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

Open AccessPublished:January 08, 2010DOI:https://doi.org/10.1074/jbc.M109.067967
      Plasminogen activator inhibitor type 1, (PAI-1) the primary inhibitor of the tissue-type (tPA) and urokinase-type (uPA) plasminogen activators, has been implicated in a wide range of pathological processes, making it an attractive target for pharmacologic inhibition. Currently available small-molecule inhibitors of PAI-1 bind with relatively low affinity and do not inactivate PAI-1 in the presence of its cofactor, vitronectin. To search for novel PAI-1 inhibitors with improved potencies and new mechanisms of action, we screened a library selected to provide a range of biological activities and structural diversity. Five potential PAI-1 inhibitors were identified, and all were polyphenolic compounds including two related, naturally occurring plant polyphenols that were structurally similar to compounds previously shown to provide cardiovascular benefit in vivo. Unique second generation compounds were synthesized and characterized, and several showed IC50 values for PAI-1 between 10 and 200 nm. This represents an enhanced potency of 10–1000-fold over previously reported PAI-1 inactivators. Inhibition of PAI-1 by these compounds was reversible, and their primary mechanism of action was to block the initial association of PAI-1 with a protease. Consistent with this mechanism and in contrast to previously described PAI-1 inactivators, these compounds inactivate PAI-1 in the presence of vitronectin. Two of the compounds showed efficacy in ex vivo plasma and one blocked PAI-1 activity in vivo in mice. These data describe a novel family of high affinity PAI-1-inactivating compounds with improved characteristics and in vivo efficacy, and suggest that the known cardiovascular benefits of dietary polyphenols may derive in part from their inactivation of PAI-1.

      Introduction

      Plasminogen activator inhibitor type 1 (PAI-1)
      The abbreviations used are: PAI-1
      plasminogen activator inhibitor type 1
      uPA
      urokinase-type plasminogen activator
      tPA
      tissue-type plasminogen activator
      PAI-1glyco
      glycosylated active human PAI-1
      mPAI-1
      murine PAI-1
      CDE
      an arbitrary designation based on the initials of one of the authors
      SPR
      surface plasmon resonance
      IVC
      inferior vena cava
      TA
      tannic acid
      EGCDG
      epigallocatechin-3,5-digallate
      EGCG
      epigallocatechin monogallate
      CCG
      Center for Chemical Genomics
      PBS
      phosphate-buffered saline.
      is the primary physiologic inhibitor of uPA and tPA with a well characterized role in fibrinolysis (
      • Yepes M.
      • Loskutoff D.J.
      • Lawrence D.A.
      ). PAI-1 also plays a role in many physiologic processes, including angiogenesis, wound healing, and cell migration (
      • McMahon G.A.
      • Petitclerc E.
      • Stefansson S.
      • Smith E.
      • Wong M.K.
      • Westrick R.J.
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      ,
      • Leik C.E.
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      ,
      • Maquerlot F.
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      ,
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      ,
      • Cao C.
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      • Von Arnim C.A.
      • Herz J.
      • Su E.J.
      • Makarova A.
      • Hyman B.T.
      • Strickland D.K.
      • Zhang L.
      ), and has been implicated in fibrotic diseases of the kidney and lung, and in tumor metastasis (
      • Andreasen P.A.
      ,
      • Huang Y.
      • Haraguchi M.
      • Lawrence D.A.
      • Border W.A.
      • Yu L.
      • Noble N.A.
      ,
      • Huang Y.
      • Border W.A.
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      • Noble N.A.
      ,
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      ,
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      • Yan S.F.
      • Chen J.
      • Carmeliet P.
      • Loskutoff D.J.
      • Stern D.M.
      ). More recently, PAI-1 has been linked to obesity and metabolic syndrome (
      • Schäfer K.
      • Fujisawa K.
      • Konstantinides S.
      • Loskutoff D.J.
      ,
      • Ma L.J.
      • Mao S.L.
      • Taylor K.L.
      • Kanjanabuch T.
      • Guan Y.
      • Zhang Y.
      • Brown N.J.
      • Swift L.L.
      • McGuinness O.P.
      • Wasserman D.H.
      • Vaughan D.E.
      • Fogo A.B.
      ,
      • De Taeye B.M.
      • Novitskaya T.
      • Gleaves L.
      • Covington J.W.
      • Vaughan D.E.
      ,
      • Shah C.
      • Yang G.
      • Lee I.
      • Bielawski J.
      • Hannun Y.A.
      • Samad F.
      ,
      • Lijnen H.R.
      ), and to the development of vascular diseases such as venous thrombosis and atherosclerosis (
      • Gohil R.
      • Peck G.
      • Sharma P.
      ,
      • Wiman B.
      • Andersson T.
      • Hallqvist J.
      • Reuterwall C.
      • Ahlbom A.
      • deFaire U.
      ,
      • Sobel B.E.
      • Taatjes D.J.
      • Schneider D.J.
      ). The prospect that PAI-1 may play a direct role in the early development of a variety of diseases has made it an attractive target for drug development (
      • Wu Q.
      • Zhao Z.
      ,
      • Vaughan D.E.
      • De Taeye B.M.
      • Eren M.
      ). However, the structural complexity of PAI-1 has made the identification and development of PAI-1 inhibitors challenging. This is due in part to the metastable structure of PAI-1, which can adopt several different conformations, including active, latent, cleaved, and protease complexed (
      • Yepes M.
      • Loskutoff D.J.
      • Lawrence D.A.
      ). These different forms of PAI-1 provide conformational control of PAI-1 interactions and dictate its localization to either matrix or the cell surface and control its activity in cell signaling events (
      • Stefansson S.
      • Muhammad S.
      • Cheng X.F.
      • Battey F.D.
      • Strickland D.K.
      • Lawrence D.A.
      ,
      • Webb D.J.
      • Thomas K.S.
      • Gonias S.L.
      ).
      Active PAI-1 inhibits protease targets and is associated with vitronectin in plasma or the extracellular matrix. In contrast, PAI-1-protease complexes shift affinity from vitronectin to receptors of the low density lipoprotein receptor family, transferring PAI-1 from vitronectin to the cell surface (
      • Stefansson S.
      • Muhammad S.
      • Cheng X.F.
      • Battey F.D.
      • Strickland D.K.
      • Lawrence D.A.
      ). Active PAI-1 is inherently unstable and undergoes a spontaneous conformational change that results in inactivation of PAI-1 to a latent form that does not bind either vitronectin or low density lipoprotein receptor family members with high affinity (
      • Stefansson S.
      • Muhammad S.
      • Cheng X.F.
      • Battey F.D.
      • Strickland D.K.
      • Lawrence D.A.
      ,
      • Lawrence D.A.
      • Palaniappan S.
      • Stefansson S.
      • Olson S.T.
      • Francis-Chmura A.M.
      • Shore J.D.
      • Ginsburg D.
      ). The flexible structure of PAI-1, the lack of a rigid active site, and its multiple functions all contribute to the difficulties in identifying and designing small-molecule PAI-1 inactivators. Despite these obstacles, several small-molecule PAI-1 inhibitors have been described (
      • Charlton P.A.
      • Faint R.W.
      • Bent F.
      • Bryans J.
      • Chicarelli-Robinson I.
      • Mackie I.
      • Machin S.
      • Bevan P.
      ,
      • Björquist P.
      • Ehnebom J.
      • Inghardt T.
      • Hansson L.
      • Lindberg M.
      • Linschoten M.
      • Strömqvist M.
      • Deinum J.
      ,
      • Neve J.
      • Leone P.A.
      • Carroll A.R.
      • Moni R.W.
      • Paczkowski N.J.
      • Pierens G.
      • Björquist P.
      • Deinum J.
      • Ehnebom J.
      • Inghardt T.
      • Guymer G.
      • Grimshaw P.
      • Quinn R.J.
      ,
      • Egelund R.
      • Einholm A.P.
      • Pedersen K.E.
      • Nielsen R.W.
      • Christensen A.
      • Deinum J.
      • Andreasen P.A.
      ,
      • Gils A.
      • Stassen J.M.
      • Nar H.
      • Kley J.T.
      • Wienen W.
      • Ries U.J.
      • Declerck P.J.
      ,
      • Crandall D.L.
      • Elokdah H.
      • Di L.
      • Hennan J.K.
      • Gorlatova N.V.
      • Lawrence D.A.
      ,
      • Liang A.
      • Wu F.
      • Tran K.
      • Jones S.W.
      • Deng G.
      • Ye B.
      • Zhao Z.
      • Snider R.M.
      • Dole W.P.
      • Morser J.
      • Wu Q.
      ,
      • Gorlatova N.V.
      • Cale J.M.
      • Elokdah H.
      • Li D.
      • Fan K.
      • Warnock M.
      • Crandall D.L.
      • Lawrence D.A.
      ,
      • Gardell S.J.
      • Krueger J.A.
      • Antrilli T.A.
      • Elokdah H.
      • Mayer S.
      • Orcutt S.J.
      • Crandall D.L.
      • Vlasuk G.P.
      ,
      • Rupin A.
      • Gaertner R.
      • Mennecier P.
      • Richard I.
      • Benoist A.
      • De Nanteuil G.
      • Verbeuren T.J.
      ,
      • Izuhara Y.
      • Takahashi S.
      • Nangaku M.
      • Takizawa S.
      • Ishida H.
      • Kurokawa K.
      • van Ypersele de Strihou C.
      • Hirayama N.
      • Miyata T.
      ,
      • Einholm A.P.
      • Pedersen K.E.
      • Wind T.
      • Kulig P.
      • Overgaard M.T.
      • Jensen J.K.
      • B⊘dker J.S.
      • Christensen A.
      • Charlton P.
      • Andreasen P.A.
      ); however, each has significant limitations that have reduced their potential for further drug development.
      One of the best characterized compounds is PAI-039, also known as tiplaxtinin, which has been shown to reduce physiologic PAI-1 activity and to be efficacious in animal models of disease (
      • Leik C.E.
      • Su E.J.
      • Nambi P.
      • Crandall D.L.
      • Lawrence D.A.
      ,
      • Hennan J.K.
      • Elokdah H.
      • Leal M.
      • Ji A.
      • Friedrichs G.S.
      • Morgan G.A.
      • Swillo R.E.
      • Antrilli T.M.
      • Hreha A.
      • Crandall D.L.
      ,
      • Crandall D.L.
      • Quinet E.M.
      • El Ayachi S.
      • Hreha A.L.
      • Leik C.E.
      • Savio D.A.
      • Juhan-Vague I.
      • Alessi M.C.
      ,
      • Baxi S.
      • Crandall D.L.
      • Meier T.R.
      • Wrobleski S.
      • Hawley A.
      • Farris D.
      • Elokdah H.
      • Sigler R.
      • Schaub R.G.
      • Wakefield T.
      • Myers D.
      ). However, PAI-039 has relatively low affinity for PAI-1, and does not inhibit vitronectin-bound PAI-1 (
      • Gorlatova N.V.
      • Cale J.M.
      • Elokdah H.
      • Li D.
      • Fan K.
      • Warnock M.
      • Crandall D.L.
      • Lawrence D.A.
      ,
      • Elokdah H.
      • Abou-Gharbia M.
      • Hennan J.K.
      • McFarlane G.
      • Mugford C.P.
      • Krishnamurthy G.
      • Crandall D.L.
      ). To develop better PAI-1 inactivators, we screened a library of known compounds for high affinity PAI-1 inhibitors with improved solubility and activity against vitronectin-bound PAI-1. A high throughput screen of the MicroSource SPECTRUM library identified five novel PAI-1 inactivating compounds. Two of the molecules identified were related natural polyphenolic compounds, which suggested a potential structure-activity relationship. Second generation compounds were designed and synthesized to probe this structure-activity relationship and tested for their ability to block PAI-1 activity in both purified systems and in vivo.

      DISCUSSION

      PAI-1 is thought to play a role in several chronic “lifestyle” diseases, including cardiovascular and fibrotic diseases, and metabolic syndrome. These pathologic associations make PAI-1 an ideal drug target; however, its metastable structure has made it a difficult candidate for drug design and study. To date most small-molecule inhibitors of PAI-1 lack high affinity for PAI-1 and are unable to inhibit PAI-1 in the presence of its plasma binding protein, vitronectin. To identify higher affinity inhibitors with better drug development potential, a high stringency screening assay was performed and a class of polyphenolic compounds was identified with anti-PAI-1 activity. A subset of these with the highest anti-PAI-1 activity contained galloyl moieties, and one, TA, demonstrated the lowest IC50 of any small-molecule PAI-1 inhibitor yet reported. One other study has identified members of the acylphloroglucinol class of polyphenols, sideroxylonals A–C, as potential PAI-1 inactivating compounds (
      • Neve J.
      • Leone P.A.
      • Carroll A.R.
      • Moni R.W.
      • Paczkowski N.J.
      • Pierens G.
      • Björquist P.
      • Deinum J.
      • Ehnebom J.
      • Inghardt T.
      • Guymer G.
      • Grimshaw P.
      • Quinn R.J.
      ). However, the reported IC50 values of these compounds (3.3–5.3 μm) are 2–3 orders of magnitude higher than TA and the novel synthetic polyphenols described here and are comparable with the IC50 of the simplest gallate compound in the current study, gallic acid (6.6 μm). This suggests that many polyphenolic compounds may share PAI-1 inactivating activity, but that the galloyl moiety may be a critical determinant in polyphenols for potent anti-PAI-1 activity.
      Despite the low IC50 of TA and its ability to inhibit PAI-1 in the high protein environment of plasma (data not shown), it is not an ideal drug candidate due to its molecular mass of nearly 2000 daltons and its relative promiscuity, interacting with other proteins as well as itself at low- to mid-micromolar concentrations. Nonetheless, the inhibition of PAI-1 by TA and other gallate-containing molecules (EGCG, EGCDG, and gallic acid) formed the basis for development of follow-up compounds with improved properties compared with these naturally occurring polyphenols. Smaller di-, tri-, and pentagallates were designed with improved solubility in physiologic buffers and greater specificity toward PAI-1. These studies determined that although two galloyl moieties were sufficient to provide potent anti-PAI-1 activity, a minimum of 3 galloyl groups was required for efficacy in plasma. This suggests the relationship between specificity for PAI-1 and nonspecific bulk protein binding is 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 (
      • Charlton P.A.
      • Faint R.W.
      • Bent F.
      • Bryans J.
      • Chicarelli-Robinson I.
      • Mackie I.
      • Machin S.
      • Bevan P.
      ,
      • Björquist P.
      • Ehnebom J.
      • Inghardt T.
      • Hansson L.
      • Lindberg M.
      • Linschoten M.
      • Strömqvist M.
      • Deinum J.
      ,
      • Neve J.
      • Leone P.A.
      • Carroll A.R.
      • Moni R.W.
      • Paczkowski N.J.
      • Pierens G.
      • Björquist P.
      • Deinum J.
      • Ehnebom J.
      • Inghardt T.
      • Guymer G.
      • Grimshaw P.
      • Quinn R.J.
      ,
      • Gils A.
      • Stassen J.M.
      • Nar H.
      • Kley J.T.
      • Wienen W.
      • Ries U.J.
      • Declerck P.J.
      ,
      • Crandall D.L.
      • Elokdah H.
      • Di L.
      • Hennan J.K.
      • Gorlatova N.V.
      • Lawrence D.A.
      ,
      • Gorlatova N.V.
      • Cale J.M.
      • Elokdah H.
      • Li D.
      • Fan K.
      • Warnock M.
      • Crandall D.L.
      • Lawrence D.A.
      ,
      • Rupin A.
      • Gaertner R.
      • Mennecier P.
      • Richard I.
      • Benoist A.
      • De Nanteuil G.
      • Verbeuren T.J.
      ,
      • Izuhara Y.
      • Takahashi S.
      • Nangaku M.
      • Takizawa S.
      • Ishida H.
      • Kurokawa K.
      • van Ypersele de Strihou C.
      • Hirayama N.
      • Miyata T.
      ,
      • Einholm A.P.
      • Pedersen K.E.
      • Wind T.
      • Kulig P.
      • Overgaard M.T.
      • Jensen J.K.
      • B⊘dker J.S.
      • Christensen A.
      • Charlton P.
      • Andreasen P.A.
      ). Another class of PAI-1 inhibitors based on diketopiperazine derivatives have been described with in vitro IC50 values reported in the 0.2–1 μm range; however, these compounds suffered from considerable physicochemical problems such as insolubility in physiologic buffer systems and were not subject to further development (
      • Folkes A.
      • Brown S.D.
      • Canne L.E.
      • Chan J.
      • Engelhardt E.
      • Epshteyn S.
      • Faint R.
      • Golec J.
      • Hanel A.
      • Kearney P.
      • Leahy J.W.
      • Mac M.
      • Matthews D.
      • Prisbylla M.P.
      • Sanderson J.
      • Simon R.J.
      • Tesfai Z.
      • Vicker N.
      • Wang S.
      • Webb R.R.
      • Charlton P.
      ). CDE-066, in contrast, is soluble in physiologic saline solution at concentrations greater than 10 mm without loss of anti-PAI-1 activity (data not shown). Two other PAI-1 inactivators have been described with IC50 values reported in the mid-nanomolar range; however, these compounds are ineffective against vitronectin-bound PAI-1, the predominant form of PAI-1 in plasma and the extracellular matrix (
      • Egelund R.
      • Einholm A.P.
      • Pedersen K.E.
      • Nielsen R.W.
      • Christensen A.
      • Deinum J.
      • Andreasen P.A.
      ,
      • Gardell S.J.
      • Krueger J.A.
      • Antrilli T.A.
      • Elokdah H.
      • Mayer S.
      • Orcutt S.J.
      • Crandall D.L.
      • Vlasuk G.P.
      ). Likewise several compounds with micromolar IC50 values are also ineffective against vitronectin bound PAI-1 (
      • Björquist P.
      • Ehnebom J.
      • Inghardt T.
      • Hansson L.
      • Lindberg M.
      • Linschoten M.
      • Strömqvist M.
      • Deinum J.
      ,
      • Gorlatova N.V.
      • Cale J.M.
      • Elokdah H.
      • Li D.
      • Fan K.
      • Warnock M.
      • Crandall D.L.
      • Lawrence D.A.
      ). 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 α-helices D and E and β-strands 1A and 2A, and directly adjacent to the vitronectin-binding site (
      • Björquist P.
      • Ehnebom J.
      • Inghardt T.
      • Hansson L.
      • Lindberg M.
      • Linschoten M.
      • Strömqvist M.
      • Deinum J.
      ,
      • Egelund R.
      • Einholm A.P.
      • Pedersen K.E.
      • Nielsen R.W.
      • Christensen A.
      • Deinum J.
      • Andreasen P.A.
      ,
      • Gorlatova N.V.
      • Cale J.M.
      • Elokdah H.
      • Li D.
      • Fan K.
      • Warnock M.
      • Crandall D.L.
      • Lawrence D.A.
      ). 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.
      The primary mechanism of action by which CDE-066 and the other synthetic polyphenols inactivate PAI-1 appears to be by binding to PAI-1 in a reversible manner and preventing stabilization of the non-covalent Michaelis complex with target proteases. This is demonstrated in Fig. 3 wherein preincubation of PAI-1 with each of the compounds inhibits its binding to the inactive protease, anhydrotrypsin. Identical data were also obtained in similar experiments using an inactive mutant of tPA (data not shown), indicating that the effect of the compounds on the initial association of PAI-1 with a protease is independent of the target protease. The SDS-PAGE analysis shown in Fig. 4 suggests that the polyphenolic compounds can also promote substrate behavior in PAI-1. However, in contrast to the loss of Michaelis complex formation (Fig. 3) and the loss of covalent complex formation (Fig. 4) the extent of cleavage observed is not dose dependent with the compounds added and varies with compound and target enzyme. It is possible that the extent of cleavage may be overestimated in these experiments due to complex dissociation during SDS-PAGE. Note, for example, that even in the absence of any compound, cleaved PAI-1 is apparent under experimental conditions where the stoichiometry of inhibition is near 1 (SI = 1.06, data not shown). Finally, consistent with the primary mechanism of action being inhibition of PAI-1:protease association, SPR experiments demonstrated that no CDE-066-dependent PAI-1 cleavage was detected when PAI-1 bound to vitronectin was reacted with active uPA (Fig. 8). This suggests that the combination of compounds and denaturants during SDS-PAGE may alter how PAI-1 is observed to behave.
      The identification of naturally occurring polyphenols as a class of PAI-1 inhibitors is intriguing because such compounds, especially polyphenols derived from teas, fruits, and cocoa, have been suggested in recent years to provide benefits against pathologies such as chronic inflammation, neurodegeneration, cancer, and cardiovascular disease (
      • Clement Y.
      ,
      • Saremi A.
      • Arora R.
      ,
      • Steinberg F.M.
      • Bearden M.M.
      • Keen C.L.
      ). Several mechanisms of action have been proposed for dietary polyphenols, characterizing these compounds as antioxidants, antiplatelet agents, and anti-inflammatory agents. Of particular relevance to PAI-1 are the proposed mechanisms by which dietary polyphenols may regulate hemostasis and prevent cardiovascular disease. In ex vivo and cell culture studies, dietary polyphenols have been shown to reduce tissue factor expression (
      • Di Santo A.
      • Mezzetti A.
      • Napoleone E.
      • Di Tommaso R.
      • Donati M.B.
      • De Gaetano G.
      • Lorenzet R.
      ), increase plasminogen activator levels (
      • Abou-Agag L.H.
      • Aikens M.L.
      • Tabengwa E.M.
      • Benza R.L.
      • Shows S.R.
      • Grenett H.E.
      • Booyse F.M.
      ), and decrease PAI-1 via changes in gene expression (
      • Pasten C.
      • Olave N.C.
      • Zhou L.
      • Tabengwa E.M.
      • Wolkowicz P.E.
      • Grenett H.E.
      ). These effects are observed at micromolar concentrations of the compounds, a dose range that is well within the effective concentrations of the polyphenols identified in our study. Thus, it is possible that a previously unrecognized direct inactivation of PAI-1 may contribute to the complex pro-fibrinolytic and cardioprotective effects associated with dietary polyphenols. Future studies will focus on improving the specificity and activity of this class of synthetic polyphenolic compounds against PAI-1 as well as clarifying the role that direct PAI-1 inactivation may play in the healthful benefits derived from dietary polyphenols.

      Acknowledgments

      We thank Martha Larsen and the Center for Chemical Genomics for drug screening, Dr. Scott Larsen of University of Michigan College of Pharmacy for the synthesis of PAI-039, and Nadine El-Ayache for assisting in the synthesis of the CDE inhibitors.

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