Structural Insight Into Inactivation of Plasminogen Activator Inhibitor-1 by a Small-Molecule Antagonist

Chemistry & Biology Article

Structural Insight into Inactivation of Plasminogen Activator Inhibitor-1 by a Small-Molecule Antagonist

Zhonghui Lin,1,2,5 Jan K. Jensen,2,3,5 Zebin Hong,1,2 Xiaoli Shi,1,2 Lihong Hu,4,* Peter A. Andreasen,2,3 and Mingdong Huang1,2,* 1State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou 350002, China 2Danish-Chinese Centre for Proteases and Cancer 3Department of Molecular Biology and Genetics Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus C, Denmark 4Shanghai Research Center for Modernization of Traditional Chinese Medicine, Chinese Academy of Sciences, Shanghai 201203, China 5These authors contributed equally to this work *Correspondence: [email protected] (L.H.), [email protected] (M.H.) http://dx.doi.org/10.1016/j.chembiol.2013.01.002

SUMMARY disease. Conditions with an increased risk of thrombosis, i.e., obesity, hypertension, and type-2 diabetes are associated with Plasminogen activator inhibitor-1 (PAI-1), a serpin, is elevated plasma PAI-1 levels (for reviews, see Booth, 1999; the physiological inhibitor of tissue-type and uroki- Vaughan, 2002). Evidence for a direct causal role of PAI-1 in nase-type plasminogen activators and thus also an thrombotic events comes from the observation that transgenic inhibitor of fibrinolysis and tissue remodeling. It is mice overexpressing PAI-1 develop thrombosis (Eren et al., a potential therapeutic target in many pathological 2002; Erickson et al., 1990), whereas removal of PAI-1 activity conditions, including thrombosis and cancer. by gene deletion (Carmeliet et al., 1993) or antagonizing antibodies (Abrahamsson et al., 1996; van Giezen et al., 1997) Several types of PAI-1 antagonist have been devel- prevents thrombus formation. In human malignant tumors, the oped, but the structural basis for their action has re- levels of uPA and PAI-1 are significantly higher than those in mained largely unknown. Here we report X-ray the corresponding normal tissues. Moreover, tumor levels of crystal structure analysis of PAI-1 in complex with uPA and PAI-1 are currently recognized as the most extensively a small-molecule antagonist, embelin. We propose validated biological prognostic factors in breast cancer (Harris a mechanism for embelin-induced rapid conversion et al., 2007; Ja¨ nicke et al., 2001; Look et al., 2002) and as of PAI-1 into a substrate for its target proteases markers of a poor prognosis in other cancers (Duffy, 2002). and the subsequent slow conversion of PAI-1 into Based on these findings, PAI-1 is a potential therapeutic target an irreversibly inactivated form. Our work provides in several pathological conditions. During the past decades, structural clues to an understanding of PAI-1 inacti- many efforts have therefore been made to develop PAI-1 antag- vation by small-molecule antagonists and an impor- onists in the form of small molecules (Bjo¨ rquist et al., 1998; Cale et al., 2010; Charlton et al., 1996; Crandall et al., 2004; tant step toward the design of drugs targeting PAI-1. Egelund et al., 2001; Einholm et al., 2003; Elokdah et al., 2004; Friederich et al., 1997; Gardell et al., 2007; Gils et al., 2002; INTRODUCTION Gorlatova et al., 2007; Izuhara et al., 2008; Liang et al., 2005; Neve et al., 1999; Rupin et al., 2008), monoclonal antibodies Plasminogen activator inhibitor-1 (PAI-1) is the primary physio- (Nielsen et al., 1986; for a review, see Gils and Declerck, 2004), logical inhibitor of tissue-type plasminogen activator (tPA) and peptides (Jensen et al., 2006; Mathiasen et al., 2008), and RNA urokinase-type plasminogen activator (uPA). In the circulation aptamers (Blake et al., 2009; Madsen et al., 2010). Several antag- and in the pericellular space, it is associated with its Mr onists were shown to function in vivo. For example, the small- 70,000 cofactor vitronectin. The PAI-1/plasminogen-activator molecule PAI-1 antagonist XR5118 (half-maximal inhibitory complexes are cleared from the circulation and the pericellular concentration [IC50] 3.5 mM) promoted endogenous fibrinolysis space by binding to endocytosis receptors of the low-density and reduced postthrombolysis thrombus growth in rabbits lipoprotein receptor family, including low-density-lipoprotein- (Friederich et al., 1997), and the small-molecule PAI-1 antagonist receptor-associated protein and very-low-density lipoprotein PAI-039 (tiplaxtinin, IC50 2.7 mM) had in vivo oral efficacy in two receptor (for reviews, see Dupont et al., 2009; Gliemann et al., different models of acute arterial thrombosis (Elokdah et al., 1994). 2004). Although several of these candidate compounds show The relative plasma levels of tPA and PAI-1 determine the promising characteristics in the laboratory, to the best of our fibrinolytic capacity of blood (for a review, see Booth, 1999). In knowledge, no PAI-1 antagonist has yet been evaluated in humans, a high plasma PAI-1 level is a risk factor in thrombotic clinical trials.

for a group of negatively charged amphipathic small-molecule Table 1. The IC50 of Embelin and Its Derivatives for Inactivation of PAI-1 inhibitors of PAI-1 (Egelund et al., 2001). Our work provides PAI-1 structural information about the mechanism behind such antagonists. Compounds Wild-Type 14-1B 1.62 ± 0.16a 4.94 ± 1.04 RESULTS

Identiﬁcation of a PAI-1 Antagonist: Embelin Embelin In order to identify small-molecule PAI-1 antagonists, we 1.04 ± 0.13 8.20 ± 2.70 adopted a chromogenic assay in which the activity of PAI-1 was evaluated by its ability to inhibit uPA-mediated hydrolysis of chromogenic substrates. By screening of a natural product library containing 1,600 compounds, we found a small-molecule Emb-d1 compound, embelin (Table 1), which inhibits wild-type (WT) 1.96 ± 0.25 3.87 ± 0.33 PAI-1 with an IC50 value of 1.6 mM. A stabilized variant of PAI-1 (N150H-K154T-Q319L-M354I) termed 14-1B (Berkenpas et al., 1995), which was required for the subsequent crystallization

Emb-d2 trials, was inhibited with an IC50 value of about 5 mM(Table 1). a IC50 mean value(mM) ± standard deviation, based on at least three inde- Similar results were obtained with tPA as the target protease pendent experiments. (Figure S1 available online). To evaluate which part of the See also Figures S2–S4. chemical structure of embelin is important for its antagonistic action, we tested two available structurally related compounds, PAI-1 belongs to the serpin family, which includes many serine Emb-d1 and Emb-d2 (Table 1). In support of a similar mode of protease inhibitors and whose members share a common fold binding, all three compounds inhibited WT and 14-1B PAI-1 with features including three b strands, nine a helixes, and a with IC50 values between 1 and 10 mM(Table 1). By comparison flexible reactive center loop (RCL). PAI-1 inhibits its target of the chemical structures, it is evident that the composition of proteases following the classical serpin mechanism of protease the aliphatic chain conjugated to the polar aromatic-ring struc- inhibition: (1) the RCL inserts into the active site of the target ture (head group) is of limited importance for the IC50 values. proteases and a reversible Michaelis complex is formed; (2) the To investigate the target specificity of embelin and the related RCL is cleaved by the target protease, leading to formation of compounds, we examined their effects on two other serpins, an acyl-enzyme intermediate, in which the P1 residue of the namely protease nexin-1 (PN-1), which is PAI-1’s closest phylo- RCL is covalently bound to the active-site serine of the protease genetic relative, and PAI-2. Both serpins are relatively fast by an ester bond; (3) the N-terminal side of the RCL rapidly inhibitors of uPA (Kruithof et al., 1995). The IC50 value for the inserts into b sheet A, thereby dragging the protease to the embelin-induced inactivation of either PAI-2 or PN-1 was more opposite pole of the serpin; and (4) the active site of the protease than 40-fold that for WT PAI-1 (Figure S2). is distorted and the P1 residue pulled out of the specificity pocket, Using PAI-1 in physiologically relevant concentrations, embe- preventing hydrolysis of the ester bond. Under some conditions, lin was shown to antagonize PAI-1 inhibition of uPA-catalyzed complex formation is abortive and the P1-P10 bond is cleaved generation of plasmin in a coupled assay (Figure S3). However, without complex formation, an event termed serpin substrate preincubation of PAI-1 with 5 mM somatomedin B-domain of behavior. Under physiological conditions, active PAI-1 can spon- the PAI-1 cofactor vitronectin (Seiffert and Loskutoff, 1991; taneously convert into an inactive, so-called latent form with Zhou et al., 2003) before the addition of embelin abolished the a half-life of 1–2 hr in which the RCL fully inserts into b sheet A inhibition of embelin against PAI-1, suggesting that SMB blocks without prior cleavage (Levin and Santell, 1987). Structurally, the binding of embelin to PAI-1 (Figure S4). thus, PAI-1 can exist in three different conformations, i.e., the active form, the latent form, and the cleaved form, in which the Embelin-PAI-1 Binding Affinity RCL is cleaved and inserted into b sheet A either in complex We next used perturbation of intrinsic tryptophan fluorescence with a target protease or as a free substrate-cleaved product to study the binding of embelin to various conformational forms (for reviews, see Dupont et al., 2009; Huntington, 2006). of PAI-1. This tryptophan-fluorescence-quenching method was Until now, structural information about the mechanism of originally developed to measure the binding of antagonists to action of small-molecule PAI-1 antagonists has been lacking. PAI-1 (Madsen et al., 2010). To be able to work with a pure The structural metastability of PAI-1, together with its propensity preparation of active PAI-1 without contamination with latent to form aggregates (Lin et al., 2011), poses challenges to material, we used PAI-1-14-1B for this analysis. Figure 1 shows X-ray crystal structure analyses of PAI-1 in complex with PAI-1 the change of tryptophan fluorescence emission of 10 mM active antagonists. Isolation from a library of natural products of PAI-1-14-1B at 328 nm when titrated with the indicated final small-molecule PAI-1 antagonist, embelin, has allowed us to concentrations of embelin at 25C. The experimental data from determine the three-dimensional structure of PAI-1 in complex at least three independent measurements were fitted to a 1:1 with a small-molecule antagonist. We have shown that embelin binding model, resulting in a KD of 24 mM, a value only slightly abolishes the inhibitory action of PAI-1 by a mechanism higher than the IC50 value for inhibition of PAI-1-14-1B by similar to that previously described by biochemical methods embelin (Table 1). This result also suggests that embelin binds

exhibiting substrate behavior, resulting in a slightly faster migration in SDS-PAGE, and subsequently, a slower irreversibly inactivated form, which causes PAI-1 to appear as a nonreactive band in SDS-PAGE (Egelund et al., 2001). Additional support for the link between the embelin-induced PAI-1 inhibition and the previously published mechanism was obtained by an assay based on a chromogenic uPA substrate. In the assay, PAI-1 was incubated with 20 mM embelin for varying periods of time, followed by assay of the remaining PAI-1 activity by a chromogenic assay in buffers with or without 0.5% bovine serum albumin (BSA). Serum albumin binds to embelin and prevents the effect of embelin on PAI-1; thus, reversibly inactivated PAI-1 will regain activity in the assay with BSA, but not in the assay without BSA, while irreversibly inactivated PAI-1 will Figure 1. Binding of Embelin to PAI-1 Perturbation of intrinsic tryptophan fluorescence of active PAI-1-14-1B remain inactive in both assays. When comparing the time (solid circles), reactive-center-cleaved PAI-1-14-1B (open circles), or latent courses of embelin inactivation of PAI-1 in the two assays, it is WT PAI-1 (triangles) at 328 nm was monitored after addition of the indicated evident that the inactivation at early time points is reversible, concentrations of embelin. The fluorescence emission values were corrected while at later time points the slower irreversible inactivation of for PAI-1 fluorescence and embelin interfilter effects and normalized to Fmax.A PAI-1 is increasingly dominant (Figure 3). KD of 24 ± 4 mM (n = 3) for binding of embelin to PAI-1-14-1B was obtained, based on a best fit (solid line) to a 1:1 binding model. Each of the plots in the figure represents one representative experiment out of three independent X-Ray Crystal Structure Analysis of PAI-1 in Complex determinations. with Embelin As embelin appears to represent a previously described group of near a tryptophan residue of PAI-1 and thus can perturb PAI-1 small-molecule antagonists of PAI-1, we decided to pursue fluorescence. When evaluating the binding of embelin to latent a structural understanding of its antagonism of PAI-1. To do WT PAI-1 and reactive-center-cleaved PAI-1-14-1B, no satu- so, we subjected the embelin-PAI-1 complex to X-ray crystal rable binding could be observed and it could be concluded structure analysis. that the binding affinities were at least 10-fold lower than that As crystallization of active PAI-1 is hampered by its tendency for binding to active PAI-1-14-1B (Figure 1). to aggregate and its short shelf life due to conversion to the latent form, and high salt and low pH conditions are typically needed to Embelin’s Antagonistic Mechanism keep PAI-1 stable in vitro (Stout et al., 2000; Zhou et al., 2001), To study embelin’s antagonistic mechanism, 0.4 mM PAI-1 was we decided to use functionally stabilized PAI-1-14-1B, which preincubated with 20 mM embelin for different lengths of time, has been commonly used in crystallographic studies (Lin et al., followed by addition of 4 mM uPA and evaluation of uPA:PAI-1 2011; Nar et al., 2000; Sharp et al., 1999; Stout et al., 2000). complex formation by SDS-PAGE. As shown in Figure 2, in the Following extensive trials with different approaches, we found absence of embelin, more than 90% of the PAI-1 was able to that PAI-1 remains stably in solution in the presence of embelin, form a covalent complex with uPA, as indicated by the appear- especially at low temperature (4C), which allows dialysis of ance of a band with a migration corresponding to a Mr of approx- the protein to low salt (150 mM NaCl) and neutral pH (pH 7.4), imately 70,000. When PAI-1 was pre-incubated with embelin, the and at this condition, we finally succeeded in cocrystallizing intensity of the complex band was reduced in a time-dependent embelin with active PAI-1-14-1B. A 2.6 A˚ data set was collected manner. Concomitantly, at short incubation times, there was an and initial phasing was obtained by molecular replacement increase in the density of a band migrating slightly faster than using existing PAI-1 structure (Protein Data Bank [PDB] 1B3K). native PAI-1, representing the reactive-center-loop-cleaved Statistics of the data collection and structure refinement are form (Jensen and Gettins, 2008)(Figure 2). At later time points, summarized in Table S1. PAI-1 reappeared at the position of the native band, representing The asymmetric unit contained four PAI-1 molecules, each a nonreactive, noncleaved species. This pattern was observed bearing the well described fold of an inhibitory active serpin. for both WT and PAI-1-14-1B, although PAI-1-14-1B was inacti- As for the majority of crystallized active serpins, the RCL appears vated more slowly by embelin. At 37C, WT PAI-1 was converted fully flexible and solvent exposed and thus, for the most part, to substrate behavior within a couple of minutes, after which time unobservable. In two of the four molecules (molecules A and the irreversibly inactivated nonreactive form began to accumu- C) in the asymmetric unit, parts of the RCL showed visible late, although the conversion to this form was not complete electron density due to crystal lattice contacts with neighboring until after more than 60 min (Figure 2A). This inactivated form molecules at their b sheet A, leading to the extension of the of PAI-1 was found to be PAI-1 oligomer. The inactivation rate b sheet A of the contacted molecule with a seventh strand. decreased strongly with decreasing temperature. At 4C, there Alignment of all four PAI-1 molecules together with the previ- was little conversion to the irreversibly inactivated, inert form ously published structure of free PAI-1-14-1B (Stout et al., within several hours (Figure 2B). These observations closely 2000) showed that none of the four molecules deviated signifi- follow those described in previous reports, in which it was shown cantly from the others with respect to overall fold (RMSD that a group of negatively charged small-molecule PAI-1 antag- between 0.40 and 0.57 A˚ ; high values include embelin). Thus, onists induces a rapid reversible conversion of PAI-1 into a form effects caused by embelin binding to PAI-1 seem to result from

Figure 2. Time Course of Inactivation of PAI-1 by Embelin, as Evaluated by SDS- PAGE PAI-1 WT or PAI-1 14-1B, at both 37C (A) and 4C (B), in both cases 478 nM, were incubated in the presence of 24.5 mM embelin. At the indicated time points, samples of the incubation mixtures were mixed with a small volume of uPA, resulting in ﬁnal concentrations of 0.4 mM PAI-1, 20mM embelin, and 4 mM uPA. After 2 min, SDS sample buffer was added to the samples, which were then subjected to reducing SDS-PAGE. The migration of uPA: PAI-1 complex and intact (—) and reactive-center- cleaved ($$$) PAI-1 is indicated to the right. The

migration of Mr markers are indicated to the left. See also Figure S1.

altered dynamics of conformational changes within the PAI-1 observe embelin in its preferred binding site in PAI-1. The embe- molecule rather than from direct structural perturbation. In lin ring must be presumed to harbor the majority of the binding addition, the crystal packing of the embelin-PAI-1 complex energy and to govern its binding-site specificity. These structural also agrees well with previously published PAI-1-14-1B struc- aspects are consistent with the binding data of embelin-related tures (PDB 1DVM and 1B3K), indicating that the presence of compounds (Table 1), which show a key role for the ring structure embelin does not impact crystal packing. Surprisingly, well- of embelin and less importance for the aliphatic tail. defined electron density of only one embelin molecule was observed in the asymmetric unit. The embelin electron density The Embelin Binding Site Evaluated by Site-Directed (Figure 4D; Table S1) and the omit map (Figure S5) of the Mutagenesis structure were of sufficient quality for unambiguous positioning To further validate the details of the interactions of PAI-1 with of the embelin molecule on the surface of PAI-1. embelin, we studied the effect of mutating the residues, which Embelin is localized in a groove bordered by a helix D, helix F, had been implicated in embelin binding by the X-ray crystal- b strand 2A, and the loop connecting b strand 1A to hE (Figure 4). structure analysis. We generated five PAI-1 mutants, Y79A, This location is close to the so-called ‘‘flexible joints region’’ (a T94A, D95A, S119A, and H143A, and used the chromogenic helix D and a helix E (Stein and Chothia, 1991) and the ‘‘shutter region’’ (the central part of b strand 5A and b strand 3A and the underlying a helix B (Carrell and Stein, 1996). As shown in Figure 4, the polar, six-member ring of embelin sits in the middle of a groove forming direct strong contacts (<3.5 A˚ ) with the side chain of Asp-95 and the main-chain atoms of residue Thr-93, both on b strand 2A, and with residue Tyr-79 on a helix D. In addition, the embelin head group makes two bifurcated hydrogen bonds (3.8 A˚ and 5.2 A˚ , respectively) with His-143 in the top of a helix F, and has weaker interactions with Thr-94, and Met-45 on the underlying a helix B. The long aliphatic carbon chain of embelin, on the other hand, stretches out of the cavity and participates in interactions with residues including Ser-119 on the hE-s1A connecting loop and Trp-139 of a helix F. Even though strong electron density for embelin molecule was seen in only one of the four PAI-1 molecules (molecule B) in the Figure 3. Time Course of Inactivation of PAI-1 by Embelin, as Evalu- asymmetric unit, we did observe weaker electron densities for ated by a Chromogenic Assay the polar embelin ring at the same location in the other three PAI-1 (20 nM) was incubated at 37C in the presence of 20 mM embelin. At the PAI-1 molecules (Figure S5). The fact that an intact embelin indicated time points, samples of the incubation mixtures were mixed with molecule is observed in only the B molecule in the asymmetric equal volumes of 20 nM uPA, either with (solid circles) or without (open circles) unit can be attributed to van der Waals crystal contacts between 1% BSA, resulting in 10 nM final concentrations of PAI-1 and uPA and a final the embelin aliphatic chain and the neighboring symmetry- concentration of BSA of 0 or 0.5%. After 2 min, the remaining uPA activity was monitored by addition of the 210 mM of S-2444. The relative PAI-1 activity at related PAI-1 molecule (residue P111 and the methylene moiety each time point was calculated from the difference in the slope of the lines of residue R115 from molecule D; Figure 4D). These contacts relating OD405 to time between mixtures with and without PAI-1 and expressed probably have a dampening effect on the presumably flexible relative to the PAI-1 activity at time zero. The inserted figure is a zoom of the embelin aliphatic tail. The reduced flexibility would allow us to early times as indicated by the broken lines.

Figure 4. Crystal Structure of PAI-1 in Complex with Embelin In all ﬁgures, secondary-structure elements of PAI-1 constituting the binding pocket of embelin are highlighted as follows: b strands 2A and 1A and the connecting loop are blue; a helixes D and F are red; and the underlying a helix B is yellow. Helixes and b strand numbers are identiﬁed. In the cartoon representation, the remaining strands of b sheet A are colored green and visible residues of the RCL connected by a hypothetical sketch of the unobserved RCL as a dashed line are highlighted in orange. (A and B) The PAI-1 molecule shown in cartoon (A) and surface (B) representation. Embelin is shown as sticks. (C) Close-up view of the embelin binding site with direct contacts shown as cyan dashed lines. Selected residues of PAI-1 binding to embelin are given in stick representation with standard atom (CPK) colors. Tyr-79, located on top of embelin, is not shown for clarity. (D) The 2Fo-Fc s-weighted electron density map wrapping the embelin molecule at a contour level of 1.0 s is shown as green mesh. Residues from the crystallographic symmetry-related PAI-1 in contact with embelin are shown in yellow stick representation and labeled SYM. See also Table S1 and Figures S5–S7.

antagonist embelin, described here, does not by itself represent a compound suitable for in vivo studies, primarily due to its modest afﬁnity, limited solubility, strong serum-albumin binding, and vitronectin competition, this study does provide structural insight into a small- assay to evaluate the inhibition of each mutant by embelin. As molecule PAI-1 antagonist using a combination of mechanisms shown in Table 2, alanine substitutions of Asp-95 (in b strand 2 and 3, thus providing an agenda for further development of

2A) and His-143 (in a helix F) increased the IC50 value about PAI-1 antagonists. 10-fold. The strong effect of His-143 mutation to alanine may The ﬁrst organochemical PAI-1 antagonist developed was stem from the bifurcated interaction of the His-143 side chain a diketopiperazine derivative (Bryans et al., 1996). A variety of with two hydroxyl groups of embelin (Figure 4C). In addition, related diketopiperazine compounds were developed later the Y79A mutation (in a helix D) caused an approximately (for a review, see Stoppelli et al., 2008). Various biochemical

2-fold increase of IC50. No signiﬁcant changes in IC50 were analyses showed that the diketopiperazine derivatives induce observed for mutations T94A and S119A. Thus, the site-directed a stable, monomeric, inactive PAI-1 conformation that is mutagenesis results were consistent with the determined crystal different from other known PAI-1 conformations (Egelund et al., structure.

DISCUSSION Table 2. The Effect of PAI-1 Mutation on the IC50 of Embelin m PAI-1 is a potential therapeutic target in several pathophysio- Mutants IC50 ( M) logical conditions. Therefore, there is great interest in developing Wild-type 1.62 ± 0.16a PAI-1 antagonists. Conceivably, there are a number of possible Y79A 3.15 ± 0.67 mechanisms for inactivation of the antiproteolytic action of T94A 1.67 ± 0.15 PAI-1 (Gils and Declerck, 2004): (1) sterically blocking the initial D95A 15.2 ± 1.87 formation of the Michae¨ lis complex between PAI-1 and its target S119A 2.23 ± 0.38 proteases; (2) preventing the conformational change in PAI-1 that is associated with complex formation, resulting in cleavage H143A 12.0 ± 1.80 a m of PAI-1 as a substrate; or (3) conversion of PAI-1 into an inactive IC50 mean value( M) ± standard deviation, based on at least three latent or otherwise inert form. Although the small-molecule independent experiments.

Figure 5. Functional Aspects of the Embelin Binding Site of PAI-1 Color coding and labels of secondary structure elements are as in Figure 4A. (A) The ﬁgure depicts the well-deﬁned hydrogen- bond network of the shutter region between b sheet A and a helix B to which embelin is connected. (B) The same region in cleaved 14-1B (PDB ID 3CVM) overlaid with embelin from the embelin- PAI-1 structure. The resulting rearrangement of the shutter-region hydrogen-bond network is shown. See also Figures S6 and S7.

2001; Einholm et al., 2003). Later, a number of other, mostly of PAI-1 (Egelund et al., 2001; Pedersen et al., 2003). Such an negatively charged, amphipathic organochemical compounds inactivation mechanism includes a fast reversible step where of diverse chemical structures were reported to inactivate embelin induces PAI-1 substrate behavior, followed by a slow PAI-1. Some of these compounds were found to induce PAI-1 irreversible induction of the PAI-1 inactive form. A shift of b strand substrate behavior within seconds, followed by PAI-1 polymeri- 3A and b strand 2A away from b strand 5A and closer to a helix D zation (Egelund et al., 2001; Pedersen et al., 2003). Also, high and a helix E is necessary to make space for the incoming RCL concentrations of nonionic detergents induced substrate during reaction of serpins with target proteases. During sheet behavior and latency transition (for a review, see Stoppelli opening, b strands 3A and 5A slide apart over the underlying et al., 2008). The most potent PAI-1 small-molecule antagonist a helix B. The shutter region (Carrell and Stein, 1996), underlying isolated to date is a polyphenolic compound, CDE-066 (IC50 b sheet A, contains a complicated hydrogen-bonding network, 10 nM), which acts by blocking the initial formation of the which is rearranged during RCL insertion (Figure 5). Accordingly, Michae¨ lis complex between PAI-1 and a target protease (Cale amino acid substitutions in the shutter region affect PAI-1 et al., 2010). latency transition and tendency to substrate behavior (Hansen Our crystal data, as well as the site-directed mutagenesis et al., 2001). In active PAI-1-14-1B, Gln-322, Asn-167, Asp-95, results, clearly show that embelin binds to the groove consti- and Ser-41 are connected by hydrogen bonds, whereas in tuted by a helixes D and F and b strand 2A. The key residues cleaved PAI-1-14-1B, Gln-322 is hydrogen-bonded to Ser-35, involved in the interaction with the antagonist have been defined while Asn-167 forms hydrogen bonds to Asp-95 and Ser-41 (Fig- as Tyr-79 (a helix D), Asp-95 (b strand 2A), and His-143 (a helix F) ure 5). A main contact between embelin and PAI-1 is the strong (Figure 4). The location of the binding site is near the binding site bifurcated hydrogen bonds to Asp-95 (Figure 4). Moreover, the suggested by in silico molecular docking and alanine scanning localization of the polar ring of embelin seen in the complex mutagenesis with other small-molecule antagonists of PAI-1. between embelin and native PAI-1-14-1B is not compatible This localization of the binding site is also in good agreement with binding after RCL insertion and b sheet A expansion, as with the fluorescence-quench data, as the most likely ‘‘reporter’’ this position is then occupied by b strand 2A. Therefore, we tryptophan side chain is Trp-139, situated less than 3.5 A˚ from propose that embelin fixes b strand 2A to the neighboring embelin (Figure 4) and thus sensing changes in the electronic a helixes, D and F. Embelin thereby may delay the RCL insertion environment upon embelin binding. Embelin occupies part of into b strand A that is a prerequisite for translocation of the a small and charged groove. This pocket is sandwiched between protease to the opposite pole of the serpin and subsequent b strand 2A and a helix B and surrounded by residues Asn-167, covalent complex formation (Dupont et al., 2009; Huntington, Thr-93, Thr-94, Asp-95, Tyr-37, Gly-38, and Ser-41 (pocket 1 in 2006). The protease will thus be allowed time to complete the Figure S6). Interestingly, an even larger and deeper pocket catalytic cycle, resulting in the cleavage of PAI-1 as a substrate. nearby is not occupied to any significant extent by embelin. Thus, our crystal structure is fully compatible with embelin- This is a larger, hydrophobic pocket surrounded by a helices induced PAI-1 substrate behavior. Also, the observed crystal A, B, and D and defined by residues Ala-12, Met-62, Phe-64, lattice contacts between the neighboring molecule at b sheet A Leu-75, Tyr-37, Ala-40, and Ser-41 (pocket 2 in Figure S6). The suggests that the facilitated crystallization in the presence of em- two pockets are separated by residue Ser-41. These two belin (and the second, irreversible inactivation step) may involve pockets represent interesting areas to target in future struc- a ‘‘strand 7A polymerization’’ (Sharp et al., 1999). ture-based studies geared toward development of PAI-1 antag- Our described binding site and antagonistic mechanism onists, and they may be utilized by some previously developed may very well be generalized to several other small-molecule inhibitors. antagonists of PAI-1, which, based on data from competition Using biochemical methods, we showed that the inactivation with monoclonal antibodies with known epitopes, site-directed of PAI-1 by embelin follows the mechanism previously described mutagenesis, and molecular modeling, bind in the same area for a group of negatively charged small-molecule antagonists (Bjo¨ rquist et al., 1998; Egelund et al., 2001; Gorlatova et al.,

2007; Pedersen et al., 2003). The identified binding site is also in 150 mM NaCl, 1% DMSO, 30 nM PAI-1, 40 nM uPA, and 80 mM S-2444. agreement with the fact that the antagonistic action of embelin The activities of the remaining free uPA were then measured by the initial and this group of compounds is rivaled by vitronectin, having velocity of cleavage of the chromogenic substrate in a microplate reader (Bio- Tek Synergy 4) at 405 nm. Compounds with 70% inhibition at 10 mM were an adjacent binding site encompassing residues in b strands selected for the confirmation screening, and their activities against WT a 1A and 2A and helix F (Jensen et al., 2002; Zhou et al., 2003). PAI-1, PAI-2, and PN-1 were also evaluated. Embelin is a natural product found in the fruit of the Embelia Data were analyzed using OriginPro 7.5 software (OriginLab). The data were ribes Burm plant (Myrsinaceae) and has been shown to have presented as mean ± SD for at least three separate determinations for each antitumor (Reuter et al., 2010) and antidiabetes (Mahendran group. et al., 2010) properties. Recently, molecular targets of embelin that contribute to its anticancer effect have been shown to Co-Crystallization of PAI-1-Embelin Complex and Structure Determination include X-chromosome-linked inhibitor-of-apoptosis protein The PAI-1-14-1B protein (2 mg/ml in buffer A: 50 mM MES, pH 6.1, and 1M k (Nikolovska-Coleska et al., 2004) and nuclear factor- B(Ahn NaCl) was incubated with 200 mM embelin for 30 min at 4C and then dialyzed et al., 2007). These observations suggest that embelin has against buffer B (20 mM Tris pH 7.4 and 150 mM NaCl). Thereafter, the protein multiple targets. In order to obtain more knowledge on the was reincubated with 200 mM embelin in a final DMSO concentration of 10% specificity of embelin’s action toward PAI-1, we tested embelin for another 30 min. The protein was then concentrated to 10 mg/ml. Crystals binding to PAI-2 (SERPINB2) and PN-1, both closely related were grown at 20 C using the sitting drop method by mixing equal volumes serpins able to inhibit uPA. We found that embelin inhibited of protein solution (10 mg/ml) with a precipitant solution (1.6 M ammonium sulfate, 0.1 M MES, pH 6.1). The crystals appeared overnight in clustered PN-1 with an IC50 more than 40-fold that with which it inhibited thin plates. Single crystals of diffraction quality were finally obtained by streak WT PAI-1, and that it did not inhibit the activity of PAI-2, even seeding method. Diffraction data were collected at BL17U of Shanghai at a concentration of 100 mM. We examined the area in the Synchrotron Radiation Facility at 160C using the precipitant solution PAI-2 crystal structure (PDB 1BY7; Harrop et al., 1999) that containing 25% glycerol as cryoprotectant. The data was processed and corresponds to the binding site in PAI-1, and found, in agree- integrated with HKL2000 (Otwinowski and Minor, 1997). The structure of the ment with the binding data, that the groove is too shallow and embelin-PAI-1 complex was solved by molecular replacement (Collaborative Computational Project 4, 1994) using the structure of active PAI-1 (PDB that the neighboring PAI-2 residues differ from those of PAI-1 1B3K; Sharp et al., 1999) as a searching model. The structure was refined to an extent that it is impossible to accommodate an embelin with iterative cycles of manual model building using Coot (Emsley and Cowtan, molecule at this pocket (Figure S7). These data suggests that 2004) and refinement with either Refmac5 (Murshudov et al., 1997) or CNS v1.3 embelin is specific enough to discriminate between structurally (Brunger, 2007). The quality of the final model was validated with PROCHECK similar serpins. Moreover, because PAI-1 also plays an impor- (Laskowski et al., 1993) and analyzed by PyMol (DeLano, 2003). tant role in tumor metastasis (Andreasen, 2007) and is currently recognized as the most extensively validated biological prog- ACCESSION NUMBERS nostic factor in breast cancer (Harris et al., 2007), our study The coordinates of the PDB were deposited in http://www.rcsb.org (3UT3). may expand the understanding of the mechanism behind the potential anticancer action of embelin. SUPPLEMENTAL INFORMATION In summary, our work provides the structural insights into the mechanism of PAI-1 antagonism by a small-molecule antago- Supplemental Information includes Supplemental Experimental Procedures, nist. Such structural information will facilitate further design one table, and seven figures and can be found with this article online at and development of highly efficacious PAI-1 antagonists. http://dx.doi.org/10.1016/j.chembiol.2013.01.002

ACKNOWLEDGMENTS SIGNIFICANCE We thank the Shanghai Synchrotron Radiation Facility and the scientists at We report the structural characterization of a small-mole- beamline BL17U for assistance with X-ray data collection. We are also grateful cule antagonist of PAI-1. This work provides structural clues to Professor Marie Ranson for providing PAI-2 DNA plasmid and to Professor to answering the long-standing questions on PAI-1 antago- Denis Monard for providing PN-1 protein. This work was supported by the nism: What is a suitable binding site in PAI-1? How can Natural Science Foundation of China (31161130356 and 30925040) and the a small molecular antagonist modulate PAI-1? These results Fujian province (2009I0029), the Danish National Research Foundation (26- 331-6), and the Lundbeck Foundation (R34.A3528). will facilitate the design and development of future PAI-1 antagonists. Received: December 27, 2011 Revised: December 24, 2012 EXPERIMENTAL PROCEDURES Accepted: December 27, 2012 Published: February 21, 2013 Screening of Natural Product Library To identify small molecules with anti-PAI-1 activity, we screened a natural REFERENCES compound library, which consists of 1,600 bioactive compounds isolated from various traditional Chinese herbal medicines. In this screening, a chromo- Abrahamsson, T., Nerme, V., Stro¨ mqvist, M., Akerblom, B., Legnehed, A., genic assay (Egelund et al., 2001) was used to measure the inhibitory activities Pettersson, K., and Westin Eriksson, A. (1996). Anti-thrombotic effect of 75 of small molecules against PAI-1. Brieﬂy, the compounds were preincubated a PAI-1 inhibitor in rats given endotoxin. Thromb. Haemost. , 118–126. with PAI-1-14-1B for 10 min, followed by the addition of human uPA. After Ahn, K.S., Sethi, G., and Aggarwal, B.B. (2007). Embelin, an inhibitor of an additional 10 min incubation, a chromogenic substrate, S-2444 (L-pyro- X chromosome-linked inhibitor-of-apoptosis protein, blocks nuclear factor- Glu-Gly-Arg-p-nitroaniline HCl, Chromogenix), was added to the mixture. kB (NF-kB) signaling pathway leading to suppression of NF-kB-regulated The ﬁnal reaction mixture (100 ml) contained 50 mM Tris–HCl, pH 7.4, antiapoptotic and metastatic gene products. Mol. Pharmacol. 71, 209–219.

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