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Targeting the Hemopexin-like Domain of Latent Matrix Metalloproteinase-9 (proMMP-9) with a Small Molecule Inhibitor Prevents the Formation of Focal Adhesion Junctions

A lack of target specificity has greatly hindered the success of inhibitor development against matrix metalloproteinases (MMPs) for the treatment of various cancers. The MMP catalytic domains are highly conserved, whereas the hemopexin-like domains of MMPs are unique to each family member. The hemopexin-like domain of MMP-9 enhances cancer cell migration through self-interaction and heterointeractions with cell surface proteins including CD44 and α4β1 integrin. These interactions activate EGFR-MAP kinase dependent signaling that leads to cell migration. In this work, we generated a library of compounds, based on hit molecule N-[4-(difluoromethoxy)phenyl]-2-[(4-oxo-6-propyl-1H-pyrimidin-2-yl)sulfanyl]-acetamide, that target the hemopexin-like domain of MMP-9. We identify N-(4-fluorophenyl)-4-(4-oxo-3,4,5,6,7,8-hexahydroquinazolin-2-ylthio)butanamide, 3c, as a potent lead (Kd = 320 nM) that is specific for binding to the proMMP-9 hemopexin-like domain. We demonstrate that 3c disruption of MMP-9 homodimerization prevents association of proMMP-9 with both α4β1 integrin and CD44 and results in the dissociation of EGFR. This disruption results in decreased phosphorylation of Src and its downstream target proteins focal adhesion kinase (FAK) and paxillin (PAX), which are implicated in promoting tumor cell growth, migration, and invasion. Using a chicken chorioallantoic membrane in vivo assay, we demonstrate that 500 nM 3c blocks cancer cell invasion of the basement membrane and reduces angiogenesis. In conclusion, we present a mechanism of action for 3c whereby targeting the hemopexin domain results in decreased cancer cell migration through simultaneous disruption of α4β1 integrin and EGFR signaling pathways, thereby preventing signaling bypass. Targeting through the hemopexin-like domain is a powerful approach to antimetastatic drug development.


M etastasis accounts for 90% of all human cancer related
deaths, yet we lack adequate drugs to target this biological process often associated with aggressive cancers. 1 Accumulating evidence suggests an important role for matrix metalloproteinases (MMPs) in promoting cancer progression whereby they modify their surrounding environment to promote the growth and spread of tumor cells. 2−5 Although MMPs represent the most prominent family of proteinases associated with tumorigenesis, drugs designed to inhibit their proteolytic activities largely failed in clinical trials due to issues with selectivity for individual MMPs. 6 The highly conserved catalytic domain within this family of zymogens required a paradigm shift to the development of novel MMP inhibitors (MMPIs) targeting less conserved, noncatalytic functional domain(s) of the proteases to increase target specificity and selectivity. 7 The biological functions of MMPs extend beyond just their proteolytic function and include induction of complex signaling cascades. 8−10 Homodimerization of secreted proMMP-9 hemopexin domains is sufficient to induce cancer cell migration independent of proteolytic activity. 11 Using an shRNA approach, the mechanism behind this phenotype was dissected and found to be dependent on an interaction between MMP-9 and CD44 at the cell surface. This interaction required an intact MMP-9 hemopexin domain (PEX-9). Interaction between CD44 and PEX-9 results in increased epidermal growth factor receptor (EGFR) phosphorylation and subsequent activation of mitogen-activated protein kinase (MAPK)/extracellular signal− regulated kinases (ERK) signaling, thereby enhancing the migratory capacity of proMMP-9 expressing cancer cells. 12 Signaling was lost upon deletion or swapping of PEX-9 with the PEX domain of its closest homologue MMP-2.
The X-ray crystal structure of the PEX domain of MMP-9 has been solved. PEX-9 forms a propeller-like structure composed of four blades, which converge and are linked between blades I and IV through disulfide bonds. 13 Each blade of the PEX domain is composed of a single α-helix and four antiparallel β-strands. They form a scaffold for substrate recognition and docking 14 Published reports have shown that PEX-9 can interact with different integrin subunits to promote enhanced cancer cell migration, invasion, and survival in various cancer cell types. 15−20 Of particular interest to our study was a recent fi n d i n g t h a t p e p t i d e s e q u e n c e F P G V P L D T H D -VFQYREKAYFC within the central cavity of PEX9/blade IV is a binding site for α4β1 integrin. Treatment with synthetic peptide was found to prevent B-cell chronic lymphocytic leukemia transendothelial cell migration and survival. 21,22 Mutation of either aspartic acid (Asp) residue (Asp-660 and Asp-663) in this peptide sequence abolished inhibition supporting the importance of Asp residues as key recognition sites for integrins. 23,24 Furthermore, the LDT motif in this peptide sequence is a highly conserved motif found in a variety of well-known α4β1 integrin ligands. 23,25,26 We previously utilized an in silico DOCKing approach for finding small molecules that bind to MMP-9 and identified a substituted pyrimidone, 1a, which docked to the noncatalytic PEX domain of MMP-9. 27 Compound 1a inhibited proMMP-9-mediated cancer cell migration and proliferation in vitro. Importantly, this compound did not inhibit MMP-9 enzymatic activity. Pyrimidone 1a specifically binds to the hemopexin domain of MMP-9 and does not bind to closely related MMPs. In addition to inhibiting proMMP9-mediated cell migration, compound 1a effectively prevented cancer metastasis in an in vivo mouse xenograft model. 27 In this current study, we generated an in silico library of analogs of compound 1a to find a more potent compound for potential clinical application. After screening for optimal DOCKing scores in silico, the top 14 compounds were synthesized and tested in a 2-D collagen dot migration assay. A high nanomolar inhibitor that maintained MMP specificity was identified (compound 3c). Our data demonstrate that PEX-9 inhibitor 3c selectively inhibits cell migration induced by proMMP-9, leading to reduced cell invasion predominately through disruption of α4β1 integrin focal adhesion complexes. Thus, PEX-9 inhibitors rectify the abnormal migratory phenotype associated with high MMP-9 expression.

Development of a Second Generation Hit Compound.
We designed analogs of compound 1a based on possible derivative syntheses that utilized an amide and a thioether coupling. Thus, three moieties were varied: the amine (R 1 ), the linker ([CH 2 ] n ), and the sulfhydrylpyrimidone (R 2 ; Scheme 1). To rationally prioritize the synthesis of compound 1a analogs, the University of California San Francisco (UCSF) DOCK (6.7) algorithm was used to rank various structures for predicted binding affinity. The proposed inhibitors were DOCKed to the PEX-9 domain pocket (PDB code 1ITV; Figure 1A and B). This pocket is at the center of the four blades of the hemopexin domain.
Chemical Synthesis of PEX-9 Parental Compound 1a and Analogs. First, the early lead compound 1a was resynthesized to confirm the chemical structure based on full characterization, using the synthetic route shown in the Scheme 1. The synthesis commenced with the reaction of aniline 5a with bromoacetic acid in the presence of EDC·HCl to afford bromoacetanilide 6a as a white solid in 67% yield. Subsequently, 6a was reacted with thiouracil 7 in aqueous NaOH and heated to 70°C for 48 h to give compound 1a in 63% yield as a white solid after recrystallization. In addition, newly designed compounds 1b and 1c were synthesized using the same synthetic process (Scheme 1).
Closely related compound 1d bearing a benzimidazole group was synthesized using a different route. In this synthesis, thiouracil 7 was converted to the corresponding thioacetic acid 8, followed by the amide coupling with 2-aminobenzimidazole to give compound 1d in modest yield. The attempted preparation of 2-bromoacetylaminobenzimidazole, following the synthetic route for compound 1a did not yield a pure compound. Thus, we judged that the synthetic strategy which coupled R 1 last would enable a better modular synthesis of analogs.
With this modular synthesis protocol in hand, we undertook synthesis of the analogs based on the original R 1 moiety and two bicyclic sulfhydrylpyrimidones, as well as the linkers with different lengths and a variety of aromatic R 2 substituents. Synthesis of compounds 2a and 2c followed the same strategy as that for compound 1d by converting 7 to the corresponding thiobutanoic acid 9, followed by amide formation by coupling with 4-substituted anilines 5a and 5c to give 2a and 2c in modest yields.
Structure−Activity Relationship Analysis of PEX-9 Derivative Compounds. COS-1 cells were chosen for this set of experiments due to their low migratory capacity and complete lack of MMP-9 expression, thereby minimizing

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Articles potential artifacts when interpreting the data. Overexpression of proMMP-9 results in enhanced cell migration in a twodimensional dot migration assay. 27 This assay was initially used to test the efficacy of analogs at a single concentration, 50 μM. This concentration was used as a maximal cutoff because concentrations above 50 μM compound 1a did not increase inhibitor effectiveness in COS-1 cells.
Cells were mixed with type I collagen solution with or without the compound, dotted onto a 96-well plate, and allowed to migrate for 18 h. They were then fixed, stained, and scored for the extent of migration (Figure 2A and B). 2-(4-Oxo-6-propyl-1,4-dihydropyrimidin-2-ylthio)-N(methyl)-phenylacetamide (1f) was used as a negative control. 27 Compound 3c was the most effective and was selected for further study. The migration assay was repeated in HT1080 fibrosarcoma Figure 2. Structure−activity relationship analysis of all synthesized PEX-9 inhibitor derivatives. (A) Representative 20× magnification DAPI images of COS-1 cells engineered to stably express either vector control or proMMP-9 cDNA vectors in a two-dimensional migration assay after treatment with 50 μM of 1f, 1a, or 3c. Scale bar = 250 μm. (B) Quantitative analysis of migrated COS-1 vector control and proMMP-9 expressing cells after treatment with 50 μM of inhibitors. (C) Tryptophan fluorescence emission of proMMP-9 or proMMP-9/PEX2 (ex, 280 nm; em, 330 nm) was monitored and inhibitor 3c titrated into the protein solution. A maximal λ Max shift of 9 nm was observed after treatment with 25 μM inhibitor. A solid line is a curve fit for K d for compound 3c that was determined. (D) K d for each inhibitor was plotted against percent inhibition of proMMP9mediated cell migration for each inhibitor. cells, which endogenously express MMP-9, and the same inhibition potency was observed for both compound 1a and 3c relative to control groups (Supporting Information Figure 1A and B).
Compound Affinity for PEX-9 Binding. A blue shift in proMMP-9 tryptophan fluorescence was monitored to determine the binding affinity of all derivative compounds as previously described. 27 The K d for compound 3c binding to proMMP-9 is 0.32 μM ( Figure 2C), four times tighter than parent compound 1a. Moreover, a clear correlation between binding affinity and inhibition potency in the migration assay was observed ( Figure 2D). Thus, the biophysical SAR is maintained in the cellular assay, and increased inhibition of cell migration is MMP9-dependent and due to optimization of the chemical structure for binding the PEX-9 domain.
PEX-9 Binding Specificity. A chimera of proMMP-9 and MMP-2 in which the MMP-9 PEX domain was replaced by that of MMP-2 (proMMP-9/MMP-2PEX) 11 was tested for analog binding. The tryptophans in the MMP-9 PEX domain are conserved in the MMP-2 PEX domain. Upon titration of compound 3c with the chimera, no shift in fluorescence was observed suggesting that 3c does not bind to MMP2-PEX ( Figure 2C).
As an additional test of binding selectivity, saturated transfer difference (STD) NMR was performed with purified recombinant proMMP-2 and proMMP-9 protein. ProMMP-2 and proMMP-9 are highly similar in structure and function, belonging to the same subfamily of MMPs known as the type IV collagenases and/or gelatinases. Thus, proMMP-2 constitutes the best control for testing the selectivity of the PEX-9 inhibitor. 28 Selective saturation of the 1.12 ppm resonance (proMMP-2 and proMMP-9) was used for STD NMR spectra. STD peaks were observed in the presence of proMMP-9, but not in the presence of proMMP-2, indicating that derivative compound 3c is specific for the PEX-9 target (Supplemental Figure 2A−F). Thus, binding specificity is maintained upon increasing binding affinity.
PEX-9 Inhibitor Target Specificity. To test whether compound 3c is specific for inhibiting only proMMP9mediated migration, a two-dimensional dot migration assay was performed using COS-1 cells engineered to stably express vector control pQCXIP, proMMP-2, proMMP-9, or proMMP-14 cDNA vectors ( Figure 3A). An immunoblot was performed MMP-9/MYC was immunoprecipitated using an anti-Myc antibody and served as an input control while α4 and β1 integrin interactions were probed via immunoblot. A decrease in MMP-9 interacting with both integrin subunits was observed as well as a (B) decrease in Src Tyr418 and downstream target proteins FAK Tyr 576/577 /PAX Tyr118 phosphorylation. (C) 60× confocal images were captured, and representative images of MMP-9 (green) and each integrin subunit (red) were taken after treatment of HT1080 cells with 50 μM of either compound 1a, 3c, or 1f. Scale bar = 100 μm. (D) 60× confocal images were captured, and representative images of p-FAK Tyr 576/577 (red) and p-PAX Tyr118 (green) were taken after treatment of HT1080 cells with 50 μM of either compound 1a, 3c, or 1f. Scale bar = 100 μm.

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Articles to confirm protein expression and proper localization of each MMP (MMP-2/-9 are secreted while MMP-14 is membrane bound; Figure 3B). As expected, expression of these three MMPs resulted in enhanced cellular migration in COS-1 cells. 11 Treatment with either compound 1a or 3c only inhibited proMMP9-mediated migration: compound 3c was significantly more effective at inhibiting migration ( Figure 3C).
Effect of PEX-9 Inhibitor on MMP-9 Catalytic Activity. Recombinant proMMP-9 protein was purified and incubated overnight in a 37°C water bath in TNC buffer + 1 mM APMA to artificially activate the protein. 29,30 The next day, the purified protein was incubated with 10 μM fluorogenic peptide (Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2), and catalytic activity was monitored over 2 h in the presence of compounds 1a, 3c, 1f, and marimastat, a broad-spectrum MMP catalytic inhibitor. No inhibition of the catalytic activity was observed with 1a or 3c, corroborating that PEX-9 inhibitors do not modulate the catalytic activity of the protease. In the presence of marimastat (K d = 80 nM), no substrate cleavage was observed ( Figure 3D). Therefore, inhibition of proMMP9-mediated cell migration by compound 1a or 3c is not due to a loss of proteolytic activity.
Cell Viability Analysis. Compound 3c was evaluated for acute cytotoxicity. COS-1 cells stably expressing either vector control or proMMP-9 cDNA were treated with 50 μM 3c for 24 h. As negative and positive controls, cells were treated with DMSO alone or 100 nM staurosporine (STS), an apoptosis inducer, respectively. 31 Significant cell death was only observed after treatment with STS. A nonstatistically significant 8% decrease in cell growth occurred after treatment with 50 μM of compound 3c ( Figure 3E). These experiments were repeated in endogenous MMP-9 expressing HT1080 fibrosarcoma cells, which were analyzed for chronic toxicity (cells were retreated every 3 days for a total of 9 days). No cell death was observed at 100 μM 3c (data not shown).
PEX-9 Inhibitors Decrease MMP-9 Dimer Formation and Association with EGFR. Human fibroscarcoma HT1080 cells were cotransfected with MMP-9 cDNAs tagged with either a Myc or HA tag as previously reported. 12 The next day, cells were switched to serum free media and treated with 50 μM of either compound 1a or 3c overnight. Using the Myc antibody for immunoprecipitation and HA antibody for immunoblotting, the formation of dimeric MMP-9 was monitored. Compound 3c significantly blocked MMP-9 dimer formation as compared to control compound 1f. In addition, inhibition of PEX-9 with 1a or 3c reduced endogenous CD44 and EGFR coimmunoprecipitation (Supporting Information Figure 3A). Moreover, these PEX-9 inhibitors reduced phosphorylation of EGFR Tyr1068 and its downstream targets AKT Ser473 and Erk1/2 Thr202/Tyr204 (Supporting Information Figure 3B).
Evaluation of MMP-9 Cell Surface Localization upon Treatment with PEX-9 Inhibitors. HT1080 cells were fixed in 4% paraformaldehyde then subsequently incubated with anti-MMP-9 in addition to anti-α4 and/or β1 integrin antibodies to monitor relative levels of localization of these proteins at the cell surface. The cells were counterstained with DAPI nuclear dye. Treatment with either compound 1a or 3c resulted in a loss of MMP-9 from the cell surface ( Figure 4C). However, treatment with either solvent control DMSO or negative control compound 1f did not disrupt the localization of MMP-9 on the cell surface.
Evaluation of Focal Adhesion Complexes in the Presence of PEX-9 Inhibitors. HT1080 cells were fixed after overnight treatment with 50 μM PEX-9 inhibitors, probed for either FAK Tyr 576/577 or PAX Tyr118 and counterstained with DAPI. A decrease in the formation of FAK and PAX adhesion junctions, normally depicted as "large punctae," was observed in cells after treatment with PEX-9 inhibitor compound 1a or 3c ( Figure 4D).
Treatment with PEX-9 Inhibitor Prevents Association of EGFR with CD44 and β1 Integrin. HT1080 cells were treated with 50 μM 3c, 1a, 1f, or DMSO alone. Cell lysates were collected and analyzed by a co-IP assay in which endogenous EGFR was captured with an anti-EGFR antibody and served as an input control. CD44 and β1 integrin were individually probed on the immunoblot. Decreased interaction between EGFR-CD44 and EGFR-β1 integrin was observed after treatment with 3c (Supporting Information Figure 5A).
Effect of PEX-9 Inhibitors on Cell Adhesion. A 96-well plate was coated with a thin layer of either collagen or fibronectin (5 μg/mL in PBS) substrate before use. HT1080 cells were then seeded onto wells and incubated at 37°C for 30 min in the presence of various inhibitors. Wells were washed and cells were fixed in 4% paraformaldehyde and stained with DAPI. Images of each 96-well plate were microscopically captured and counted for the number of cells still adherent to the coated surface using automated computer software. Compound 1a or 3c inhibited cellular adhesion, whereas treatment with Marimastat, a broad-spectrum MMP catalytic inhibitor, had no observable effect on cell attachment. Solvent DMSO and compound 1f also had no effect on cell adhesion (Supporting Information Figure 6A).
Effect of Broad-Spectrum MMP Catalytic Inhibitor Marimastat on Focal Adhesion Complexes. HT1080 cells treated with 80 nM of broad-spectrum MMP inhibitor marimastat were analyzed by immunofluorescence microscopy

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Articles for both p-FAK Tyr 576/577 and p-PAX Tyr118 . Treatment with marimastat did not prevent the formation of focal adhesion contact sites in HT1080 cells (Supporting Information Figure  6B).
Treatment with PEX-9 Inhibitor Prevents Both Angiogenesis and Invasion in a Chorioallantoic Membrane (CAM) Assay. A CAM assay was carried out to evaluate angiogenic and invasive potential of HT1080 cells after treatment with 3c. HT1080 cells were adsorbed onto a gelatin sponge and implanted onto the surface of the chicken embryo CAM followed by treatment with a 100 μM stock solution of compound 1f, 1a, or 3c (final drug concentration is estimated to be 0.5 μM). After a 4-day incubation, neovascularization was imaged and quantified. Treatment with either compound 1a or 3c significantly reduced the angiogenic potential of HT1080 cells as compared to solvent DMSO and negative compound 1f control treated cells ( Figure 5A,B).
HT1080 cells stably expressing GFP were mixed with a type I collagen solution (3 mg mL −1 ) and seeded atop the CAM to ensure the cells localized to a specific area. Then, they were treated with a 100 μM stock solution of compound 1f, 1a, or 3c (final drug concentration is estimated to be 0.5 μM). The invasion of cancer cells through the epithelium and basement membrane of the upper CAM into connecting tissue was examined by hematoxylin and eosin staining ( Figure 5C) in addition to fluorescence microscopy for GFP + cells ( Figure  5D). Treatment of HT1080 cells with either compound 1a or 3c reduced invasion of CAM tissue compared to treatment with either DMSO or compound 1f control.

■ DISCUSSION
MMPs are crucial for the degradation of ECM and blood vessel linings. Degradation allows tumor cells to escape their primary location and enter the circulatory system where they can travel to seed secondary tumors elsewhere in the body. 32,33 Therefore, targeting MMPs with inhibitors is of interest for cancer drug development. 34 Original inhibition attempts focused on

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Articles synthetic collagen-mimicking hydroxamate inhibitors that chelate the zinc ion in the active site upon binding. 35 These drugs largely failed in clinical trials due to lack of selectivity for individual MMPs. Due to the specificity issues inherent in generating inhibitors of MMP proteolytic activity, we developed inhibitors which target MMP-9's hemopexin domain. MMP hemopexin domains share a common tertiary structure, yet the surface residues of MMP hemopexin domains are distinct, and one hemopexin domain cannot functionally substitute for another.
Identification of a More Potent PEX-9 Inhibitor. To identify compounds that bind more tightly to the hemopexin domain of MMP-9 than our original hit, 27 structural diversity was introduced at three positions and computational DOCKing utilized to prioritize synthesis and testing of a small subset. The similarities between their predicted and measured binding affinities indicate that the DOCKed structures may be used to infer important binding modalities. Compounds that displayed the greatest inhibition of proMMP-9 mediated migration had longer linkers with three methylenes between the amide and the sulfide moieties and at least one fluorine in the R 1 group. A cluster of derivative compounds (2c, 3a−3c, 4a, 4d) shared a similar binding geometry within the hemopexin domain of MMP-9 ( Figure 1C). In all the tightest binding PEX-9 inhibitor derivatives, the aryl fluorine atoms were found deep within the binding pocket in contrast to the original hit 1a, in which the difluoromethoxyphenol moiety remained solvent exposed. The STD-NMR data support the binding pose in which the aniline is deeply buried in the pocket (Supporting Information Figure  2A−F). In addition, fusion of a second ring to the pyrimidone moiety to form a quinazolinone significantly improved inhibitor efficiency and affinity. The length of the aliphatic chain between the two ring moieties is key for optimizing binding interactions. Comparison of DOCKed compounds 1a and 3c highlights differences in binding orientation that may contribute to the higher affinity of 3c ( Figure 1D).
Binding affinity for PEX-9 correlates with inhibition of proMMP9-mediated migration ( Figure 2D) across our 16compound series that spans 1.5 logs of binding affinity. These structure−activity results support the mechanism of inhibition of proMMP9-dependent cell migration being through targeting PEX-9 on the cell surface.
proMMP-9 homodimerization is a critical step for interaction with CD44 at the cell surface, which results in decreased phosphorylation of EGFR Tyr1068 and its downstream targets AKT Ser473 and Erk 1/2 Thr202/Tyr204 phosphorylation. 12 Compound 3c demonstrated enhanced efficacy of blocking proMMP-9 homodimerization and activation of downstream protein targets (Figure 4, Supporting Information Figure 3). Moreover, our most potent compound, 3c, retained specificity for a single MMP as demonstrated by the lack of inhibition in proMMP-2 or proMMP-14 mediated cell migration ( Figure  3C). Our structure−activity correlation ( Figure 2D) and the specificity of inhibition for proMMP-9 mediated cell migration corroborate that compound 3c targets PEX-9 in vitro and in vivo Figure 6. Overview of the mechanism by which MMP-9 regulates formation of focal adhesion junctions. (A) During exocytosis and upon being secreted into the extracellular space, proMMP-9 forms a large complex with itself that can then act as a scaffold for promoting outside-in signaling. In our model, PEX-9 scaffolding promotes the association of β1 integrin-EGFR-CD44. Interaction with CD44 results in enhanced EGFR activation in addition to increased phosphorylation of its downstream targets AKT and Erk 1 + 2 (through the MAPK/Erk pathway). While complexed to β1 integrin, EGFR then goes on to transactivate Src kinase after it is recruited to the α4 integrin subunit during the generation of a new focal adhesion contact site. After activation, Src can then directly phosphorylate FAK currently associated with the β1 integrin subunit resulting in its maximal catalytic activity. This active Src−FAK complex can then bind and activate PAX, resulting in a mature FAK-PAX complex necessary for the formation of focal adhesion junctions. Formation of a complex between FAK and PAX results in final translocation to ECM−integrin junctions at the cell surface where they regulate cytoskeletal interactions resulting in enhanced cellular adhesion, migration, and invasion of cancer cells. (B) Treatment with PEX-9 inhibitor (depicted as red triangle) prevents MMP-9 scaffolding, thereby preventing downstream signaling driven by EGFR activation.

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Articles and not a downstream signaling target common to other mediators of cell migration.
Recently, Garcia-Pardo and co-workers reported that the central cavity of PEX-9 interacts with α4β1 integrin as a mechanism for cross-talk between MMPs and integrin focal adhesion formation. 17,18,21 In turn, Src kinase interacts with the cytoplasmic tail of α4β1 integrin subunits, which is critical for integrin function to promote and regulate focal adhesion turnover. 36−42 Interestingly, PEX9-α4β1 integrin downstream signaling increases activation of Fyn, a Src family kinase, and drives chronic lymphocytic leukemia B cell survival. 17 Therefore, we examined the consequences of binding 3c to PEX-9 on α4β1 integrin-mediated Src signaling.
Using a coimmunoprecipitation approach, we found that PEX-9 inhibitors interfere with binding between proMMP-9 and α4β1 integrin and can block cross-talk signaling. This disruption is accompanied by decreases in phosphorylation levels of Src Tyr418 and its downstream substrates FAK Tyr 576/577 and PAX Tyr118 ( Figure 4B). Immunofluorescence imaging in HT1080 cells further supported the idea that focal adhesion junctions ("large punctae") were lost upon treatment with the PEX-9 inhibitor ( Figure 4C,D). We also demonstrated that overexpression of proMMP-9 in MCF-7 breast cancer cells enhanced Src activation. Overexpression of proMMP-9/ MMP2-PEX in MCF-7 breast cancer cells or silencing of endogenous MMP-9 expression in HT1080 attenuated Src activation. Taken together, the data suggest that the proMMP-9 hemopexin domain is required for enhanced cellular adhesion and migration.
Potential Mechanism by Which Src Is Activated. Our data are in agreement with the literature showing that α4β1 integrin clustering is sufficient for activation of Src kinase. 37,43 However, in addition to integrin clustering, EGFR can directly activate Src kinase, and EGFR interacts with β1 integrin. 44−46 It is proposed that proMMP-9 forms a large charged surface for complex interactions with multiple surface proteins involved in outside-in signaling. 47 The hemopexin domain of proMMP-9 may act as a scaffold to enhance clustering of α4β1 integrin-EGFR-CD44 at the cell surface and result in aberrant Src activation. 48 Garcia-Pardo and co-workers demonstrated that a peptide mimicking blade 1 of PEX-9 inhibits interaction with integrin and that a second peptide mimicking blade 4 of PEX-9 inhibits interaction with CD44. 21,22 We reasoned that our inhibitor, which binds directly to PEX-9, could simultaneously disrupt both interactions. Indeed, our coimmunoprecipitation experiments demonstrate that the addition of 3c selectively disrupts proMMP-9 interaction with both α4β1 integrin ( Figure 4) and CD44 (Supporting Information Figure S3). Importantly, our inhibitor is specific for MMP-9. Targeting the hemopexin domain to investigate the role of MMP-9 in promoting cellular adhesion has identified a connection to integrin and Src in a complex adhesion network as depicted in Figure 6. There are currently no effective cancer therapeutics designed to target adhesion receptors because cancer cells can change the mechanism by which they migrate depending upon their environmental constraints. 49 Our in vivo CAM experiments demonstrate that we can inhibit two key determinants of tumor cell metastatic potential, angiogenesis and invasion ( Figure 5), through PEX domain targeting. Our small molecule inhibitor paves the way for designing new therapeutic strategies for the treatment of metastatic disease and opens the door for the future development of new technologies and tools for understanding the role of individual MMPs in cancer progression. Development of PEX inhibitors for other MMPs will provide understanding of their noncatalytic functions and a strategy for reducing undesirable off-target effects previously observed in broad-spectrum MMP catalytic inhibitor clinical trials.
General Methods. 1 H and 13 C NMR spectra were measured on a Bruker 300, 400, 500, or 700 MHz spectrometer. Melting points were measured on a Thomas-Hoover capillary melting point apparatus and are uncorrected. TLC was performed on Sorbent Technologies aluminum-backed Silica G TLC plates (Sorbent Technologies, 200 μm, 20 cm × 20 cm), and column chromatography was carried out on silica gel 60 (Merck, 230−400 mesh ASTM). High-resolution mass spectrometry analysis was carried out on an Agilent LC−UV−TOF mass spectrometer at the Mass Spectrometry facility of the Institute of Chemical Biology and Drug Discovery, Stony Brook University.
Two-Dimensional Dot Migration Assay. Transfected cells were mixed with an equal volume of neutralized type I collagen (3 mg mL −1 ) on ice. The cell−collagen mixture (1 μL of 1 × 10 7 cell/mL) was then dotted onto a 96-well plate. After solidification of cell− collagen dots, the cell−collagen hemispheres were covered with 100 μL of complete media and incubated for 8−18 h (incubation time varies by cell type), followed by staining with DAPI nuclear dye and counting of the migrated cells using a Nikon Elements Basic Research Software analysis tools. 50 Co-immunoprecipitation. Twenty-four hours following transfection, the conditioned medium in the absence of serum was collected from the transfected HT1080 cells. The cells were then lysed in RIPA buffer containing 10 mM Tris-Cl (pH 8.0), 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl, and a protease inhibitors mixture (Sigma). Both the conditioned medium and the cell lysates were incubated with specific antibodies overnight (18 h) at 4°C. Antigen antibody complexes were precipitated with protein A-agarose beads followed by a brief centrifugation, washing, and then electrophoresis in a 10% SDS-polyacrylamide gel. Western blotting was then followed using a corresponding antibody as previously described. 12 Immunofluorescence. Cultured cells were fixed with 4% paraformaldehyde/phosphate-buffered saline (PBS) followed by blocking with 5% bovine serum albumin (BSA)/PBS. Both FAK and PAX were detected with an anti-p-FAK Tyr 576/577 and anti-p-PAX Tyr118 antibody followed by secondary antibodies conjugated with antirabbit Alexa 568 or Alexa 488, respectively (Invitrogen). For colocalization experiments, MMP-9 was detected with an anti-MMP-9 antibody followed by a secondary antibody conjugated with antirabbit Alexa 488, while α4 and β1 integrins were detected with an anti-α4 integrin and anti-β1 integrin, respectively, followed by a secondary antibody conjugated with antimouse Alexa 568. Nuclei were counterstained with DAPI (Invitrogen). Images were captured using a Zeiss LSM 510 META NLO Two-Photon Laser Scanning Confocal Microscope System (Central Microscopy Imaging Center, Stony Brook University).
Fluorogenic Assay of Enzyme Activity. Mca-PLGL-Dpa-AR-NH 2 fluorogenic peptide substrate (final concentration of 10 μM in DMSO) was incubated with the compounds and p-aminophenylmercuric acetate (APMA)-activated proMMP-9 for 30 min at 25°C before detection. Fluorescence emission at 405 nm with excitation at 320 nm was measured in a fluorescent plate reader (Gemini EM; Molecular Devices). 51 Cell Viability. HT1080 cells were cultured in complete media with or without compound 3c. Media and drugs were changed on a bidaily basis, and cell viability was monitored by MTT assay [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (Promega, Madison, WI). Each day, cells were exposed to MTT and incubated at 37°C for 4 h. The reaction was stopped, and formazan crystals were solubilized; the resultant solution was subject to colorimetric spectrophotometry and read at a wavelength of 570 nm. 52 Chorioallantoic Membrane Angiogenesis and Invasion Assay. The chorioallantoic membrane (CAM) assay was performed as previously described. 53 Fertilized white chicken eggs (SPF Premium, Charles River Laboratory, North Franklin, CT) were incubated at 37°C in 70% humidity for 3 days. The embryos were then incubated ex ovo in a sterile Petri dish for 7 days. Gelatin sponges adsorbed with HT1080 cells treated with or without compound 3c were implanted on the CAM surface, and neovasculature was counted on day 4 postimplantation. 54,55 For histochemical analysis of the chorioallantoic membrane, embryos were treated as for the angiogenesis assay, except at day 10 the embryos were inoculated with pretreated HT1080 cells mixed with a type IV collagen solution (3 mg mL −1 ). After a 7-day incubation, CAM segments containing the cell dot collagen mixture were isolated, formalin fixed, and sectioned by microtome into 6-μm sections after embedding in OCT. Sections were then stained with hematoxylin and counterstained with eosin. 56 proMMP-9 Protein Purification. For purification of proMMP-2 and proMMP-9 protein, COS-1 cells stably expressing each respective MMP were incubated overnight in 10 mL of serum-free medium until 500 mL was collected for purification via a gelatin-sepharose column. Both proMMP-2 and proMMP-9 have three fibronectin repeats

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Articles present in their gelatin binding domain, which allowed us to capture and collect the secreted protein present in the serum-free medium fraction. Protein was then eluted off the gelatin sepharose column using 5% DMSO and then dialyzed to remove any organic contaminants. A coomassie blue gel was then run to check the purity of the samples to ensure no protein contamination was present in the final solution. All methods were followed as previously described. 57 Fluorescence Spectroscopy. Binding of compound 3c to proMMP-9 was assayed by observing the change of tryptophan emission upon binding. Purified recombinant proMMP-9 (0.5 μM) or MMP-9/MMP-2PEX (0.5 μM) was diluted in TNC buffer (50 mM Tris-HCl at pH 7.5, 150 nM NaCl, 10 mM CaCl 2 ; pH 7.5) in the presence or absence of compound 3c. As a control for protein stability and loss, an analogous buffer solution was added to the protein. The protein sample was excited at 280 nm, and emission scans were collected from 300 to 425 nm, using slit widths of 0.3 nm on a QM-4/ 200SE spectrofluorimeter with double excitation and emission monochromators. Between each titration, the protein/inhibitor mixture was gently stirred for 1 min at RT before collecting each reading. Three emission scans were collected and averaged at each concentration. The K d was determined using the Prism software package (GraphPad V5) to fit the data to the equation ΔF/ΔF Max , in which ΔF is the nanometer shift for a given titration and ΔF Max is the maximal nanometer shift observed overall.
Small Molecule DOCKing. To DOCK the MMP-9 PEX domain inhibitors and predict the binding mode, the inhibitors were first created via the Advanced Chemistry Development Chemsketch software (ACD Laboratories, Toronto, Canada) in the MOL format. The molecules were then loaded into the program Avogadro to undergo energy minimization and were then saved as PDB format files. Once in PDB format, the molecules were individually loaded into the