Characterization of a Novel Class of Polyphenolic Inhibitors of Plasminogen Activator Inhibitor 1

Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2010/01/08/M109.067967.DC1.html

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 11, pp. 7892–7902, March 12, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

Characterization of a Novel Class of Polyphenolic Inhibitors
of Plasminogen Activator Inhibitor-1*□ S

Received for publication, September 18, 2009, and in revised form, January 4, 2010 Published, JBC Papers in Press, January 8, 2010, DOI 10.1074/jbc.M109.067967
Jacqueline M. Cale‡1, Shih-Hon Li‡1, Mark Warnock‡, Enming J. Su‡, Paul R. North§, Karen L. Sanders§,
Maria M. Puscau§, Cory D. Emal§, and Daniel A. Lawrence‡2
From the ‡Department of Internal Medicine, Division of Cardiovascular Medicine, University of Michigan Medical School, Ann
Arbor, Michigan 48109-0644 and the §Department of Chemistry, Eastern Michigan University, Ypsilanti, Michigan 48197

Plasminogen activator inhibitor type 1, (PAI-1) the primary terized role in fibrinolysis (1). PAI-1 also plays a role in many
inhibitor of the tissue-type (tPA) and urokinase-type (uPA) physiologic processes, including angiogenesis, wound healing,
plasminogen activators, has been implicated in a wide range of and cell migration (2– 6), and has been implicated in fibrotic
pathological processes, making it an attractive target for phar- diseases of the kidney and lung, and in tumor metastasis (7–11).
macologic inhibition. Currently available small-molecule inhib- More recently, PAI-1 has been linked to obesity and metabolic
itors of PAI-1 bind with relatively low affinity and do not inac- syndrome (12–16), and to the development of vascular diseases
tivate PAI-1 in the presence of its cofactor, vitronectin. To such as venous thrombosis and atherosclerosis (17–19). The
search for novel PAI-1 inhibitors with improved potencies and prospect that PAI-1 may play a direct role in the early develop-
new mechanisms of action, we screened a library selected to

Downloaded from www.jbc.org by guest, on July 1, 2010
ment of a variety of diseases has made it an attractive target for
provide a range of biological activities and structural diversity. drug development (20, 21). However, the structural complexity
Five potential PAI-1 inhibitors were identified, and all were of PAI-1 has made the identification and development of PAI-1
polyphenolic compounds including two related, naturally inhibitors challenging. This is due in part to the metastable
occurring plant polyphenols that were structurally similar to structure of PAI-1, which can adopt several different conforma-
compounds previously shown to provide cardiovascular benefit tions, including active, latent, cleaved, and protease complexed
in vivo. Unique second generation compounds were synthesized (1). These different forms of PAI-1 provide conformational
and characterized, and several showed IC50 values for PAI-1 control of PAI-1 interactions and dictate its localization to
between 10 and 200 nM. This represents an enhanced potency of either matrix or the cell surface and control its activity in cell
10 –1000-fold over previously reported PAI-1 inactivators. Inhi- signaling events (22, 23).
bition of PAI-1 by these compounds was reversible, and their Active PAI-1 inhibits protease targets and is associated with
primary mechanism of action was to block the initial association vitronectin in plasma or the extracellular matrix. In contrast,
of PAI-1 with a protease. Consistent with this mechanism and in PAI-1-protease complexes shift affinity from vitronectin to
contrast to previously described PAI-1 inactivators, these com- receptors of the low density lipoprotein receptor family, trans-
pounds inactivate PAI-1 in the presence of vitronectin. Two of ferring PAI-1 from vitronectin to the cell surface (22). Active
the compounds showed efficacy in ex vivo plasma and one PAI-1 is inherently unstable and undergoes a spontaneous con-
blocked PAI-1 activity in vivo in mice. These data describe a formational change that results in inactivation of PAI-1 to a
novel family of high affinity PAI-1-inactivating compounds with latent form that does not bind either vitronectin or low density
improved characteristics and in vivo efficacy, and suggest that lipoprotein receptor family members with high affinity (22, 24).
the known cardiovascular benefits of dietary polyphenols may The flexible structure of PAI-1, the lack of a rigid active site, and
derive in part from their inactivation of PAI-1. its multiple functions all contribute to the difficulties in identi-
fying and designing small-molecule PAI-1 inactivators. Despite
these obstacles, several small-molecule PAI-1 inhibitors have
Plasminogen activator inhibitor type 1 (PAI-1)3 is the pri- been described (25–36); however, each has significant limita-
mary physiologic inhibitor of uPA and tPA with a well charac- tions that have reduced their potential for further drug
development.
* This work was supported, in whole or in part, by National Institutes of Health One of the best characterized compounds is PAI-039, also
Grants HL55374, HL54710, and HL089407 (to D. A. L.). known as tiplaxtinin, which has been shown to reduce physio-
□
S
The on-line version of this article (available at http://www.jbc.org) contains logic PAI-1 activity and to be efficacious in animal models of
supplemental “Methods” and Figs. S1–S4.
1
Both authors contributed equally to this work.
disease (3, 37–39). However, PAI-039 has relatively low affinity
2
To whom correspondence should be addressed: 7301 MSRB III, 1150 W. for PAI-1, and does not inhibit vitronectin-bound PAI-1 (32,
Medical Center Dr., Ann Arbor MI 48109-0644. Tel.: 734-763-7838; Fax: 734- 40). To develop better PAI-1 inactivators, we screened a library
936-2641; E-mail: dlawrenc@umich.edu.
3 of known compounds for high affinity PAI-1 inhibitors with
The abbreviations used are: PAI-1, plasminogen activator inhibitor type 1;
uPA, urokinase-type plasminogen activator; tPA, tissue-type plasminogen improved solubility and activity against vitronectin-bound
activator; PAI-1glyco, glycosylated active human PAI-1; mPAI-1, murine PAI-1; PAI-1. A high throughput screen of the MicroSource SPEC-
CDE, an arbitrary designation based on the initials of one of the authors; SPR, TRUM library identified five novel PAI-1 inactivating com-
surface plasmon resonance; IVC, inferior vena cava; TA, tannic acid; EGCDG,
epigallocatechin-3,5-digallate; EGCG, epigallocatechin monogallate; CCG, pounds. Two of the molecules identified were related natural
Center for Chemical Genomics; PBS, phosphate-buffered saline. polyphenolic compounds, which suggested a potential struc-

7892 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 11 • MARCH 12, 2010


A Novel Class of PAI-1 Inactivating Compounds
ture-activity relationship. Second generation compounds were pounds either had intrinsic absorbance at 405 nm or insolubil-
designed and synthesized to probe this structure-activity rela- ity in the buffer system. Of the remaining compounds, 5 directly
tionship and tested for their ability to block PAI-1 activity in inhibited PAI-1 activity.
both purified systems and in vivo. Enzymatic Assays—Recombinant nonglycosylated or glyco-
sylated active human PAI-1 (PAI-1 and PAI-1glyco, respectively)
EXPERIMENTAL PROCEDURES or recombinant murine PAI-1 (mPAI-1) was incubated at 2 nM
Primary Screen—In conjunction with the Center for Chemi- for 15 min at 23 °C with increasing concentrations of each com-
cal Genomics (CCG) at the University of Michigan, we devel- pound in assay buffer (40 mM HEPES, pH 7.8, 100 mM NaCl,
oped a PAI-1 activity assay to screen for compounds with anti- 0.005% Tween 20, 0.1% Me2SO), followed by the addition of
PAI-1 activity in the MicroSource SPECTRUM compound uPA (Molecular Innovations) or tPA (Genentech) to 3 nM and
collection. This collection consists of known drugs, compounds further incubation for 30 min at 23 °C. At each drug concentra-
approved for agricultural use, natural products, and other bio- tion, parallel control reactions without PAI-1 were assembled.
active compounds. A chromogenic assay was used with a 2:1 Residual enzymatic activity was determined by addition of an
molar ratio of PAI-1 to uPA. We selected uPA because it is equal volume of 100 M Z-Gly-Gly-Arg-AMC (Calbiochem)
considerably more active toward low molecular weight sub- fluorogenic substrate for uPA or Pefafluor tPA (Centerchem)
strates than tPA, allowing for more than 10-fold lower concen- for tPA, and the rate of AMC release monitored at 23 °C (exci-
trations of uPA and PAI-1 in this screen (5 nM uPA and 10 nM tation 370 nm and emission 440 nm). The percent change in
PAI-1) compared with assays using tPA (70 nM tPA and 140 nM PAI-1 activity was determined according to Equation 1,
PAI-1) (41). The screen was performed in 384-well microti-
ter plates in the CCG lab as follows: recombinant active human Ei P i /E i / E 0 P 0 /E 0 (Eq. 1)

PAI-1 (final 10 nM) was incubated for 60 min at 23 °C either
with or without 10 M of each compound in high throughput where Ei is the enzyme activity at drug concentration i; Pi is the
screen buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 0.005% enzyme activity in the presence of PAI-1 at drug concentration
Tween 20), uPA was added (final 5 nM) to each reaction well, i; E0 is the enzyme activity in the absence of drug; and P0 is the
and the incubation continued for an additional 30 min at 23 °C. enzyme activity in the presence of PAI-1 in the absence of drug.
Residual uPA activity in each reaction mixture was then deter- The effect of the compounds on 2 nM anti-thrombin III in the
mined with p-Glu-Gly-Arg p-nitroanilide chromogenic sub- presence of 3 units/ml of heparin was also determined using 3
strate (Sigma) (final 0.25 mM) measured spectrophotometri- nM -thrombin. The reactions were assembled as above except
cally at 405 nm after 60 min. Compounds that inactivated PAI-1 that 10% Me2SO was included in the assay buffer to ensure
were identified by the restoration of uPA activity. The extent of compound solubility at the higher concentrations used. Resid-
uPA activity restoration was determined by comparing each ual -thrombin activity was measured using an equal volume of
drug-containing sample to wells with untreated PAI-1 (100% 100 M benzoyl-Phe-Val-Arg-AMC (Calbiochem).
PAI-1 activity) and to wells with uPA only (0% PAI-1 activity). Synthesis of New Inhibitors—Synthetic procedures and spec-
The data from this screen were then uploaded to the CCG troscopic data for CDE compounds are provided in supplemen-
informatics system and positive hits were identified as any com- tal “Methods”.
pound that increased uPA activity by more than 3 S.D. above Surface Plasmon Resonance (SPR) Analysis—Direct binding
control and compound wells on each plate. Using these selec- of PAI-1 treated with vehicle or inhibitor to anhydrotrypsin
tion criteria, the primary screen of 2000 compounds yielded an (Molecular Innovations) was monitored using a Biacore 2000
initial total of 23 compounds deemed positive hits. Each of optical biosensor. Bovine anhydrotrypsin was immobilized to
these hits was then re-assayed by dose-response testing using CM5 SPR chips at a level of 2000 response units in 10 mM
the same chromogenic assay with the compounds at the follow- sodium acetate, pH 5.0. The reference flow cell surface was left
ing concentrations (0.1, 0.32, 1, 3.2, 10, 32, and 100 M) in blank to serve as a control. Remaining binding sites were
duplicate by the CCG. In this secondary analysis 19 of the 23 blocked by 1 M ethanolamine, pH 8.5. All binding reactions
compounds were deemed positive; however, 3 of these com- were performed in assay buffer. PAI-1 at 2 nM was first incu-
pounds were known to have significant toxicity and therefore bated with the indicated concentrations of inhibitor in assay
were not analyzed further. Samples of the 16 remaining com- buffer for at least 15 min at 23 °C. Binding of PAI-1 to anhydro-
pounds were then obtained from the CCG for further analysis trypsin was then monitored at 25 °C at a flow rate of 30 l/min
in our laboratory. These more detailed analyses first investi- for 2.5 min, followed by 2 min of dissociation. Chip surfaces
gated whether each compound had intrinsic absorbance at 405 were regenerated with a 1-min pulse of 10 mM glycine, pH 1.5,
nm that would give false positive absorbance readings, or was followed by a 1-min wash of assay buffer. Injections were per-
not completely soluble in the assay buffer system used because formed using the Wizard Customized Application program in
insolubility and compound precipitation could likewise lead to automated mode. Binding experiments were performed in
false positive absorbance readings. Each compound was also duplicate and corrected for background and bulk refractive
tested for its ability to directly block PAI-1 complex formation index by subtraction of the reference flow cell, and data were
with uPA by SDS-PAGE analysis. For this latter analysis each analyzed with BIAevaluation 3.1 (Biacore) by linear fitting of
compound was incubated at 10 M with 1 g of PAI-1 for 15 the initial association phase. Compound-induced alterations in
min at 23 °C followed by the addition of 1 g of uPA for an PAI-1 binding to anhydrotrypsin were determined by compar-
additional 5 min at 37 °C. Approximately half of the 16 com- ing the initial slopes of the association phases because there is a

MARCH 12, 2010 • VOLUME 285 • NUMBER 11 JOURNAL OF BIOLOGICAL CHEMISTRY 7893


linear relationship between the slope and the concentration of (median setting, 50- l sample size, 100 events/bead). Mean fluo-
available active PAI-1 (supplemental Fig. S1 and Ref. 32). These rescence intensities of unknown samples were converted to
data were then fit to an exponential association equation to picograms/ml of base on a standard curve of mPAI-1 in mPAI-
determine the apparent affinity between PAI-1 and compound. 1-depleted plasma using a five-parameter regression formula
To monitor the inhibition of vitronectin-bound PAI-1, (Masterplex QT version 4.0, Miraibio).
human vitronectin purified under non-denaturing conditions Plasma Enzymatic Assay—Citrated blood was collected from
was coupled to a CM5 sensor chip to a surface density of 1000 the inferior vena cava (IVC) of C57Bl6J mice that were either
response units (32). Five nM PAI-1 was injected over the chip at PAI-1 null or vitronectin/PAI-1 null and plasma were prepared
a rate of 20 l/min at 25 °C for 4 min, followed by assay buffer by centrifugation (15 min at 1500 g). The plasma was treated
alone or 100 nM CDE-066 in assay buffer for 10 min, and then with 10 g/ml aprotinin (Roche Applied Science) for 15 min at
100 nM uPA for 5 min. After injections of PAI-1 or CDE-066, 23 °C before reconstituting with 20 nM PAI-1. Plasma (10 l,
the chip was washed with assay buffer for 4 min. Results with or without PAI-1) was placed in microtiter wells with 80 l
were corrected for background and bulk refractive index in of CDE-066 or PAI-039, synthesized as described (40) in assay
BIAevaluation 3.1. buffer containing 10% Me2SO and incubated for 15 min at
SDS-PAGE/Western Blotting—Human PAI-1 at 2 nM was 23 °C, followed by addition of 10 l of 25 nM uPA, and a further
incubated with the indicated concentrations of the compound incubation for 30 min. Residual enzymatic activity was moni-
for 15 min at 23 °C in assay buffer, followed by a 30-min incu- tored as above using the fluorogenic uPA substrate, and PAI-1
bation with 3 nM uPA or tPA. Samples were analyzed via reduc- activity was determined using Equation 1.
ing SDS-PAGE with 10% Tris-HCl gels (Bio-Rad) and trans- Inhibition of PAI-1 in Vivo—Transgenic mice heterozygous
ferred onto polyvinylidene difluoride overnight. PAI-1 was for murine PAI-1 overexpression (10) were weighed, then anes-

detected using polyclonal high-titer sheep anti-human PAI-1 thetized with isoflurane for the duration of the experiment. The
antibody (Molecular Innovations), horseradish peroxidase- IVC was isolated and 50 l of citrated blood was collected as
conjugated donkey anti-sheep IgG (Jackson ImmunoResearch pre-treatment samples. The syringe was replaced with a syringe
Laboratories), and Pierce ECL Western blotting substrate containing vehicle or CDE-066 (in lactated Ringers) and 100 l
(Thermo Scientific). was injected for doses of 3, 10, and 30 mg/kg. After 1 h, 300 l of
Reversibility Assay—The reversibility of PAI-1 inactivation citrated blood was collected via IVC, after which the mice were
by the compounds was performed essentially as described (32). euthanized. Plasma was isolated by centrifugation at 1500 g
PAI-1 (2 nM) was incubated with each compound at 3–5-fold its for 15 min at 23 °C. All animal experiments were approved by
IC50 value as determined using uPA, followed by serial 2-fold the Institutional Animal Care and Use Committee of Unit for
dilutions into assay buffer and further incubation to allow dis- Laboratory Animal Medicine at the University of Michigan. To
sociation of the compounds from PAI-1. UPA (3.5 nM) was then determine active murine PAI-1 levels in the plasma, 10 l of
added and PAI-1 activity was determined. The PAI-1 activity in plasma, diluted in PAI-1-depleted murine plasma (Molecular
compound-containing samples recovered at each dilution was Innovations), 10 l of buffer (PBS, pH 7.4, 1% bovine serum
calculated as a percentage of the activity in PAI-1 dilutions car- albumin), and 25 l of uPA-coupled SeroMAP beads were
ried out in parallel without compound. Initial concentrations of added to a filter plate and incubated by shaking overnight at
compounds were 150 nM CDE-008, 75 nM CDE-031, 400 nM 4 °C in the dark, and the reactions were analyzed as above in the
CDE-034, 300 nM CDE-056, 50 nM CDE-066, and 50 nM ex vivo plasma assay.
CDE-082. Data and Statistical Analysis—Data were analyzed and IC50
Inhibition of mPAI-1 in ex Vivo Plasma—Murine PAI-1 was values were calculated using Grafit 5. Apparent KD values for
added to PAI-1-depleted murine plasma (Molecular Innova- the binding of compounds to PAI-1 were determined using
tions) at 5000 pg/ml. Ten microliters of increasing concentra- GraphPad Prism 4. Data were analyzed for significance with a
tions of compound in assay buffer containing 10% Me2SO and Student’s t test using non-diluted samples in the reversibility
10 l of mPAI-1-reconstituted plasma were incubated for 15 assays and 0 mg/kg of CDE-066 treatment in the in vivo assays
min at 23 °C in a filter plate (Millipore), followed by the addition as the control groups, with p 0.05 considered significant.
of 25 l of SeroMAP beads (Luminex) coupled to uPA (2500
beads/well), and further incubated in the dark on a microtiter RESULTS
plate shaker for 2 h. The plate was vacuum washed 3 times with High Throughput Screen—The MicroSource SPECTRUM
wash buffer (PBS, pH 7.4, 0.05% Tween 20), then 50 l of PBS, compound library was screened under stringent conditions
pH 7.4, 1% bovine serum albumin, and 50 l of 4 g/ml of such that PAI-1 was present at a 2-fold molar excess over uPA,
biotin-labeled rabbit anti-mPAI-1 (Molecular Innovations) was and each compound was tested at a concentration of 10 M.
added to each well and the plate incubated at 23 °C in the dark The statistical criteria of 3 S.D. above the control and com-
on a microtiter plate shaker for 1 h. After vacuum washing 3 pound means on each plate resulted in 23 hits. These com-
times, 50 l of PBS, pH 7.4, 1% bovine serum albumin, and 50 l pounds were further tested by dose-response analysis, and 19
of 4 g/ml of streptavidin-R-phycoerythrin conjugate (Molec- remained positive in this secondary screen. Of these, 16 were
ular Probes) was added to each well and incubated with shaking deemed safe and subjected to further study including SDS-
at 23 °C for 30 min in the dark. After washing another 3 times, PAGE analysis of complex formation between PAI-1 and uPA.
100 l of sheath fluid (Luminex) was added to each well, shaken Based on these analyses, 5 compounds were confirmed as PAI-1
for 5 min in the dark at 23 °C, and read on a Luminex100 inhibitors in both enzymatic and SDS-PAGE assays, yielding a



final hit rate of 0.25%. The structures and IC50 values of these 5 compounds was seen when the physiologic substrate of tPA,
compounds along with two related compounds are shown in plasminogen, was used (supplemental Fig. S2), suggesting that
Fig. 1. the compounds may be interacting with the low molecular
Each of these five compounds contain polyphenolic moieties, weight tPA substrates.
and three of them, tannic acid (TA), epigallocatechin-3,5-digal- It is also apparent from these data that although a single
late (EGCDG), and sennoside A, are naturally occurring plant gallate (gallic acid, 6.6 M) is a relatively poor inhibitor of PAI-1,
polyphenols with reported biological activities (42– 46). The a minimum of two galloyl units translates into significant anti-
former two compounds, TA and EGCDG, have highly related PAI-1 activity (20 –116 nM, Fig. 2 and Table 1). Compound
structures that both contain galloyl or gallo-galloyl moieties CDE-008 was compared with several similar digallates with
suggesting the possibility of a structure-activity relationship linkers of different lengths between the gallate moieties, and
between polyphenols in general, and more specifically gallic CDE-008 was found to have the optimal distance between the
acid moieties and PAI-1 inactivation. We therefore examined galloyl units (data not shown). To further explore structural
two additional galloyl-containing compounds, epigallocatechin requirements for digalloyl compound inhibition of PAI-1, we
monogallate (EGCG) and gallic acid (Fig. 1, B and F). Mono- examined 1,2-disubstituted galloyl units on different ring struc-
meric gallic acid was 1000-fold less active toward PAI-1 than tures to determine whether cis (CDE-031), trans (CDE-034), or
TA, whereas EGCG inhibited PAI-1 only 10-fold less well planar (CDE-056) relationships between galloyl units inhibited
than TA. Thus, each of the galloyl-containing compounds was PAI-1 more effectively. All of these compounds were active
able to inhibit PAI-1, but the efficacy of inhibition appears de- against PAI-1 with the cis form (CDE-031) being 2-fold more
pendent on the number of galloyl units in each compound and active against PAI-1 than the acyclic CDE-008. These data
their relative orientation or context. demonstrate that the relative orientation of the gallates is

Synthetic Compounds—The IC50 value obtained with 7 nM important for anti-PAI-1 activity, with the cis form inhibiting
TA was 1000-fold lower than our previously reported IC50 PAI-1 4-fold better than the planar form and 6-fold better
value for the PAI-1 inactivator, PAI-039 (32), and is markedly than the trans form (Table 1).
lower than any previously reported small-molecule PAI-1 inac- SPR Analysis—To establish binding constants for the drugs
tivating compound (25–36). Likewise the IC50 values obtained to PAI-1, an indirect approach using SPR was employed. Vary-
with EGCG and EGCDG were also significantly better than ing concentrations of each drug were preincubated with PAI-1
PAI-039 and most other PAI-1 inactivators, suggesting that gal- in solution and then passed over immobilized anhydrotrypsin,
loyl-containing compounds may represent a potent new family and the loss of PAI-1 binding to anhydrotrypsin was quantified.
of PAI-1 inactivating compounds. However, TA is not an ideal We have previously shown for PAI-1 binding to vitronectin (32)
drug candidate as its molecular mass of nearly 2000 daltons is that the slope of the association phase of PAI-1 binding to an
considered too large, and it was subject to aggregation at micro- immobilized ligand has a linear relationship with the concen-
molar concentrations. Therefore, we synthesized a series of tration of available active PAI-1 in solution. This relationship is
novel compounds containing different numbers of galloyl moi- also true for PAI-1 binding to immobilized anhydrotrypsin
eties in different structural configurations and compared their (supplemental Fig. S1). Thus, when the slopes of the association
activity against PAI-1 to determine a potential structure-activ- phase are plotted as a percent of control PAI-1 binding in the
ity relationship between galloyl-containing compounds and absence of compound versus the concentration of the com-
PAI-1 inhibition. In addition, to make this analysis sensitive to pound, an IC50 can be calculated for the drug-induced inhibi-
inactivators with low nanomolar IC50 values, the PAI-1 concen- tion of PAI-1 interaction with anhydrotrypsin (Fig. 3). From a
tration in the assay was lowered from 10 to 2 nM. Using these transformation of these data, the apparent KD values for all six
optimized assay conditions, we were able to accurately deter- compounds binding to PAI-1 can be calculated (Table 2). The
mine IC50 values for several novel compounds. Six of these apparent KD values range from 3.1 to 67 nM and are significantly
compounds, four digallates, one trigallate, and one pentagal- tighter than the previously reported values for other PAI-1
late, are shown in Fig. 2. Comparison of the IC50 values of these inactivators (28, 32, 33, 40). These values are also similar to the
6 compounds demonstrated IC50 values ranging from 10 to 174 IC50 values calculated in PAI-1 inhibition assays (Table 1).
nM for inactivation of PAI-1 (Fig. 2 and Table 1). The activity of These data indicate that drug binding interferes with the initial
each compound against glycosylated human PAI-1 (PAI-1glyco) association of PAI-1 with the protease and can block formation
and murine PAI-1 (mPAI-1) was also compared with nongly- of the PAI-1-protease Michaelis-like complex.
cosylated recombinant human PAI-1 (PAI-1) (Table 1). In gen- SDS-PAGE—Each compound was also tested for its ability to
eral the compounds inhibited PAI-1glyco as well as the nongly- block complex formation between PAI-1 and PAs, and exam-
cosylated form; however, most inhibited mPAI-1 less well than ples of CDE-008, CDE-066, and CDE-082 are shown in Fig. 4.
human PAI-1. The two exceptions were the pentagallate, CDE- For these studies each compound was incubated with PAI-1,
066, and TA, which inhibited all forms of PAI-1 equally well. then either uPA or tPA was added and the formation of SDS-
The inactivation of PAI-1 by the polyphenolic compounds stable complexes was monitored by SDS-PAGE. The concen-
was specific, because only TA and CDE-082 (IC50 10 M) trations of PAI-1 and PAs were the same as those used in the
showed any inhibition of the related serpin anti-thrombin III. enzyme assays (see Fig. 1 and Table 1), and we observed com-
Some of the gallate-containing compounds tested did show an parable IC50 values between the two techniques. Inhibition of
apparent inhibition of tPA in assays with a chromogenic or covalent complex formation also closely mirrored inhibition of
fluorogenic substrate; however, little inhibition of tPA by these PAI-1 binding to anhydrotrypsin (see Fig. 3 and Table 2). An



reduce the compound concentra-
tions, incubated for an additional 30
min, and the mixtures tested for res-
toration of PAI-1 activity. Fig. 5
shows that for each synthetic poly-
phenol, PAI-1 activity increased
upon dilution, indicating that PAI-1
inactivation by the compounds is
reversible. The extent of PAI-1
activity recovered with each com-
pound was slightly less than pre-
dicted, suggesting that the rate of
dissociation between PAI-1 and
these novel compounds is relatively
slow and the samples may not have
reached a new equilibrium after 30
min. Consistent with this mecha-
nism, incubation of PAI-1 for vari-
ous times with CDE-066, the most
potent synthetic compound, dem-

onstrated that the IC50 remained
unchanged from 15 min until termi-
nation of the experiment at 4 h (sup-
plemental Fig. S3). These data indi-
cate that the compounds do not
irreversibly modify PAI-1, and are
consistent with a high affinity
reversible mechanism of action.
Inhibition of mPAI-1 in Plas-
ma—Each of the new compounds
was tested for anti-PAI-1 activity in
ex vivo plasma. This tests the ability
FIGURE 2. Structures of six synthetic compounds. A, the two-dimensional structures of the 6 synthetic of the drugs to inhibit mPAI-1 in the
polyphenolic compounds are pictured. The inhibition curves of each compound against PAI-1 in the presence
of either uPA (B) or tPA (C) are shown. The data were plotted using the Grafit IC50 fit and are based on three presence of plasma proteins, includ-
independent experiments, points represent the mean S.E. For comparison PAI-039 has a reported IC50 in a ing vitronectin. None of the newly
similar assay system of 10 M (32). generated digallate compounds
increase in PAI-1 cleavage was also noted with each compound were active against mPAI-1 in the
primarily at compound concentrations just below the IC50; plasma activity assay (Fig. 6). This was likely due to high non-
however, this was modest compared with the near complete specific protein binding of these digallates in plasma because
loss of covalent complex, and much less cleavage was observed the digallates were also ineffective against mPAI-1 in buffers
at compound concentrations above the IC50. Together with the containing high concentrations of bovine serum albumin (data
SPR studies, these data suggest that the principal mechanism of not shown). In contrast, all of the compounds with at least 3
PAI-1 inactivation by these compounds is the inhibition of the galloyl moieties inhibited mPAI-1 in the plasma, including the
PAI-1 Michaelis-like complex formation with PAs, but that at trigallate (CDE-082), pentagallate (CDE-066), and TA. Overall,
concentrations near the IC50 some increase of PAI-1 substrate TA had the lowest IC50 against mPAI-1 in plasma (data not
behavior may be induced. shown) but it was less specific than the novel polyphenols as it
Inactivation of PAI-1 Is Reversible—To test whether the inhi- also inhibited normal plasma clotting, whereas CDE-066 and
bition of PAI-1 by the synthetic polyphenols was reversible, CDE-082 did not (supplemental Fig. S4). CDE-066 exhibited
PAI-1 and each synthetic compound was incubated at a con- the lowest IC50 of the new compounds in plasma, and was also
centration where most of the anti-proteolytic activity of PAI-1 significantly more specific than TA, therefore CDE-066 was
was abolished. The mixtures were then serially diluted to used in further studies in plasma and in vivo.

FIGURE 1. IC50 values of PAI-1 inactivating compounds from high throughput screen and related compounds. The two-dimensional structures of the five
hits from the screen (A, C, D, E, and G) and two related compounds (B and F) are shown with IC50 values from the enzyme assays. Recombinant active human
PAI-1 (final 2 nM) was incubated for 15 min at 23 °C with increasing concentrations of each compound in assay buffer. Next uPA (final 3 nM) was added to each
reaction well and incubated for an additional 30 min at 23 °C. Activity of uPA in each reaction mixture was determined with the Z-Gly-Gly-Arg-AMC fluorogenic
substrate (final 50 M). UPA activity was measured fluorometrically (excitation 370 nm and emission 440 nm) for 15 min. The IC50 values were calculated using
Grafit IC50 fit and the mean S.E. are based on three independent experiments. The asterisk indicate compounds identified in the original screen, and the
dagger indicates related compounds not identified in the original screen.



TABLE 1
The IC50 values of TA and six synthetic compounds against various forms of PAI-1 or anti-thrombin III using the indicated target enzymes
IC50 values (nM) S.E. were determined using the Grafit IC50 fit. Values are based on three independent experiments.
PAI-1 PAI-1glyco mPAI-1 Hep:anti-thrombin IIIa
Compound
uPA tPA uPA uPA -Thrombin
TA 6.6 1.1 8.0 0.3 4.8 1.2 4.1 1.2 11,800 300
CDE-008 44 5 53 4 28 2 162 27 10,000b
CDE-031 20 1 28 1 18 1 132 14 10,000b
CDE-034 116 11 174 26 169 21 644 53 300,000
CDE-056 74 4 86 8 152 28 758 26 300,000
CDE-066 10 1 12 2 13 2 10 1 300,000
CDE-082 14 1 18 1 56 2 79 4 15,400 4,400
a
Values represent measured IC50 values or the highest concentration of compound tested.
b
20% of Hep:anti-thrombin III was inactivated at the highest compound concentration used.

The ability of CDE-066 to inactivate PAI-1 bound to purified
vitronectin was verified in vitro via BIAcore. To be certain that
the PAI-1 was in complex with vitronectin, PAI-1 was injected
over immobilized vitronectin and complex formation was
detected by changes in relative response units. These data dem-
onstrate that as expected active PAI-1 binds vitronectin with
high affinity and dissociates very slowly from immobilized
vitronectin; however, upon reaction with uPA, PAI-1 affinity

for vitronectin is reduced by several orders of magnitude (22,
24) and the PAI-1 rapidly dissociates from vitronectin, (Fig. 8,
large dots), In contrast, PAI-1 bound to vitronectin and then
exposed to 100 nM CDE-066 does not dissociate from the
FIGURE 3. Apparent affinity between PAI-1 and synthetic inhibitors immobilized vitronectin following the uPA injection (Fig. 8,
assessed by SPR. Two nM PAI-1 was incubated with the concentrations indi- small dots). These data indicate that CDE-066 blocks the asso-
cated of each synthetic compound and the mixtures injected over an anhy- ciation of uPA with PAI-1 even when in complex with vitronec-
drotrypsin-conjugated CM5 sensor chip. The compound-dependent change
in the initial association rates for PAI-1 binding to anhydrotrypsin, which is tin, and are consistent with the hypothesis that the primary
directly proportional to the amount of free PAI-1 in the analyte, is plotted mechanism by which CDE-066 inactivates PAI-1 is to prevent
against the compound dose to determine the apparent KD values of each
compound for PAI-1. Data are based on two independent experiments; non-covalent complex formation with target proteases. These
points represent the mean S.E. For comparison PAI-039 has a reported data also demonstrate that CDE-066 is not inducing PAI-1
affinity for PAI-1 of 15 M in a direct SPR binding assay system (32). cleavage as a substrate, or latency, because both cleaved and
TABLE 2 latent PAI-1 also exhibit low affinity for vitronectin (22, 24), and
Affinity between PAI-1 and synthetic compounds as measured by would likewise result in loss of PAI-1 signal from the chip.
SPR Finally, consistent with the reversibility studies shown in Fig. 5,
The data from Fig. 3 were fit to an exponential association curve in GraphPad Prism these data indicate that the dissociation of CDE-066 from
4 to calculate the apparent KD. Shown are the mean S.E. of two independent
experiments. PAI-1 is relatively slow because even after 240 s of wash PAI-1 is
Compound Apparent KD still inhibited by CDE-066.
nM PAI-1 Inactivation in Vivo—Finally, to examine whether
CDE-008 23 1 CDE-066 inhibits murine PAI-1 in vivo, mice overexpressing
CDE-031 31 2
CDE-034 67 3 PAI-1 were treated acutely with either vehicle or increasing
CDE-056 51 6 concentrations of CDE-066. Plasma samples were removed
CDE-066 3.1 0.2
CDE-082 5.3 0.2 from each mouse before treatment and then 1 h following intra-
venous infusion of CDE-066 at the indicated concentrations
(Fig. 9). Plasma samples were then tested for active PAI-1 levels.
Our previous studies (32) with PAI-039, the most widely Although a small increase in active PAI-1 was observed in the
studied PAI-1 small-molecule inhibitor, indicated that it is vehicle-treated animals, a dose-dependent decrease in active
unable to inhibit PAI-1 bound to vitronectin, and one of the PAI-1 was observed after 1 h of treatment with CDE-066. These
main objectives of the current study was to identify compounds data indicate that CDE-066 can significantly inhibit PAI-1 in
that could inhibit PAI-1 in the presence of vitronectin. This was vivo.
examined by adding a known amount of PAI-1 to murine
plasma from either PAI-1 null mice or from mice doubly null DISCUSSION
for PAI-1 and vitronectin. After incubating the PAI-1 in these PAI-1 is thought to play a role in several chronic “lifestyle”
plasmas, samples were incubated with dilutions of either CDE- diseases, including cardiovascular and fibrotic diseases, and
066 or PAI-039 and then tested for PAI-1 inhibition of uPA. Fig. metabolic syndrome. These pathologic associations make
7 demonstrates that unlike PAI-039, which is only inhibitory in PAI-1 an ideal drug target; however, its metastable structure
plasma that lacks vitronectin, CDE-066 inhibited PAI-1 equally has made it a difficult candidate for drug design and study. To
well in plasma with or without physiologic vitronectin. date most small-molecule inhibitors of PAI-1 lack high affinity




FIGURE 6. Inhibition of mPAI-1 by synthetic compounds in ex vivo plasma.
Murine plasma depleted of PAI-1 was reconstituted with 5000 pg/ml of
mPAI-1 and treated with each compound, and residual active mPAI-1
detected by Luminex. Curves were generated with the Grafit IC50 fit and the
IC50 S.E. are indicated, NI indicates no detectable inhibition. The data are
based on three independent experiments performed in duplicate.

FIGURE 4. CDE compounds inhibit complex formation between PAI-1 and
uPA or tPA. PAI-1 (2 nM) was incubated with 10-fold dilutions of CDE-008 (A),
-066 (B), and -082 (C) for 15 min at 23 °C in assay buffer. Then uPA (left panels)
or tPA (right panels) was added (3 nM final) and complexes were formed at
23 °C for 30 min. Samples were analyzed by reducing SDS-PAGE followed by
transfer to polyvinylidene difluoride membranes and immunoblotting for
PAI-1. SDS stable complexes (asterisk), unreacted PAI-1 (open arrowhead), and
cleaved PAI-1 (closed arrowhead) were detected.

FIGURE 7. CDE-066 but not PAI-039 inhibits PAI-1 in the presence of
vitronectin. Plasma collected from PAI-1 null or PAI-1/vitronectin null mice
were reconstituted with 20 nM PAI-1, and then vehicle or PAI-1 inactivators,
CDE-066 (A) or PAI-039 (B) were added at the concentrations indicated, and
the samples incubated. Residual PAI-1 activity was determined using uPA and
Z-Gly-Gly-Arg-AMC as described under “Experimental Procedures.” The data
FIGURE 5. Inactivation of PAI-1 by the synthetic inhibitors is reversible. are shown as the mean S.E. and are based on three independent experi-
PAI-1 (2 nM) was incubated with the compounds shown at 3–5-fold excess ments performed in duplicate.
concentrations over the IC50 of each compound for 15 min, then serially
diluted 1:1 three times and further incubated for 30 min. PAI-1 activity was
determined as described under “Experimental Procedures” and is shown as a
percentage of control activity without compound. The data represent the subset of these with the highest anti-PAI-1 activity contained
mean S.E. of at least three independent experiments and were evaluated galloyl moieties, and one, TA, demonstrated the lowest IC50 of
against the activities of the undiluted samples using a Student’s t test (*, p any small-molecule PAI-1 inhibitor yet reported. One other
0.05; **, p 0.01).
study has identified members of the acylphloroglucinol class of
polyphenols, sideroxylonals A–C, as potential PAI-1 inactivat-
for PAI-1 and are unable to inhibit PAI-1 in the presence of its ing compounds (27). However, the reported IC50 values of these
plasma binding protein, vitronectin. To identify higher affinity compounds (3.3–5.3 M) are 2–3 orders of magnitude higher
inhibitors with better drug development potential, a high strin- than TA and the novel synthetic polyphenols described here
gency screening assay was performed and a class of polyphe- and are comparable with the IC50 of the simplest gallate com-
nolic compounds was identified with anti-PAI-1 activity. A pound in the current study, gallic acid (6.6 M). This suggests



complex and is not dependent on only the number of galloyl
subunits.
The synthetic polyphenolic derivatives demonstrate clear
advantages over previous pharmacologic inactivators of PAI-1.
For example most of the existing PAI-1 inhibitors exhibit IC50
values in the low- to mid-micromolar range in comparable in
vitro assays, which is several orders of magnitude less potent
than the best novel synthetic polyphenolic derivatives
described here (25–27, 29, 30, 32, 34 –36). Another class of
PAI-1 inhibitors based on diketopiperazine derivatives have
been described with in vitro IC50 values reported in the 0.2–1
M range; however, these compounds suffered from consider-
FIGURE 8. Inactivation of vitronectin-bound PAI-1 by CDE-066 assessed
by SPR. PAI-1 (5 nM) was injected over a vitronectin-conjugated CM5 chip, able physicochemical problems such as insolubility in physio-
followed by 100 nM CDE-066 (small dots) or vehicle (large dots). Residual logic buffer systems and were not subject to further develop-
vitronectin-bound PAI-1 activity was assessed by injection of 100 nM uPA,
with active PAI-1 binding to the uPA and rapidly dissociating from the chip ment (47). CDE-066, in contrast, is soluble in physiologic saline
resulting in loss of surface response units, whereas CDE-066-inactivated PAI-1 solution at concentrations greater than 10 mM without loss of
remains on the chip surface after the uPA injection. The starts of injections anti-PAI-1 activity (data not shown). Two other PAI-1 inacti-
and washes are indicated by black arrows.
vators have been described with IC50 values reported in the
mid-nanomolar range; however, these compounds are ineffec-
tive against vitronectin-bound PAI-1, the predominant form of

PAI-1 in plasma and the extracellular matrix (28, 33). Likewise
several compounds with micromolar IC50 values are also inef-
fective against vitronectin bound PAI-1 (26, 32). The resistance
of vitronectin-bound PAI-1 to these inhibitors is thought to be
due to the location of the binding site for these compounds, in a
hydrophobic cavity on PAI-1 that is defined by -helices D and
E and -strands 1A and 2A, and directly adjacent to the
vitronectin-binding site (26, 28, 32). In contrast, the CDE-066
compound shows vitronectin-independent anti-PAI-1 activity
in a purified system, in ex vivo plasma, and in vivo in PAI-1
transgenic mice.
FIGURE 9. CDE-066 reduces endogenous active PAI-1 in mouse plasma. The primary mechanism of action by which CDE-066 and
Citrated blood was removed via the IVC from mice overexpressing PAI-1 the other synthetic polyphenols inactivate PAI-1 appears to be
before, and 1 h following treatment with the indicated dose of CDE-066.
Active murine PAI-1 was measured by Luminex assay and compared with by binding to PAI-1 in a reversible manner and preventing sta-
standards of known murine PAI-1 concentrations. The data are expressed as a bilization of the non-covalent Michaelis complex with target
percentage of active PAI-1 present in the plasma relative to active PAI-1 at proteases. This is demonstrated in Fig. 3 wherein preincubation
time 0 for each mouse. The data represent the mean S.E., n 5 at each dose,
and were evaluated against the 0 mg/kg treatment using a Student’s t test (*, of PAI-1 with each of the compounds inhibits its binding to the
p 0.05; **, p 0.01). inactive protease, anhydrotrypsin. Identical data were also
obtained in similar experiments using an inactive mutant of
that many polyphenolic compounds may share PAI-1 inactivat- tPA (data not shown), indicating that the effect of the com-
ing activity, but that the galloyl moiety may be a critical deter- pounds on the initial association of PAI-1 with a protease is
minant in polyphenols for potent anti-PAI-1 activity. independent of the target protease. The SDS-PAGE analysis
Despite the low IC50 of TA and its ability to inhibit PAI-1 in shown in Fig. 4 suggests that the polyphenolic compounds can
the high protein environment of plasma (data not shown), it is also promote substrate behavior in PAI-1. However, in contrast
not an ideal drug candidate due to its molecular mass of nearly to the loss of Michaelis complex formation (Fig. 3) and the loss
2000 daltons and its relative promiscuity, interacting with other of covalent complex formation (Fig. 4) the extent of cleavage
proteins as well as itself at low- to mid-micromolar concentra- observed is not dose dependent with the compounds added and
tions. Nonetheless, the inhibition of PAI-1 by TA and other varies with compound and target enzyme. It is possible that the
gallate-containing molecules (EGCG, EGCDG, and gallic acid) extent of cleavage may be overestimated in these experiments
formed the basis for development of follow-up compounds due to complex dissociation during SDS-PAGE. Note, for
with improved properties compared with these naturally example, that even in the absence of any compound, cleaved
occurring polyphenols. Smaller di-, tri-, and pentagallates were PAI-1 is apparent under experimental conditions where the
designed with improved solubility in physiologic buffers and stoichiometry of inhibition is near 1 (SI 1.06, data not
greater specificity toward PAI-1. These studies determined that shown). Finally, consistent with the primary mechanism of
although two galloyl moieties were sufficient to provide potent action being inhibition of PAI-1:protease association, SPR
anti-PAI-1 activity, a minimum of 3 galloyl groups was required experiments demonstrated that no CDE-066-dependent PAI-1
for efficacy in plasma. This suggests the relationship between cleavage was detected when PAI-1 bound to vitronectin was
specificity for PAI-1 and nonspecific bulk protein binding is reacted with active uPA (Fig. 8). This suggests that the combi-



nation of compounds and denaturants during SDS-PAGE may 13. Ma, L. J., Mao, S. L., Taylor, K. L., Kanjanabuch, T., Guan, Y., Zhang, Y.,
alter how PAI-1 is observed to behave. Brown, N. J., Swift, L. L., McGuinness, O. P., Wasserman, D. H., Vaughan,
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The identification of naturally occurring polyphenols as a
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class of PAI-1 inhibitors is intriguing because such compounds, Vaughan, D. E. (2006) J. Biol. Chem. 281, 32796 –32805
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pathologies such as chronic inflammation, neurodegeneration, 16. Lijnen, H. R. (2009) Thromb. Res. 123, Suppl. 4, S46 –S49
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nisms of action have been proposed for dietary polyphenols,
18. Wiman, B., Andersson, T., Hallqvist, J., Reuterwall, C., Ahlbom, A., and
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the complex pro-fibrinolytic and cardioprotective effects asso- 25. Charlton, P. A., Faint, R. W., Bent, F., Bryans, J., Chicarelli-Robinson, I.,
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polyphenolic compounds against PAI-1 as well as clarifying the 26. Bjorquist, P., Ehnebom, J., Inghardt, T., Hansson, L., Lindberg, M., Lin-
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Chemical Genomics for drug screening, Dr. Scott Larsen of University 28. Egelund, R., Einholm, A. P., Pedersen, K. E., Nielsen, R. W., Christensen,
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Characterization of a Novel Class of Polyphenolic Inhibitors of Plasminogen Activator Inhibitor 1