;
Goto, M.; Maruyama, T. J. Am. Chem. Soc. 2015, 137, 770.
6) Silva, G. A.; Czeisler, C.; Niece, K. L.; Beniash, E.; Harrington,
D. A.; Kessler, J. A.; Stupp, S. I. Science 2004, 303, 1352.
7) Rudra, J. S.; Tian, Y. F.; Jung, J. P.; Collier, J. H. Proc. Natl.
(
28) Zheng, Z.; Chen, P.; Xie, M.; Wu, C.; Luo, Y.; Wang, W.;
5
Jiang, J.; Liang, G. J. Am. Chem. Soc. 2016, 138, 11128.
(
(
(
Acad. Sci. U. S. A. 2010, 107, 622; Wang, H. M.; Luo, Z.; Wang, Y. C.
Z.; He, T.; Yang, C. B.; Ren, C. H.; Ma, L. S.; Gong, C. Y.; Li, X. Y.;
Yang, Z. M. Adv. Funct. Mater. 2016, 26, 1822.
(
8) Zhao, F.; Ma, M. L.; Xu, B. Chem. Soc. Rev. 2009, 38, 883;
Cheetham, A. G.; Zhang, P.; Lin, Y.-a.; Lock, L. L.; Cui, H. J. Am.
Chem. Soc. 2013, 135, 2907.
(
9) Salick, D. A.; Kretsinger, J. K.; Pochan, D. J.; Schneider, J. P. J.
Am. Chem. Soc. 2007, 129, 14793; Xing, B. G.; Yu, C. W.; Chow, K. H.;
Ho, P. L.; Fu, D. G.; Xu, B. J. Am. Chem. Soc. 2002, 124, 14846.
(10) Julien, O.; Kampmann, M.; Bassik, M. C.; Zorn, J. A.;
Venditto, V. J.; Shimbo, K.; Agard, N. J.; Shimada, K.; Rheingold, A.
L.; Stockwell, B. R.; Weissman, J. S.; Wells, J. A. Nat. Chem. Biol.
2014, 10, 969.
(
11) Driver, J. A.; Beiser, A.; Au, R.; Kreger, B. E.; Splansky, G. L.;
Kurth, T.; Kiel, D. P.; Lu, K. P.; Seshadri, S.; Wolf, P. A. Br. Med. J.
2012, 344.
(
12)
Gallardo, R.; Ramakers, M.; De Smet, F.; Claes, F.;
Khodaparast, L.; Khodaparast, L.; Couceiro, J. R.; Langenberg, T.;
Siemons, M.; Nystrom, S.; Young, L. J.; Laine, R. F.; Young, L.;
Radaelli, E.; Benilova, I.; Kumar, M.; Staes, A.; Desager, M.; Beerens,
M.; Vandervoort, P.; Luttun, A.; Gevaert, K.; Bormans, G.;
Dewerchin, M.; Van Eldere, J.; Carmeliet, P.; Vande Velde, G.;
Verfaillie, C.; Kaminski, C. F.; De Strooper, B.; Hammarstrom, P.;
Nilsson, K. P. R.; Serpell, L.; Schymkowitz, J.; Rousseau, F. Science
2016, 354, 720.
(
13) Gustafsson, L.; Leijonhufvud, I.; Aronsson, A.; Mossberg, A.-
K.; Svanborg, C. N. Engl. J. Med. 2004, 350, 2663.
(14) Du, X.; Zhou, J.; Shi, J.; Xu, B. Chem. Rev. 2015, 115, 13165.
(
15) Kuang, Y.; Shi, J.; Li, J.; Yuan, D.; Alberti, K. A.; Xu, Q.; Xu, B.
Angew. Chem., Int. Ed. 2014, 53, 8104.
ACS Paragon Plus Environment

Page 5 of 5
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
Insert Table of Contents artwork here
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment