Fluorescent water soluble polymers for isozyme-selective interactions with matrix metalloproteinase-9

Article abstract of DOI:10.1016/j.bmcl.2011.02.020

Matrix metalloproteinases (MMPs) are overexpressed in various pathological conditions, including cancers. Although these isozymes have similar active sites, the patterns of exposed amino acids on their surfaces are different. Herein, we report the synthes

Full text of DOI:10.1016/j.bmcl.2011.02.020

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2007–2010
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Fluorescent water soluble polymers for isozyme-selective interactions
with matrix metalloproteinase-9
Rinku Dutta a, Michael D. Scott a, Manas K. Haldar a, Bratati Ganguly b, D. K. Srivastava b,
Daniel L. Friesner c, Sanku Mallik a,
⇑
a Department of Pharmaceutical Sciences, North Dakota State University, Fargo, ND 58102, USA
b Department of Chemistry and Biochemistry, North Dakota State University, Fargo, ND 58102, USA
c Department of Pharmacy Practice, North Dakota State University, Fargo, ND 58102, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Matrix metalloproteinases (MMPs) are overexpressed in various pathological conditions, including
cancers. Although these isozymes have similar active sites, the patterns of exposed amino acids on their
surfaces are different. Herein, we report the synthesis and molecular interactions of two water soluble,
fluorescent polymers which demonstrate selective interactions with MMP-9 compared to MMP-7
and -10.
Received 10 January 2011
Revised 3 February 2011
Accepted 4 February 2011
Available online 28 February 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Polymers
Matrix metalloproteinases
Enzymes
Selective interactions
Fluorescence spectroscopy
Water soluble, flexible polymers have been used by various
groups to selectively bind to different proteins.1–3 The flexibility
of the polymer backbone aids in the formation of multiple, comple-
mentary interaction sites between the polymer and the amino acid
residues on the protein surface. These polymers have been demon-
strated to be useful as affinity membranes, as switches to ‘turn on’
enzyme activity, as selective protein immobilization agents, as pro-
tein sensors, etc.1–3 However, the proteins used in these selective
recognition experiments are structurally very different (e.g., lyso-
zyme, bovine serum albumin, cytochrome c, etc.). To the best of
our knowledge, selective binding of polymers to different isozymes
of an enzyme family has not been demonstrated.
enzyme can be as high as 100–200 nM in the bronchial lavage flu-
ids.13 The levels of MMP-9 serve as diagnostic and prognostic
markers for these diseases.12,13
Since isozymes catalyze the same chemical reaction, their active
sites are remarkably similar.14 However, the pattern of amino acid
residues on the surface of the isozymes are not under evolutionary
pressure and are usually not conserved.15 We reasoned that this
difference can be exploited for selective binding of polymers to
one isozyme in preference to the others. Herein, we report our re-
sults on selective binding to recombinant human matrix metallo-
proteinase-9 (MMP-9) by a set of water soluble, flexible polymers.
We synthesized two sets of monomers using two different poly-
merizable moieties. The first set included the use of a commonly
used water soluble compound methacrylic acid. For the second
set, we choose to examine the effects of a benzene ring incorpo-
rated into the structure and 4-vinylbenzoic acid was used. We con-
jugated the polymerizable moieties to an alcohol (for increased
water solubility and hydrogen bonding with amino acid residues
on the surface of MMPs), to the fluorogenic dansyl group and to
a non-selective MMP inhibitor. Different amino acids such as lysine
(positively charged), aspartic acid (negatively charged) and b-ala-
nine (non-polar) were also linked to the polymerizable groups to
impart electrostatic and hydrophobic properties to the polymers
(Scheme 1, synthetic details are provided in Supplementary data).
Random copolymers were prepared from these synthesized
monomers using azoisobutyronitrile (AIBN) as the free-radical
Matrix metalloproteinases (MMPs) are a group of Zn2+ contain-
ing metalloenzymes capable of hydrolyzing the extracellular ma-
trix.4,5 These enzymes are involved in a number of different
physiological processes, for example, cell proliferation, apoptosis,
differentiation, angiogenesis, chemokine/cytokine activation and
the expression levels of these enzymes are strictly regulated at
multiple levels.6–9 Various MMP isozymes (in particular MMP-9)
have been shown to be upregulated in degenerative diseases, for
example, arthritis, multiple sclerosis, metastatic cancers, etc.9–11
In healthy individuals, the serum concentration of MMP-9 is about
5–10 nM.12 For lung cancer patients, the concentration of this
⇑
Corresponding author. Tel.: +1 701 231 7888; fax: +1 701 231 8333.
E-mail address: Sanku.Mallik@ndsu.edu (S. Mallik).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.02.020

2008
R. Dutta et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2007–2010
(1) R'COOH,
HOBT, HBTU,
iPr2NEt
R
R: -CH2COOH
-(CH2)4NH2
R
O
R':
BuOt
HO
NH2
R'
N
H
(2) CF3COOH,
iPr3SiH
O
R: -CH2COOH
-(CH2)4NH2
O
R':
H3C
Amino acid
monomer
O
R'COOH,
OH
HOBT, HBTU,
OH
H2N
R'
N
H
Alcohol monomer
iPr2NEt
OH
OH
O
O
H
(1) H2NCH2CH2NHBoc
HBTU, HOBT, iPr2NEt
O
N
S
CH3
R'
N
H
N
R'
OH
(2) CF3COOH
(3) Dansyl chloride, iPr2NEt
CH3
O
Fluorescent monomer
(1) H2N(CH2CH2O)2CH2CH2NHBoc
HBTU, HOBT, iPr2NEt
(2) CF3COOH
O
O
O
O
NHOH
R'
N
H
N
R'
OH
2 H
O
O
OCPh3
(3)
HO2C
HBTU, HOBT, iPr2NEt
(4) CF3 COOH
N
H
Inhibitor monomer
Scheme 1. Synthesis of methacrylamide and 4-vinylbenzamide based monomers.
initiator following a literature protocol.2,3 Because of flexibility, we
decided to prepare a small set of polymers and screen for selective
interactions with MMP-9. We systemically varied the starting
monomer compositions (Table 1) and studied the interactions of
the resultant polymers with recombinant human MMP-7, -9 and
-10. The molecular weights of the polymers were determined by
gel permeation chromatography (GPC). The molecular weights
were in the range of 83–129 kDa with polydispersities in the range
1.2–2.5 (Table 2).
Table 2
The weight average (Mw) and number average (Mn) molecular weights, polydisper-
sities (P.I.) of the polymers R1–R11, M1–M7 and the concentrations used during the
titration experiments with MMP isozymes
Polymers
Mw
Mn
66,623
P.I.
Concn used (nM)
R1
128,920
107,291
99,486
119,800
96,232
94,514
116,450
93,405
117,010
106,802
117,191
106,622
128,790
83,618
115,498
114,428
89,801
1.93
2.44
1.97
2.27
2.59
1.78
2.44
1.98
1.54
1.59
1.81
1.59
1.23
2.04
1.58
1.46
2.36
1.65
27
34
31
28
32
34
26
36
27
30
31
29
24
36
26
27
34
24
R2
43,945
50,436
52,727
37,139
52,959
47,670
47,258
76,187
67,273
64,577
66,942
104,510
40,895
72,963
78,161
37,978
77,145
R3
R4
R5
R6
R7
R8
Table 1
R9
The starting monomer amounts (mol %) for the synthesized methacrylamide (R1–R11
and 4-vinylbenzamide-based polymers (M1–M7)
)
R10
R11
M1
M2
M3
M4
M5
M6
M7
Polymer
Monomers (mol %)
Inhibitor
Dansyl
Alcohol
Asp
Lys
Ala
R1
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
18
12
11
80
90
80
80
70
60
70
70
80
70
50
80
70
80
70
45
64
78
10
—
—
—
—
10
10
10
—
10
20
—
10
—
10
19
—
—
—
—
10
10
10
10
—
—
—
10
—
—
10
10
9
12
—
—
—
—
—
—
—
—
—
—
—
10
10
—
—
—
—
—
—
—
11
R2
R3
10
—
10
10
—
10
—
—
10
10
10
—
—
9
R4
127,656
R5
R6
R7
R8
To study the interactions of the synthesized polymers with the
MMP isozymes, nanomolar solutions of the polymers (25–35 nM in
30 mM phosphate buffer, pH 7.4, Table 1) were prepared and the
MMP isozymes were added to make 200 nM of the final enzyme
concentration (details are provided in Supplementary data). The
changes in the emission spectra of the polymer-incorporated dan-
syl group were recorded in the region 350–750 nm (kex = 325 nm;
Fig. 1 and Supplementary data). The emission intensity was found
to decrease in the presence of added enzymes. This possibly
reflects that the polymer-incorporated dansyl groups are experi-
R9
R10
R11
M1
M2
M3
M4
M5
M6
M7
12
—
—

R. Dutta et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2007–2010
2009
where i indexes each observation; P() denotes the cumulative logis-
tic distribution; D is a binary indicator of each (j = 1,...,J) polymer, k
denotes each isozyme (MMP-7, -9, and -10), F is the fluorescence of
each isozyme; J denotes the number of polymers (11 and 7, respec-
20000
15000
10000
5000
0
tively) and b,
c are parameter estimates. While this is estimated as
single maximum likelihood estimation, the stacked data matrix al-
lows separate intercept and slope estimates for each polymer in-
cluded in the regression. That is, each of the polymer-specific
response functions in a given group is ‘stacked’ and estimated to-
gether to preserve adequate degrees of freedom to run the regres-
sion. As noted earlier, Eq. (1) is estimate twice, once for each
random copolymer group.
Because the goal of the analysis was to indentify which poly-
mers selectively interacts with MMP-9, Eq. (1) was estimated as
a binary logit model, where the dependent variable takes a value
of one for a MMP-9 isozyme, and zero if the isozyme is in the
remaining categories (MMP-7, -10). This ensures a parsimonious
estimation procedure without being forced to estimate Eq. (1) mul-
tiple times for each isozyme-copolymer grouping (MMP-9 vs
MMP-7, MMP-9 vs MMP-10, etc.). Additionally, because each poly-
mer has a separate response function with two parameter esti-
mates (a slope and an intercept), results are presented using an
odds ratio (with accompanying 95% profile confidence intervals)
capturing the joint effects of these intercept and slope parameters
for a given polymer on the MMP-9 isozyme. Values greater than
unity (and whose confidence intervals do not contain a value of
one) indicate that the polymer significantly predicts or identifies
the given MMP relative to its alternatives.
400
500
600
700
Wavelength (nm)
Figure 1. Fluorescence emission spectra of the polymer R11 (31 nM in 30 mM
phosphate buffer, pH 7.4, kex = 325 nm, black trace) in presence of 200 nM of
recombinant human MMP-7 (red trace), MMP-9 (blue trace) and MMP-10 (green
trace).
encing more hydrophilic microenvironments in the presence of re-
combinant enzymes.20 We observed that the relative decrease in
emission intensity (541 nm for polymethacrylamide polymers
R1–R11 and 510 nm for polyvinylbenzamide polymers M1–M7) de-
pends on the polymer used and the MMP isozyme tested. The ra-
tios of the fluorescence emission intensities (at 541 nm and
510 nm) in the absence and in the presence of the MMP isozymes
were calculated and subjected to statistical analyzes to determine
if they are significantly different.
Table 3 identifies those polymers whose odds ratios are signif-
icantly greater than unity. This statistical analysis revealed that it
is possible to design polymers that are able to selectively interact
with isozymes within the MMP family. In particular, note that
polymer R11 uniquely and significantly interacts with the MMP-9
isozyme in the methacrylamide copolymer group. Polymer M5 un-
iquely and significantly interacts with MMP-9 among the 4-vinylb-
enzamide copolymer group.
The primary objective of the statistical analysis was to deter-
mine whether (and to what extent) each of the polymer selectively
interacts with MMP-9 isozyme. Because each of the random
copolymer groups (methacrylamide and 4-vinylbenzamide, see Ta-
ble 1) is fundamentally different in structure, a decision was made
to analyze each group separately. A common approach to identify
these relationships is linear discriminant analysis.16,17 However,
the large number of polymers (11 in the methacrylamide random
copolymer group and 7 in the 4-vinylbenzamide group, including
one control for each group) relative to the small number of replica-
tions (6) for each enzyme–MMP pair made this approach infeasi-
ble.18 A commonly used alternative, employed in this analysis, is
(binary) logistic regression.19 More specifically, each of the
18 ꢀ 1 data matrices containing the fluorescence intensities for a
given polymer (6 replications and 3 MMPs per polymer) were
‘stacked’ or ‘blocked’ together to form a larger (198 ꢀ 1 for the
methacrylamide random copolymer group and 144 ꢀ 1 in the 4-
vinylbenzamide group) vector of fluorescence intensity readings
(11 polymers with 18 observations per polymer for the meth-
acrylamide group and 7 polymers with 18 observations for the 4-
vinylbenzamide group, respectively).19 Additional columns in the
data matrix were appended by creating a series of binary variables
that identify each polymer, and these binary indicator variables
were subsequently interacted with the fluorescence data to create
a fully interacted data matrix (198 ꢀ 22 and 144 ꢀ 16 for each
copolymer group, respectively). Lastly, three columns of binary
variables were appended, where each new column identified a par-
ticular MMP (-7, -9 or -10). The full data matrix are provided in the
Supplementary data accompanying this manuscript.
Both of R11 and M5 polymers contain the aspartic- and the ly-
sine monomers (10 mol % each) and 20 mol % of the inhibitor
monomer. These two polymers also contain the least amounts of
the alcohol monomers. Decreasing the amount of the inhibitor
monomer to 10 mol % (i.e., polymers R6), omitting any charged
monomers (polymers R8, M2 and M4) or the inhibitor monomer
from the polymers (i.e., R5, M6) led to the loss of selective interac-
tions of the polymers with MMP-9 (Supplementary data, Table S3).
Reducing the amount of inhibitor monomer (to 10 mol %) (i.e.,
polymer R6) or incorporation of more hydrophobic alanine-based
monomer in the polymer (i.e., R10 and M7) also had negative effect
on the selective interactions with MMP-9 (Supplementary data,
Table S3). We also observed that the polymers R11 and M5
(100 nM each) are effective in inhibiting the activity of the enzyme
MMP-9 (Supplementary data). Although we did not systematically
Table 3
Estimates and p-values from the logit regression analyzes
Methacrylamide-
based polymer
4-Vinylbenzamide-
based polymer
Polymers
R11
M5
Intercept
Intercept p-value
Slope
Slope p-value
Odds Ratio (OR)
95% OR lower confidence
interval limit
43.644
0.017
101.8
0.025
The value of this approach is that it allows for the estimation of
a fully interacted logit model of the following form:
ꢁ32.235
ꢁ85.875
0.016
0.026
187.598
1.347
>999.999
15.387
J
X
PðMMPki ¼ 1 j D; FÞ ¼
Djðbj þ
c
jFjÞ
i
i
j¼1
95% OR upper confidence
interval limit
>999.999
>999.999
PðMMPki ¼ 0 j D; FÞ ¼ 1 ꢁ PðMMPki ¼ 1 j D; FÞ
ð1Þ

2010
R. Dutta et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2007–2010
vary the steric effects in the monomers, these observations suggest
that possibly the inhibitor on the polymers is interacting with the
active site of MMP-9 and the charged amino acids are forming
additional interactions with the amino acid residues on the surface
of the enzyme. Incorporation of hydrophobic monomers was detri-
mental to the selectivity of interactions with MMP-9. However,
increasing the amount of inhibitor monomer to 30 mol % in the
polymers did not improve the selective binding to MMP-9 (data
not shown). We do not have an explanation for this observation
yet.
Next we proceeded to determine if the selective interactions of
the polymers R11 and M5 are maintained in a complex mixture of
proteins. The fluorescence emission from the polymer-incorpo-
rated dansyl group (kex = 325 nm) was found to increase and
blue-shift (about 100 nm; Fig. 2) in the presence of dilute (less than
5% by volume) human serum in phosphate buffer (pH 7.4). When
this dilute human serum contained 200 nM of MMP-9 (levels of
this enzyme in bronchial lavage fluid from lung cancer patients13),
the emission intensity was substantially increased and blue-
shifted (Fig. 2, blue trace). The same trends were observed when
the human serum contained either 200 nM MMP-7 (Fig. 2, olive
trace) or 200 nM MMP-10 (Fig. 2, magenta trace). This indicates
that the polymer-incorporated dansyl fluorophore was experienc-
ing a more hydrophobic microenvironment in the presence of di-
lute human serum and the MMPs.20 We note that with the
recombinant MMPs in buffer, the reverse was observed, that is,
the emission intensities decreased in the presence of added en-
zymes. This may indicate that the conformations of the polymers
change substantially when the buffer contains small amounts of
human serum. Clearly, the emission intensity in the presence of
MMP-9 was more pronounced in the presence of MMP-9 compared
to MMP-7 and -10. Increasing the amount of human serum to more
than 5% (by volume) led to the loss of the selective enhancements
of the polymer emission intensity in the presence of MMP-9 (data
not shown).
In conclusion, we have synthesized flexible, water soluble poly-
mers containing charges and a weak inhibitor for the MMPs. Two
of these polymers demonstrated selective interactions to the iso-
zyme MMP-9 compared to MMP-7 and -10, even in a complex mix-
ture of proteins (e.g., dilute human serum). We anticipate that by
incorporating more potent and selective MMP inhibitors in the
polymer and by optimizing the polymer structures, the selectivity
of the interactions with MMP-9 can be further enhanced and main-
tained in more concentrated human serum samples. These studies
are currently in progress and the results will be reported in the
future.
Acknowledgments
This research was supported by NIH grants 1R01 CA 113746,
1R01 CA 132034 to S.M. and D.K.S. and NSF grant DMR 1005011
to S.M.
Supplementary data
Supplementary (experimental details of the syntheses of the
monomers and the polymers; details of the fluorescence experi-
ments and detailed results of the statistical analysis) data associ-
ated with this article can be found, in the online version, at
doi:10.1016/j.bmcl.2011.02.020.
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