Abiotic Mimic of Endogenous Tissue Inhibitors of Metalloproteinases: Engineering Synthetic Polymer Nanoparticles for Use as a Broad-Spectrum Metalloproteinase Inhibitor

Article abstract of DOI:10.1021/jacs.9b11481

We describe a process for engineering a synthetic polymer nanoparticle (NP) that functions as an effective, broad-spectrum metalloproteinase inhibitor. Inhibition is achieved by incorporating three functional elements in the NP: a group that interacts with the catalytic zinc ion, functionality that enhances affinity to the substrate-binding pocket, and fine-tuning of the chemical composition of the polymer to strengthen NP affinity for the enzyme surface. The approach is validated by synthesis of a NP that sequesters and inhibits the proteolytic activity of snake venom metalloproteinases from five clinically relevant species of snakes. The mechanism of action of the NP mimics that of endogenous tissue inhibitors of metalloproteinases. The strategy provides a general design principle for synthesizing abiotic polymer inhibitors of enzymes.

Full text of DOI:10.1021/jacs.9b11481

Subscriber access provided by Queen Mary, University of London
Article
An Abiotic Mimic of Endogenous Tissue Inhibitors of
Metalloproteinases. Engineering Synthetic Polymer Nanoparticles
for use as a Broad-Spectrum Metalloproteinase Inhibitor
Masahiko Nakamoto, Di Zhao, Olivia Rose Benice, Shih-Hui Lee, and Kenneth J. Shea
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b11481 • Publication Date (Web): 10 Jan 2020
Downloaded from pubs.acs.org on January 15, 2020
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 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 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.
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 10
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
An Abiotic Mimic of Endogenous Tissue Inhibitors of Metallopro-
teinases. Engineering Synthetic Polymer Nanoparticles for use as a
Broad-Spectrum Metalloproteinase Inhibitor.
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
Masahiko Nakamoto†, Di Zhao†, Olivia Rose Benice†, Shih-Hui Lee†, Kenneth J. Shea†*
†Department of Chemistry, University of California, Irvine, Irvine, California 92697, USA
Supporting Information Placeholder
ABSTRACT: We describe a process for engineering a synthetic polymer nanoparticle (NP) that functions as an effective, broad-
spectrum metalloproteinase inhibitor. Inhibition is achieved by incorporating three functional elements in the NP; a group that in-
teracts with the catalytic zinc ion, functionality that enhances affinity to the substrate-binding pocket and by fine-tuning the chemi-
cal composition of the polymer to strengthen NP affinity for the enzyme surface. The approach is validated by synthesis of a NP
that sequesters and inhibits the proteolytic activity of snake venom metalloproteinases from five clinically relevant species of
snakes. The mechanism of action of the NP mimics that of endogenous tissue inhibitors of metalloproteinases. The strategy pro-
vides a general design principle for synthesizing abiotic polymer inhibitors of enzymes.
Introduction:
Enzyme inhibition is a therapeutically important approach to
regulate many biological processes. To realize efficacy it is
often necessary to incorporate multipoint interactions between
enzyme and inhibitor. For example small molecule inhibitors
often include combinations of interactions with both the cata-
lytic group and the substrate-binding site (S-pocket), as bind-
ing to either individual element often will not provide suffi-
cient selectivity/potency.1-3 In addition to small molecule in-
hibitors, multi-point interactions with peptides, proteins and/or
enzymes can also be realized with functional copolymers or
nanoparticles (NPs).4-18 Polymers/NPs may also inhibit target
enzymes by binding in the vicinity of the active site, thereby
blocking access to the active site8, 10-11, 17 or by an allosteric
mechanism.14-15 Conjugation of enzyme inhibitors/receptors to
polymers/NPs represents another approach.16, 19 In one exam-
ple the inhibitor benzamidine was incorporated in a trypsin
imprinted polymer particle to regulate its activity.16 Polymer
inhibitors can offer an additional advantage over small mole-
cule inhibitors because of their molecular weight, binding to
the enzyme can take place over a large surface area to enhance
selectivity11 and/or potency16. The inhibitory action of poly-
mers can be switched on/off10, 12 and/or influence enzyme con-
formation.8-9, 15 or in other cases coupled with enzyme seques-
tration.16-18 In addition, polymers allow for alteration of their
chemical and/or physical properties to provide tailored circula-
tion time and/or routing under biophysical conditions.20-25
synthetic polymer NP inhibitor that targets snake venom met-
alloproteinases (SVMPs). Our polymer inhibitor design took
inspiration from the endogenous tissue inhibitors of metallo-
proteinases (TIMPs), proteins that are responsible for regulat-
ing the proteolytic activity of matrix metalloproteinases
(MMPs).26-27 An X-ray crystal structure of an MMP-TIMP
complex established inhibition arises from collective interac-
tions that include binding to the catalytic zinc cation (Zn) and
S-pocket in addition to multiple contacts with the enzyme
surface over a contact area >1,300Å2.28-29 The biomimetic
TIMP described in this report is realized by incorporating the-
se three elements in the NP.
SVMPs are a diverse family of proteins responsible for the
myotoxic, hemorrhagic and dermonecrotic effects of enven-
omation, and are found in multiple species of venomous
snakes.30-31 Traditional immunological antivenom can be an
effective treatment if administrated in a timely manner. How-
ever delays in this process are often unavoidable which con-
tributes to 250,000-500,000 debilitating morbid injuries each
year from snake envenomation victims.32 A fast acting trans-
dermal intervention at the site of envenomation could serve to
mitigate this damage. Since the intra- and inter-species com-
position of snake venom varies within families of protein tox-
ins, inhibition of the entire SVMP family requires a broad-
spectrum inhibitor.33 A recent report described a polymer NP
that inhibits phospholipase A2 variants from snake venom.18, 34
Drawing on this background we undertook an effort to create a
1
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 2 of 10
1
2
3
4
5
6
7
8
9
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
Figure 1. Overall process described in this work. (a) The candidates for binding groups to the catalytic zinc cation, substrate pocket
and controls, and (b) enzyme surface were independently screened. (c) Then, the selected candidate groups were combined then
evaluated.
An abiotic SVMP inhibitor would complement the PLA2 se-
questrant and together could function as a broad-spectrum
antidote to mitigate tissue damage at the site of envenomation.
the increased efficacy of the inhibitor to interactions at the S-
pocket.3
Resunts and Discussion:
The SVMP family is subdivided into PI to PIII classes accord-
ing to their domain organization. Each class often includes
variants/isoforms and post-translational modifications.35
SVMPs belong to a superfamily of metalloproteases, the
metzincins which include, in addition to SVMP, MMP and a
disintegrin and metalloproteinase (ADAM). Most in this fami-
ly share a similar catalytic domain that contains a zinc ion
coordinated by three histidine residues.36 The role of mamma-
lian members of this family in cancer/inflammation has pro-
duced an extensive literature of small molecule inhibitors for
these enzyme families.37-40 We identified a hydroxamate group
from this literature as a candidate for the Zn-binding group.
However, the reported IC50 of acetohydroximate for MMP is
~25 mM.41 This alone would be insufficient for effective inhi-
bition. A common design feature of metzincin inhibitors is to
introduce a hydrophobic group in proximity to the hydrox-
amate functionality. X-ray crystal structures have attributed
Identification of the binding group to the catalytic zinc
cation and substrate pocket.
Cross-linked NIPAm (2%) NPs containing 20% of (6-
methacrylamidohexanoyl)-S-phenylalanine
hydroxamate
(PHX) displaying proximate hydroxamate and benzyl groups
as binding elements for the Zn and S-pocket with a six-carbon
spacer were synthesized using general procedures described in
the literature (Figure 1a, NP1 in table S1).42-44 To reveal the
importance of a carbon spacer, NPs with 20% of methac-
rylamidohexanoyl-S-phenylalanine hydroxamate (C1PHX)
was also synthesized (NP2). The contribution of the combina-
tion of Zn and S-pocket binding was investigated using NPs
with 6-methacrylamidohexanoyl hydroxamiate (HX) or (6-
methacrylamidohexanoyl)-S-phenylalanine-methyl
ester
(MHPM) displaying either a hydroxamate or a benzyl group
(NP3 and NP4). A NP without active site binding groups was
2
ACS Paragon Plus Environment

Page 3 of 10
Journal of the American Chemical Society
known to be relatively hydrophobic proteins.47, 53-54
also synthesized as a control (NP5). Acrylic acid (AAc) 5%
1
2
3
4
5
6
7
8
was incorporated in all NPs. In the absence of AAc NPs were
observed to aggregate in PBS. Since the hydroxamate is an
effective radical inhibitor,45 it was protected with a 2-
tetrahydropyranyl group (THP) during the polymerization
(THP-PHX, THP-C1PHX and THP-HX). The THP group
was removed by acid treatment following polymerization. The
inhibitory effect on SVMP was evaluated by the azocasein
assay46 using venom from Crotalus atrox, a SVMP rich ven-
om47 (Figure 2a and b, SI4) (final concentration of NPs and
venom were 0.5 mg/mL and 0.05 mg/mL, respectively). Inhi-
bition (%) was quantified using EDTA as a positive control,
which irreversibly removes metal ions from proteins (Figure
2c). NPs with PHX showed 28±7% of inhibition, while NP
with C1PHX showed significantly lower inhibitory effect,
indicating the importance of the carbon spacer for the hydrox-
amate and benzyl group binding to the Zn and S-pocket at the
buried active site cavity of the enzyme. In addition, NPs with
comparable amounts of either HX or MHPM did not inhibit
SVMPs even though these functional groups contain the six-
carbon spacer. 48-51 These results revealed that incorporation of
both hydroxamate and benzyl groups to the Zn and S-pocket
are required for inhibition. The absence of inhibition by NPs
with 5% AAc alone indicates that AAc itself is not a strong
candidate for binding to and deactivating the enzyme active
site.
A library of 2% crosslinked NIPAm NPs containing 40% of
candidate groups was screened for inhibition of Crotalus atrox
venom using the azocasein assay (Figure 1b and 2f, NP6-NP10 in
table S1). NPs with IBAm and PAm were synthesized with 5%
AAc for the the same reason mentioned above. NPs incorporating
bifunctional groups, N-acrylamido-S-leuicine (ALeu) and N-
acrylamido-S-phenylalanine (APhe) showed higher potency than
NPs having the same hydrophobic group but without a carboxylic
acid, N-isobutylacrylamide (IBAm) and N-phenylacrylamide
(PAm), indicating the importance of bifunctional structures for
SVMP binding and inhibition.55-57 Interestingly a NP having N-
benzamidineacrylamide (ABA) showed comparable potency with
a NP having APhe, indicating that electrostatic interactions be-
tween the NPs and SVMPs alone may not dominate the NP-
protein interaction and support the proposition that the NP-protein
interactions are ‘local.’ This result also indicates that the dom-
inate locus of binding of these functional groups is not at the cata-
lytic zinc cation site, but rather at the enzyme surface.
9
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
Bifunctional monomers such as APhe are more effective at
enzyme inhibition than monofunctional ones (PAm). To un-
derstand this result we measured the diameter of NPs with
APhe 40% and PAm 40% in water and PBS by dynamic light
scattering (Figure S2). APhe was significantly larger in PBS
pH 7.5) than that in water where most carboxylic acids are not
charged. This indicates that APhe containing NPs are highly
hydrated in PBS due to the intra-particle electrostatic repulsion
between charged carboxylate anions. On the other hand, PAm
containing NPs in PBS was comparable in size to those in
water indicating this NP is significantly dehydrated in PBS.
Note that NPs with PAm also contain 5% of AAc for colloidal
stability. In aqueous solution, hydrophobic groups incorpo-
rated in polymer NPs can form hydrophobic clusters within
polymer networks.58 This would alter the functional group
presentation of the NP and perhaps suppress the accessibility
of proteins into the interior of the NP. Therefore, there appears
to be a critical role of bifunctional monomers that contain both
hydrophobic and charged groups in proximity by allowing
polymer networks to swell providing greater protein access to
the functional groups of the NPs. It is important to note that
enzyme inhibition is used to evaluate NP-protein affinity.
Binding and inhibition are not strictly linked since NP binding
to the enzyme could occur remote from the active site. Despite
this caveat, the inhibition by APhe NPs would suggest some
fraction of the NP binding occurs in the vicinity of the active
site blocking access to the active site. These results identified
APhe as an effective binding group to the enzyme surface.
A docking simulation using analogue ligands supported the
experimental results (SI5).52 The binding free energies of cal-
culated conformations was evaluated by the RMSD based
cluster histogram and indicate a higher affinity of PHX for the
active site of SVMP than HX and MHPM (Figure 2d). The
docked model revealed the PHX group binds to both Zn and
S-pocket (Figure 2e). The buried PHX analogue in the active
site cavity also confirms the importance of the six-carbon
spacer (Figure S5). From these results, we can conclude that
PHX can function by binding to the Zn and S-pocket at the
enzyme active site.
Identification of groups that contribute to binding to the
enzyme surface
Although modest inhibition was achieved by PHX containing
NPs, further improvement of the inhibitory effect was sought
by enhancing the intrinsic affinity of the NP to SVMPs. To
that end, we evaluated enzyme surface binding formulations.
These were examined independent of active site binding
groups. Functional monomers that included aliphatic or aro-
matic hydrophobic groups were chosen since SVMPs are
3
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 4 of 10
1
2
3
4
5
6
7
8
9
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
Figure 2 (a) Protein composition of Crotalus atrox venom47 (b) Crystal structures of P-I (PDB ID: 2W13) and P-III (PDB ID:
2DW0) class SVMP. The conserved catalytic domain and catalytic zinc cation are green and light blue in both structures, respec-
tively. (c) Inhibition of SVMPs from Crotalus atrox venom (0.05 gL-1) by NPs (0.5 gL-1) with 20% PHX (NP1), C1PHX 20%
(NP2), 20% HX (NP3), 20% MHPM (NP4) or without candidate groups for binding to the active site (NP5). All NPs contain 5%
AAc. (d) RMSD based cluster histogram of free energy of ligand-enzyme complex. (e) Docking results of the active site of P-I
SVMP, BaPI and ligand analogues of PHX (black), HX (white) and MHPM (gray) (e) The docked model of PHX analogue (ma-
genta) and active site of BaPI. Residues in S-pocket and Zn are green and light blue sphere, respectively. His142, His 146, His
152 and hydroxamate form
a
pyramid like structure around the catalytic zinc cation, indicating the coordi-
nation of hydroxamate with the zinc cation. (f) Inhibition of SVMPs from Crotalus atrox venom (0.05 gL-1) by NPs (0.5 gL-1) with
candidate groups for the enzyme surface binding, IBAm (NP6), PAm (NP7), ALeu (NP8), APhe (NP9) and ABA (NP10). NPs
without
charged
groups,
NP6
and
NP7
contain
5%
AAc.
Optimization of the three functional elements in the NP.
cates that PHX and APhe play different roles for enzyme
binding and inhibition. PHX selectively binds to the active
site, and the contribution of APhe arises in part from restrict-
ing access to the active site of the bound enzyme. Since APhe
interacts with the enzyme surface, increasing the amount of
APhe could enhance multivalent binding to the enzyme. We
established the optimal composition to be a 2% BIS cross-
linked NIPAm NP with PHX 20% and APhe 40%, NP16
(Figure 3b and 3c). In addition to the direct combination of
Zn, S-pocket and surface binding, the hydrophobic nature of
the NPs provided by APhe may also enhance the intrinsic
affinity of PHX to the catalytic zinc cation since the lower
Finally, a NP library was generated to find the optimum com-
bination and population of the functional elements that include
active site binding groups, PHX, and a group that enhances
enzyme surface-binding, APhe (Figure 1c, NP11-NP16 in
table S1). The comparison of potency of NPs having either
20% of PHX or 20% of APhe revealed that the PHX group
alone is more effective than APhe alone. Importantly the in-
hibitory effect was dramatically improved by the combination
of PHX and APhe (Figure 3a). The potency of NPs having the
same feed ratio of APhe reached a plateau when PHX was
10%. Whereas, the potency increased with an increase in the
percentage of APhe when PHX was fixed. This behavior indi-
dielectric medium favors the binding of small molecule lig-
59
ands
to
the
zinc
cation.
4
ACS Paragon Plus Environment

Page 5 of 10
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
10
11
Figure 3 (a) Inhibition of SVMPs from Crotalus atrox venom (0.05 gL-1) by NPs (0.5 gL-1) with combinations of PHX and APhe.
(b) Chemical structure of optimized NP with PHX 20% and APhe 40% (NP16). (c) Illustration of the binding interface between
SVMP and NP with the active site binding group, PHX displaying hydroxamate (red) functionalized with proximate benzyl
(green) and enzyme surface binding groups, APhe (blue). The catalytic zinc cation, substrate pocket and exposed hydrophobic
surface of the enzyme are shown as a cyan sphere, green and blue, respectively.
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
Broad-spectrum SVMPs inhibitory effect of NP with PHX
20% and APhe 40% (NP16)
fectively inhibited all SVMP venoms (102±12% of inhibition)
with comparable IC50 values (101±17 µg/mL) despite the di-
versity of the SVMP population and composition of these
venoms (Figure 4f). Despite a slight reduction in inhibition
(75±10%) the NP was effective in the presence of whole hu-
man serum (Figure 4g). We also observed that the inhibitory
effect was independent of the pre-incubation period of the NP-
venom mixture indicating the fast action of NP (Figure S3).
The broad-spectrum SVMPs inhibitory effect of NP16 was
confirmed by using venom from Bitis arietans, Bitis gabonica,
Echis ocellatus and Echis carinatus in addition to Crotalus
atrox (Figure 4a-e and SI4). The choices represent a group of
medically relevant venoms all rich in SVMPs but with dis-
tinctly different variant compositions.30, 47, 54, 60-62 The NP ef-
Figure 4. (a)-(e) SVMP inhibitory effect of NPs containing PHX 20% and APhe 40% (NP16) for (a) Crotalus atrox, (b) Bitis ari-
etans, (c) Bitis gabonica, (d) Echis ocellatus and (e) Echis carinatus venom as a function of NP concentration. (f) IC50 of NPs with
PHX 20% and APhe 40% for SVMPs from five species of venomous snakes. (g) Effect of human serum (0 (white), 12.5 (gray)
and 25 vol% (black)) on the inhibitory effect of NP16 (0.5 gL-1) for SVMPs. (a)-(g) The concentration of venom was 0.05 gL-1.
5
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 6 of 10
SVMPs toxin affinity and selectivity of PHX 20% and SVMP even at a ten-fold excess of venom/NP (g/g) indicating
1
APhe 40% NPs (NP16),
a large binding capacity of NP16 (Figure S6).
2
3
4
5
6
7
8
9
The SVMP affinity and selectivity of NP16 was investigated
by a pull-down filter experiment (Figure 5 and SI6). After
filtration of a mixture of NPs and Crotalus atrox venom, the
enzymatic activity of unbound SVMPs in the filtrate was
quantified. Approximately 70% of SVMPs were sequestrated
by NP16, a value substantially larger than a NP with 40% of
APhe alone (NP9). The NP-protein complexes were repeated-
ly washed and eluted to assess the stability of the complex.
Interestingly, little SVMP was released from the NP16 (black
circles in Figure 6a), whereas almost all SVMP proteins were
released from the NP9 (78±10%) after several washings
(white circles in Figure 6a). From these results, we conclude
that the combination of binding groups led to the increase of
binding capacity and affinity of the NP for the target enzymes.
It was also revealed that NP16 sequestrated more than 50% of
After the repeated washings and filtration of the complex of
venom and NP16, strongly bound proteins from Crotalus
atrox venom by the NP were run and visualized on an SDS-
PAGE gel to assess the selectivity of NP for the target en-
zymes (Figure 6b). Despite the complexity of whole venom,
the gel revealed only two dominant bands. Following trypsin-
digestion and analysis by mass spectrometry of the bands, we
established the protein composition of the two bands was
comprised of SVMPs, members of the PI family, atroxase, Ht-
e and atrolysin-B in the lower MW band (~23KD) and mem-
bers of the PIII family, VAP2B and VAP2A in the higher MW
band (~68KD) (Figure S8 and Table S3). The results confirm
that the inhibitory effect of NP16 was due to the highly selec-
tive sequestration and inhibition of PI and PIII components of
SVMP.
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
Figure 5. Procedure of pull-down filter experiment. (1) The mixture of NP (1 gL-1) and Crotalus atrox venom (1 or 5 gL-1) was
incubated in PBS at 37°C. The mixture was filtrated by ultrafiltration (MWCO: 300 kDa, 5 kG, 20 min) to remove the NP-protein
complex from solution. (2) Unbound SVMPs in the filtrate was quantified by enzymatic assay. The NP-protein complex on the
filter was repeatedly washed and filtrated to elute weakly bound proteins. Eluted proteins after each washing was quantified by
enzymatic assay. (3) Proteins strongly bound by the NP were analyzed by SDS-PAGE and (4) in-gel tryptic digestion followed by
mass spectrometric analysis.
Figure 6. (a) Accumulated proteolytic activity of SVMPs (%) in flow-through and eluted fractions after washings. Black circles
and white circles indicate non-bond and eluted SVMPs from NPs with PHX 20% and APhe 40% (NP16 ●) and NP with APhe
40% alone (NP9 ❍), respectively. (b) SDS-PAGE visualization of Crotalus atrox venom and strongly bound proteins by NP16.
●
6
ACS Paragon Plus Environment

Page 7 of 10
Conclusion
Journal of the American Chemical Society
(5) Koide, H.; Yoshimatsu, K.; Hoshino, Y.; Lee, S. H.; Okajima, A.;
Ariizumi, S.; Narita, Y.; Yonamine, Y.; Weisman, A. C.; Nishimura,
Y.; Oku, N.; Miura, Y.; Shea, K. J. A polymer nanoparticle with en-
gineered affinity for a vascular endothelial growth factor (VEGF165).
Nat. Chem. 2017, 9, 715−722.
1
By mimicking the mechanism of action of endogenous tissue
inhibitors of metalloproteinases (TIMPs), we have developed
an effective broad-spectrum synthetic polymer NP inhibitor of
a family of metalloproteinases. The mimic utilizes the collec-
tive interactions of three variables, binding to the catalytic zinc
cation (Zn) and substrate-binding pocket (S-pocket) in addi-
tion to modulating binding of the NP to the enzyme surface.
The pull-down filter experiment followed by proteomic analy-
sis confirms that inhibition arises from sequestration of PI and
PIII, the two important classes of the SVMP family with high
selectivity and affinity, from the complex mixture of Crotalus
atrox venom. The synthetic polymer inhibitor provides broad
coverage of SVMPs from multiple species of medially im-
portant venomous snakes. Our results demonstrate the poten-
tial of NPs to be engineered to function as an abiotic antidote
of snake envenoming. We believe this strategy is general for
the systematic and rational design of effective polymer inhibi-
tors for target enzymes.
2
3
4
5
6
7
8
9
(6) Renner, C.; Piehler, J.; Schrader, T. Arginine- and Lysine-Specific
Polymers for Protein Recognition and Immobilization. J. Am. Chem.
Soc. 2006, 128, 620−628.
(7) Latza, P.; Gilles, P.; Schaller, T.; Schrader, T. Affinity Polymers
Tailored for the Protein A Binding Site of Immunoglobulin G Pro-
teins Chem. Eur. J. 2014, 20, 11479−11487.
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
(8) Hong, R.; Fischer, N. O.; Verma, A.; Goodman, C. M.; Emrick,
T.; Rotello, V. M. Control of Protein Structure and Function through
Surface Recognition by Tailored Nanoparticle Scaffolds. J. Am.
Chem. Soc. 2004, 126, 739-743
(9) You, C.; De, M.; Han, G. Rotello, V. M. Tunable Inhibition and
Denaturation of α-Chymotrypsin with Amino Acid-Functionalized
Gold Nanoparticles. J. Am. Chem. Soc. 2005, 127, 12873-12881
(10) Wenck, K. Koch, S. Renner, C. Sun, W. Schrader, T. A Non-
covalent Switch for Lysozyme. J. Am. Chem. Soc. 2007, 129, 16015-
16019
ASSOCIATED CONTENT
Supporting Information
Experimental procedures and supporting data. This material is
available free of charge via the Internet at http://pubs.acs.org.
(11) Gilles, P.; Wenck, K.; Stratmann, I.; Kirsch, M.; Smolin, D. A.;
Schaller, T.; de Groot, H.; Kraft, A.; Schrader, T. High-Affinity Co-
polymers Inhibit Digestive Enzymes by Surface Recognition. Biom-
acromolecules 2017, 18, 1772−1784.
AUTHOR INFORMATION
Corresponding Author
(12) Ganguli, S.; Yoshimoto, K.; Tomita, S.; Sakuma, H.; Matsuoka,
T.; Shiraki, K.; Nagasaki, Y. Regulation of lysozyme activity based
on thermotolerant protein/smart polymer complex formation. J. Am.
Chem. Soc. 2009, 131, 6549−6553.
* kjshea@uci.edu
Author Contributions
All authors approved the final version of the manuscript.
(13) Cha, S.-H.; Hong, J.; McGuffie, M.; Yeom, B.; VanEpps, J.
S.;Kotov, N. A. Shape-Dependent Biomimetic Inhibition of Enzyme
by Nanoparticles and Their Antibacterial Activity. ACS Nano 2015, 9,
9097−9105.
ACKNOWLEDGMENT
We are grateful to Professor José María Gutiérrez, Universidad de
Costa Rica for helpful discussions and advice about this work. We
thank the Laser Spectroscopy Laboratory and Mass Spectrometry
Facility at University of California, Irvine. NM thanks the Japan
Society for the Promotion of Science 15J04705 for partial sup-
port. ORB thanks the UC Irvine Undergraduate Research Oppor-
tunities Program for a research grant.
(14) Lira, A., L.; Ferreira, R. S.; Torquato, R. J. S.; Oliva, M. L. V.;
Schuck, P.; Sousa, A. A. Allosteric inhibition of α-thrombin enzymat-
ic activity with ultrasmall gold nanoparticles. Nanoscale Adv. 2019, 1,
378–388
(15) Hijazi, M.; Krumm, C.; Cinar, S.; Arns, L.; Alachraf, W.; Hiller,
W.; Schrader, W.; Winter, R; Tiller, J. C. Entropically driven Poly-
meric Enzyme Inhibitors by End-Group directed Conjugation. Eur. J.
2018, 24, 4523−4527.
REFERENCES
(1) Farady, C. J.; Craik, C. S. Protease Inhibitors, Mechanisms of.
Wiley Encyclopedia of Chemical Biology, John Wiley & Sons 1555-
1574. doi:10.1002/9780470048672.wecb474
(16) Cutivet, A.; Schembri, C.; Kovensky, J.; Haupt, K. Molecularly
Imprinted Microgels as Enzyme Inhibitors. J. Am. Chem. Soc. 2009,
131, 14699−702.
(2) Natesh, R.; Schwager, S. L. U.; Sturrock, E. D.; Acharya, K. R.
Crystal structure of the human angiotensin-converting enzyme-
lisinopril complex. Nature 2003, 421, 551−554.
(17) Tran, H.; Kitov, P. I.; Paszkiewicz, E.; Sadowska, J. M.; Bundle,
D. R. Multifunctional multivalency: a focused library of polymeric
cholera toxin antagonists. Org. Biomol. Chem. 2011, 9, 3658.
(3) Orth, P.; Reichert, P.; Wang, W.; Prosise, W. W.; Yarosh-
Tomaine,T.; Hammond, G.; Ingram, R. N.; Xiao, L.; Mirza, U. A.;
Zou, J.; Strickland, C.; Taremi, S. S.; Le, H. V.; Madison, V. Crystal
structure of the catalytic domain of human ADAM33. J. Mol. Biol.
2004, 335, 129-137.
(18) O’Brien, J.; Lee, S. H.; Onogi, S.; Shea, K. J. Engineering the
Protein Corona of a Synthetic Polymer Nanoparticle for Broad-
Spectrum Sequestration and Neutralization of Venomous Biomacro-
molecules. J. Am. Chem. Soc. 2016, 138, 16604−16607.
(4) Hoshino, Y.; Koide, H.; Furuya, K.; Haberaecker, W. W.; Lee, S.-
H.; Kodama, T.; Kanazawa, H.; Oku, N.; Shea, K. J. The rational
design of a synthetic polymer nanoparticle that neutralizes a toxic
peptide in vivo. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 33.
(19) Stiti, M.; Cecchi, A.; Rami, M.; Abdaoui, M.; Barraga-Montero,
V.; Scozzafava, A.; Guari, Y.; Winum, J. Y.; Supuran, C. T. Carbonic
Anhydrase Inhibitor Coated Gold Nanoparticles Selectively Inhibit
7
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 8 of 10
the Tumor-Associated Isoform IX over the Cytosolic Isozymes I and
(33) Wagstaff, S. C.; Laing, G. D.; Theakston, R. D. G.; Papaspyridis,
II. J. Am. Chem. Soc. 2008, 130, 16130-16131.
C.; Harrison, R. A. Bioinformatics and Multiepitope DNA Immuniza-
tion to Design Rational Snake Antivenom. PLoS Med. 2006, 3, e184.
1
2
3
4
5
6
7
8
(20) Nel, A. E.; mädler, L.; Velegol, D.; Xia, T.; Hoek, E. M. V.;
Somasundaran, P.; Klaessig, F.; Castranova, V.; Thompson, M. Un-
derstanding biophysicochemical interactions at the nano-bio interface.
Nature Materials 2009, 8, 543–557.
(34) O’Brien, J.; Lee, S. H.; Gutiérrez, J. M.; Shea, K. J. Engineered
nanoparticles bind elapid snake venom toxins and inhibit venom-
induced dermonecrosis. PLoS Neglected Trop. Dis. 2018, 12, No.
e0006736.
(21) Monopoli, M. P.; Aberg, C.; Salvati, A.; Dawson, K. A. Bio-
molecular coronas provide the biological identity of nanosized mate-
rials. Nat. Nanotechnol. 2012, 7, 779−786.
(35) Takeda, S.; Takeya, H.; Iwanaga, S. Snake venom metallopro-
teinases: structure, function and relevance to the mammalian
ADAM/ADAMTS family proteins. Biochimica et Biophysica Acta
1824, 2012, 164–176.
9
(22) Tenzer, S.; Docter, D.; Kuharev, J.; Musyanovych, A.; Fetz,
V.;Hecht, R.; Schlenk, F.; Fischer, D.; Kiouptsi, K.; Reinhardt,
C.;Landfester, K.; Schild, H.; Maskos, M.; Knauer, S. K.; Stauber, R.
H. Rapid formation of plasma protein corona critically affects nano-
particle pathophysiology. Nat. Nanotechnol. 2013, 8, 772−781.
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
(36) Gomis-Ruth, F. X. Catalytic domain architecture of metzincin
metalloproteases. THE JOURNAL OF BIOLOGICAL CHEMISTRY
2009, 284, 15353–15357.
(23) Davis, M. E.; Chen, Z.; Shin, D. M. Nanoparticle therapeutics: an
emerging treatment modality for cancer. Nat. Rev. Drug Discovery
2008, 7, 771-782.
(37) Rucavado, A.; Escalante, T.; Gutiérrez, J. M. Effect of the metal-
loproteinase inhibitor batimastat in the systemic toxicity induced by
Bothrops asper snake venom: understanding the role of metallopro-
teinases in envenomation. Toxicon 2004, 43, 417–424.
(24) Walkey, C. D.; Olsen, J. B.; Guo, H.; Emili, A.; Chan, W. C. W.
Nanoparticle size and surface chemistry determine serum protein
adsorption and macrophage uptake. J. Am. Chem. Soc. 2012, 134,
2139−2147
(38) Hu, J.; Van den Steen, P. E.; Sang, Q. X. A.; Opdenakker, G.
Matrix metalloproteinase inhibitors as therapy for inflammatory and
vascular diseases. Nat. Rev. Drug Discovery 2007, 6, 480−498.
(25) Anselmo, A. C.; Zhang, M.; Kumar, S.; Vogus, D. R.; Menegatti,
S.; Helgeson, M. E.; Mitragotri, S. Elasticity of nanoparticles influ-
ences their blood circulation, phagocytosis, endocytosis, and target-
ing. ACS Nano 2015, 93, 3169-3177.
(39) Vandenbroucke, R. E.; Libert, C. Is there new hope for therapeu-
tic matrix metalloproteinase inhibition? Nat. Rev. Drug Discovery
2014, 13, 904−927.
(40) Chen, A. Y.; Adamek, R. N.; Dick, B. L.; Credille, C. V.; Morri-
son, C. N.; Cohen, S. M. Targeting Metalloenzymes for Therapeutic
Intervention. Chem. Rev. 2019, 119, 1323−1455.
(26) Bourboulia, D.; Stetler-Stevenson, W. G. Matrix metalloprotein-
ases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs):
Positive and negative regulators in tumor cell adhesion. Seminars in
Cancer Biology 2010, 20, 161–168.
(41) Puerta, D. T.; Lewis, J. A.; Cohen, S. M. New Beginnings for
Matrix Metalloproteinase Inhibitors: Identification of High-Affinity
Zinc-Binding Groups. J. Am. Chem. Soc. 2004, 126, 8388–8389.
(27) Huang, W.; Meng, Q.; Suzuki, K.; Nagase, H.; Brew, K. Muta-
tional study of the amino-terminal domain of human tissue inhibitor
of metalloproteinases 1 (TIMP-1) locates an inhibitory region for
matrix metalloproteinases. J. Biol. Chem. 272, 22086−22091.
(42) Pelton, R. Temperature-sensitive aqueous microgels. Adv. Col-
loid Interface Sci. 2000, 85, 1−33.
(28) Batra, J.; Robinson, J.; Soares, A. S.; Fields, A. P.; Radisky, D.
C.; Radisky, E. S. Matrix Metalloproteinase-10 (MMP-10) Interaction
with Tissue Inhibitors of Metalloproteinases TIMP-1 and TIMP-2
BINDING STUDIES AND CRYSTAL STRUCTURE.
JOURNAL OF BIOLOGICAL CHEMISTRY 2012, 287, 15935–15946
(43) Nayak, S.; Lyon, L. A. Soft nanotechnology with soft nanoparti-
cles. Angew. Chem., Int. Ed. 2005, 44, 7686-7708.
THE
(44) Hu, X.; Tong, Z.; Lyon, L. A. Control of Poly(N-
isopropylacrylamide) Microgel Network Structure by Precipitation
Polymerization near the Lower Critical Solution Temperature. Lang-
muir 2011, 27, 4142–4148
(29) Gomis-Ruth, F. X.; Maskos, K.; Betz, M.; Bergner, A.; Huber,R.;
Suzuki, K.; Yoshida, N.; Nagase, H.; Brew, K.; Bourenkov, G.
P.;Bartunik, H.; Bode, W. Mechanismof inhibition of the humanma-
trix metalloproteinase stromelysin-1 by TIMP-1. Nature 1997, 389,
77−81.
(45) Maimon, E.; Samuni, A.; Goldstein, S. Nitrogen Dioxide Reac-
tion with Nitroxide Radical Derived from Hydroxamic Acids: The
Intermediacy of Acyl Nitroso and Nitroxyl (HNO). J. Phys. Chem. A,
2018, 122, 3747−3753.
(30) Gutiérrez, J. M.; Rucavado, A. Snake venom metalloproteinases:
Their role in the pathogenesis of local tissue damage. Biochimie,
2000, 82, 841−850.
(46) Gutiérrez, J. M.; Sanz, L.; Escolano, J.; Fernández, J.; Lomonte,
B.; Angulo, Y.; Rucavado, A.; Warrell, D. A.; Calvete, J. J. Snake
Venomics of the Lesser Antillean Pit VipersBothropscaribbaeus and
Bothrops lanceolatus: Correlation with ToxicologicalActivities and
Immunoreactivity of a Heterologous Antivenom. Journal of Proteome
Research 2008, 7, 4396–4408
(31) Gutiérrez, J. M.; Escalante, T.; Rucavado, A.; Herrera, C.; Fox, J.
A Comprehensive View of the Structural and Functional Alterations
of Extracellular Matrix by Snake Venom Metalloproteinases
(SVMPs): Novel Perspectives on the Pathophysiology of Envenom-
ing. Toxins 2016, 8, 304
(47) Calvete, J. J.; Fasoli, E.; Sanz, L.; Boschetti, E.; Righetti, P. G.
Exploring the venom proteome of the western diamondback rattle-
snake, Crotalus atrox, via snake venomics and combinatorial peptide
ligand library approaches. Journal of Proteome Research 2009, 8,
3055–3067
(32) Gutierrez, J. M.;Calvete, J. J.; Habib, A. G.; Harrison, R. A.;
Williams, D. J.; Warrell, D. A. Snakebite envenoming. Nat. Rev. Dis.
Primers. 2017, 3, 17063.
8
ACS Paragon Plus Environment

Page 9 of 10
Journal of the American Chemical Society
(48) Fruehauf, K., R.; Kim, T. I.; Nelson, E. L.; Patterson, J. P.;
Wang, S-W.; Shea, K. J. Metabolite Responsive Nanoparticle–Protein
Complex. Biomacromolecules 2019, 20, 2703−2712.
1
2
3
4
5
(49) O’Carra, P.; Barry, S. Affinity chromatography of lactic acid
dehydrogenase on N-(6-aminohexyl)oxamate-Sepharose. FEBS Lett.
1972, 21, 281−285.
6
7
8
9
(50) Roop-ngam, P.; Thongboonkerd, V. Development of an oxalate-
affinity chromatographic column to isolate oxalate-binding proteins.
Anal. Methods 2010, 2, 1051−1055.
(51) Vaidya, A. A.; Lele, B. S.; Kulkarni, M. G.; Mashelkar, R. A.
Enhancing ligand–protein binding in affinity thermoprecipitation:
Elucidation of spacer effects. BIOTECHNOLOGY AND
BIOENGINEERING 1999, 64, 418-425.
(62) Patra, A.; Kalita, B.; Chanda, A.; Mukherjee, A. K. Proteomics
and antivenomics of Echis carinatus carinatus venom: Correlation
with pharmacological properties and pathophysiology of enven-
omation. Sci. Rep. 2017, 7, 17119.
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
(52) Santos-Martins, D.; Forli, S.; Ramos, M. J.; Olson, A. J.
AutoDock4(Zn): an improved AutoDock force field for small-
molecule docking to zinc metalloproteins. J. Chem. Inf. Model. 2014,
54, 2371– 2379.
(53) Calvete, J. J.; Marcinkiewicz, C.; Sanz, L. Snake venomics of
Bitis gabonica gabonica. Protein family composition, subunit organi-
zation of venom toxins, and characterization of dimeric disintegrins
bitisgabonin-1 and bitisgabonin-2. Journal of Proteome Research
2007, 6, 326-336.
(54) Wagstaff, S. C.; Sanz, L.; Juárez, P.; Harrison, R. A.; Calvete, J.
J. Combined snake venomics and venom gland transcriptomic analy-
sis of the ocellated carpet viper, Echis ocellatus. JOURNAL OF
PROTEOMICS 2009, 71, 609–623.
(55) Wang, X.; Gan, H.; Sun, T. Chiral Design for Polymeric Bioin-
terface: The Influence of Surface Chirality on Protein Adsorption.
Adv. Funct. Mater. 2011, 21, 3276–3281.
(56) Wang, X.; Gan, H.; Zhang, M.; Sun, T. Modulating cell behav-
iors on chiral polymer brush films with different hydrophobic side
groups. Langmuir 2012, 28, 2791−2798.
(57) Raczkowska, J.; Ohar, M.; Stetsyshyn, Y.; Zemła, J.; Awsiuk,
K.; Rysz, J.; Fornal, K.; Bernasik, A.; Ohar, H.; Fedorova, S.;
Shtapenko, O.; Polovkovych, S.; Novikov, V.; Budkowski, A. Tem-
perature-responsive peptide-mimetic coating based on poly(N-
methacryloyl-l-leucine): properties, protein adsorption and cell
growth. Colloids and Surfaces B: Biointerfaces 2014, 118, 270–279.
(58) Chou, B.; Mirau, P.; Jiang, T.; Wang, S.; Shea, K. J. Tuning
Hydrophobicity in Abiotic Affinity Reagents: Polymer Hydrogel
Affinity Reagents for Molecules with Lipid-like Domains. Biom-
acromolecules, 2016, 17, 1860−1868.
(59) Šramko, M.; Šille, J.; Ježko, P.; Garaj, V. Effect of dielectric
medium on angiotensin converting enzyme inhibitors binding to Zn2+.
Chemical Papers, 2010, 64, 395–404.
(60) Lavonas, E. J.; Tomaszewski, C. A.; Ford, M. D.; Rouse, A. M.;
Kerns II, W. P. Severe Puff Adder (Bitis arietans) Envenomation with
Coagulopathy. Journal of Toxicology: Clinical Toxicology 2002, 40,
911-918.
(61) Marsh, N.; Gattullo, D.; Pagliaro, P.; Losano, G. The Gaboon
viper, Bitis gabonica: Hemorrhagic, metabolic, cardiovascular and
clinical effects of the venom. Life Sci. 1997, 61, 763–769.
9
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 10 of 10
1
2
3
Insert Table of Contents artwork here
4
5
6
7
8
9
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
10
ACS Paragon Plus Environment