MMP-9/matrix metalloproteinase-9 (Kd = 2.1 μM)

MMP-9-IN-1 (compound 2; 100 μM; 14 hours) does not cause notable cytotoxicity[1].
MMP-9-IN-1 (compound 2; 10 μM) significantly prevents MDA-MB-435 and HT-1080 cells from proliferating[1].

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Inhibition of MMP-9-induced cell migration by the identified compounds [1]
COS-1 cells expressing MMP-9 cDNA, or GFP cDNA as a control, were pre-incubated with or without the compounds (doses ranging from 100 nM to 100 μM) for 30 minutes, and examined by a Transwell chamber migration assay. Compounds 1, 2, 3 and 5 inhibited the migration of MMP-9-expressing COS-1 cells, whereas compound 4 showed no activity (Fig. 3A–E). Compounds 3 and 5, but not 1 and 2/MMP-9-IN-1, inhibited the migration of control cells (GFP-transfected) as well as MMP-9 transfected cells (Fig. 3C & E).

To rule out the possibility that the reduction of cell migration by these compounds was due to cytotoxicity, a cell viability assay was performed. COS-1 cells were treated with the compounds for 24 hours followed by a cytotoxicity assay. Thapsigargin, an ER stress inducer that inhibits intracellular Ca2+-ATPases, was used as a positive control to trigger cell death. Treatment with compounds 1, 2 and 4 did not cause notable cytotoxicity at the maximum concentration used, whereas treatment with compounds 3 and 5 induced cell death (Fig. 3F). Compounds 3 and 5 were therefore excluded from further evaluation.

The lethal dose (LD50) of compounds 1 and 2/MMP-9-IN-1 was determined in COS-1 cells. Cells were treated with increasing doses of the compounds for 24 hours followed by cell viability assay (Suppl. Fig. 1). The LD50 of compounds 1 and 2 were 360 ± 2 μM and 3.5 ± 0.3 mM, respectively, suggesting that their inhibition of MMP-9-induced cell migration was not due to cytotoxicity.

To further determine the specificity and selectivity of compound 1 and 2/MMP-9-IN-1 for MMP-9-induced cell migration, we examined the effect of compounds 1 and 2 on cell migration induced by other MMPs in which the PEX domain has been reported to play a critical role in enhanced cell migration, e.g., MMP-2 and MT1-MMP (MMP-14). In contrast to MMP-9 expressing cells, neither compound inhibited the migration of MMP-2 or MT1-MMP ectopically expressing COS-1 cells (Fig. 4A). In addition, compound 2 did not interfere with MT1-MMP-mediated cancer cell invasion examined by a 3D invasion assay (Suppl. Fig. 2). Thus, small synthetic compounds that potentially bind to the PEX domain of MMP-9, inhibit MMP-9-induced cell migration with enhanced-specificity and -selectivity.

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Inhibition of migration in cancer cells that produce endogenous MMP-9 by compounds 1 and 2/MMP-9-IN-1 [1]
We next investigated whether compounds 1 and 2 inhibit migration of cells producing a pathologically relevant level of endogenous MMP-9. Two human invasive cancer cell lines, HT-1080 and MDA-MB-435, expressing high endogenous levels of MMP-9 were employed. Treatment of the cells with compounds 1 and 2 significantly reduced cell migratory abilities. Furthermore, both compounds inhibited the migration of HT-1080 and MDA-MB-435 cells in a dose-dependent manner (Fig. 4B & C).

Cell migration is a critical determinant of cancer cell invasiveness. Therefore, HT-1080 cells were assessed in the 3D type I collagen invasion assay. As anticipated, the cell invasive ability of HT-1080 cells was significantly inhibited in cells treated with compounds 1 and 2 (Fig. 4D & E). Inhibition of MDA-MB-435 cell invasion was also observed (data not shown). These data suggest that inhibition of MMP-9-mediated cell migration by the compounds results in suppressed cancer cell invasion.

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Compounds 1 and 2/MMP-9-IN-1 do not affect MMP-9 expression or proteolytic activity [1]
Cell lysates from HT-1080 cells treated with and without compounds 1 and 2 were examined for MMP-9 expression levels by Western blot using an anti-MMP-9 antibody. Western blotting using an antibody to tubulin was employed as a control. No effect on MMP-9 expression by the compounds was observed (Fig. 5A).

Activated MMP-9 was obtained by incubating purified proMMP-9 with p-aminophenyl mercuric acetate (APMA). Addition of compounds 1 and 2 to APMA-activated MMP-9 did not inhibit the catalytic activity of MMP-9 as measured by cleavage of the fluorescent Mca-P-L-G-L-Dpa-A-R-NH2 peptide (Fig. 5B). These data suggest that inhibition of MMP-9-induced cell migration by compounds 1 and 2 is not due to inhibition of MMP-9 expression or proteolytic activity.

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Binding of compound 2/MMP-9-IN-1 to the MMP-9 PEX domain [1]
We titrated the binding of compound 2 to MMP-9 by monitoring MMP-9 tryptophan fluorescence. Saturation of purified proMMP-9 with compound 2 resulted in a 7 nm blue shift in the λmax of MMP-9 emission (Fig. 5C). No effect on the protein fluorescence occurred in the buffer only control. The Kd for MMP-9 binding to compound 2 is 2.1 ± 0.2 μM.

To further characterize the binding between compound 2 and MMP-9, we employed a previously generated chimera of MMP-9 in which the PEX domain of MMP-9 was replaced with that of MMP-2 (MMP-9/MMP-2PEX). Upon addition of compound 2 to MMP-9/MMP-2PEX, no shift in fluorescence was detected (Fig. 5D). Likewise, compound 2 did not bind to purified recombinant soluble MT1-MMP. These data confirmed that compound 2 binds specifically to the PEX domain of MMP-9. The absorption of compound 1 at 280 nm precluded evaluation of its binding properties.

To test whether compound 2 interferes with proMMP-9 homodimerization, co-immunoprecipitation of COS-1 cells transfected with both proMMP-9/Myc and proMMP-9/HA cDNAs in the presence or absence of compounds 2 and 4 was utilized. Treatment of the transfected cells with compound 2, but not inactive compound 4, resulted in blocked MMP-9 homodimer formation (Fig. 5E). This defect was not due to inhibition of expression of MMP-9 by compound 2 as evidenced by Western blotting of the cell lysate from HT-1080 cells (Fig. 5A). Similar results were obtained in reciprocal co-immunoprecipitation assays (Fig. 5E). This experiment confirms that compound 2 specifically inhibits MMP-9 homodimerization

Homodimerized MMP-9 interacts with cell surface adhesion molecule, CD44, which leads to activation of EGFR and downstream MAPK (ERK1/2) pathway. To explore this network, the activity status of downstream effector ERK1/2 was examined. COS-1 cells ectopically expressing MMP-9 cDNA were serum starved in the presence or absence of compounds for 18 hours followed by Western blotting using anti-phospho-ERK1/2 and total ERK1/2 antibodies. As depicted in Fig. 5F, decreased activation of ERK1/2 was observed in compound 2 treated cells. Taken together, these data suggest that abrogation of MMP-9-mediated cell migration by compound 2 is due to disruption of MMP-9 homodimerization, which results in failure to cross-talk with CD44 and the EGFR-MAPK signaling pathway.

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Effect on MMP-9-mediated cell proliferation by compound 2/MMP-9-IN-1 [1]
COS-1 cells transfected with MMP-9 or GFP cDNA (control) were monitored for cell proliferation in the absence or presence of compound 2/MMP-9-IN-1 or 4 with a CellTiter-Glo® Luminescent assay. In agreement with previous observations, the rate of cell proliferation increased significantly (P < 0.05) in COS-1 cells expressing MMP-9 as compared to GFP expressing COS-1 cells (Fig. 6A). MMP-9-induced cell proliferation was not affected by compound 4, consistent with its lack of effect on MMP-9-induced cell migration. In contrast, compound 2 significantly decreased MMP-9-induced cell proliferation (Fig. 6A), but did not affect the proliferation of COS-1 cells transfected with GFP cDNA (Fig. 6B). To determine if compound 2 also affects the proliferation of cancer cells producing endogenous MMP-9, HT-1080 and MDA-MB-435 cancer cells were treated with 10 μM compound 2. Significant inhibition of cell proliferation was observed for HT-1080 and MDA-MB-435 cells treated with compound 2, but not with compound 4 or with DMSO controls (Fig. 6C & D).

MMP-9-IN-1 (compound 2; 20 mg/kg; intraperitoneal and intratumoral injection alternately; 6 days/week; for 14 weeks) causes a significant tumor growth delay in NCR-Nu mice that have an MDA-MB-435/GFP tumor[1]. MMP-9-IN-1 inhibits cancer cell metastasis in vivo[1].

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Decreased tumor growth and lung metastases in MMP-9-IN-1/compound 2-treated mice [1]
MDA-MB-435 cells are highly metastatic in nude mice (30, 31) and produce high levels of MMP-9, thus serving as an appropriate experimental model to explore the in vivo inhibitory activity of compounds exhibiting anti-MMP-9 activity. To facilitate in vivo analysis of tissues and visualization of the lung metastases, MDA-MB-435 cells were stably transfected with GFP cDNA and implanted subcutaneously within the mammary fat pad of female immunodeficient mice. Treatment of mice with compound 2/MMP-9-IN-1 resulted in a profound delay in tumor growth, whereas treatment with the inactive control compound 4 or the vehicle alone failed to inhibit tumor growth (Fig. 7A & B). Tumor incidence was unaffected by compound 2.

The lungs of tumor-bearing mice were removed and slices of the lungs (3 mm thickness) were examined under a fluorescent microscope (Fig. 7C). In the vehicle control and compound 4-treated groups, multiple large nodules were evident in MDA-MB-435/GFP tumor-bearing mice, whereas the extent of lung metastasis was dramatically reduced in mice treated with compound 2/MMP-9-IN-1 (Fig. 7C). Also, dimensions of tumor foci area in the lung and the percent of mice displaying lung metastases were significantly decreased in these mice (Fig. 7D & E). Thus, treatment with compound 2 impaired the in vivo effect of MMP-9 on both primary tumor growth and metastasis. No significant change in body weight nor other signs of toxicity during the 14-week period were observed in compound 2-treated mice.

Fluorogenic Assay of Enzyme Activity [1]
Fluorogenic peptide substrate (50 μM) (18) was incubated with the compounds either in the presence or absence of latent MMP-9 and APMA-activated MMP-9 for 30 min at 25 °C before detection. Fluorescence emission at 393 nm with excitation at 328 nm was measured in a fluorescent plate reader.

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Fluorescence Spectroscopy [1]
Binding of compound 2 to MMP-9 was assayed by observing the change of tryptophan emission upon binding. Purified recombinant MMP-9 (50 nM) or MMP-9/MMP-2PEX (50 nM) was diluted in buffer (50 mM Tris-HCl, 60 mM KCl and 0.05% Tween 20, pH 7.4) in the presence or absence of compound 2. 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 290 to 400 nm, using slit widths of 0.3 nm on a QM-4/200SE spectrofluorimeter with double excitation and emission monochromators. Three emission scans were collected and averaged at each concentration. The Kd was determined using the Prism software package (GraphPad V5) to fit the data to equation (1).

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Fluorogenic Assay of Enzyme Activity [2]
Mca-PLGL-Dpa-AR-NH2 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.

Cell Viability [1]
Compound cytotoxicity was determined using the CellTiter-Glo™ Luminescent Cell Viability Assay .. 2.5 × 104 COS-1 cells were plated to a 96-well plate and incubated for 18 h with compounds 1–5. Luminescence was recorded using a SpectraMax Microplate Reader. LD50’s of the compounds were measured over a 100 pM to 10 mM concentration range. The LD50 was determined using the Prism software package (GraphPad V5) and fitting to equation (2).
Δ⁢L=(Δ⁢Lmax∗⁢[𝐢𝐧𝐡𝐢𝐛𝐢𝐭𝐨𝐫])/(LD50+[𝐢𝐧𝐡𝐢𝐛𝐢𝐭𝐨𝐫])(2) in which L = the measured luminescence.

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Cell Proliferation [1]
Cell proliferation was determined using the CellTiter-Glo™ Luminescent Assay. Cells (5 × 103) were added to a 96-well plate in the presence or absence of the compounds and monitored for 9 days by luminescence assay.

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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-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. 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

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Chorioallantoic Membrane Angiogenesis and Invasion Assay [2]
The chorioallantoic membrane (CAM) assay was performed as previously described.53 Fertilized white chicken eggs 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.

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Two-Dimensional Dot Migration Assay [2]
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 × 107 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.

4-5 week-old female NCR-Nu mice bearing MDA-MB-435/GFP tumor[1]
20 mg/kg
Intraperitoneal and intratumoral injection alternately; 6 days/week; for 14 weeks, In vivo Study [1]
Human MDA-MB-435 cancer cells (2 × 106) expressing green fluorescent protein (GFP) cDNA were inoculated subcutaneously into 4–5 week-old female NCR-Nu mice with 5 mice per group (Taconic). Once palpable, tumors were measured twice/week and volume was calculated using the following formula: length × width × height × 0.5236. Mice were treated with a vehicle control (DMSO/PBS), compound 2/MMP-9-IN-1, or 4 (20 mg/kg) via intraperitoneal and intratumoral injection alternately (6 days/week). At 14 weeks, the mice were sacrificed and the tumors and lungs were dissected. Fresh lung sections were cut (~3 mm thick) and examined for the presence of GFP-expressing tumor foci. The area of metastatic foci per field of examination was quantified from 10 random sites of three different slides for each mouse using NIH ImageJ software.

[1]. Small-molecule anticancer compounds selectively target the hemopexin domain of matrix metalloproteinase-9. Cancer Res. 2011 Jul 15;71(14):4977-88.

[2]. Targeting the Hemopexin-like Domain of Latent Matrix Metalloproteinase-9 (proMMP-9) with a Small Molecule Inhibitor Prevents the Formation of Focal Adhesion Junctions. ACS Chem Biol. 2017;12(11):2788-2803.

MMP-9-IN-1 is a secondary amide formed by the condensation of the carboxyl group of [(4-oxo-6-propyl-1,4-dihydropyrimidin-2-yl)thio]acetic acid with the amino group of 4-(difluoromethoxy)aniline. It is a specific inhibitor of matrix metalloproteinase-9 (MMP-9). It functions as an EC 3.4.24.35 (gelatinase B) inhibitor and an antitumor drug. It is an organofluorine compound, aromatic compound, pyrimidinone, organosulfur compound, and secondary amide. Existing matrix metalloproteinase (MMP) inhibitors lack target specificity, hindering the development of drugs against metastatic cancer. Inhibitors that bind to the non-catalytic sites of MMPs and disrupt protease signaling function have higher specificity and selectivity. This study used computer-simulated docking to screen compounds targeting the heme-binding protein (PEX) domain of MMP-9 and evaluated them using biochemical and biological methods. Two of the screened compounds were able to interfere with MMP-9-mediated migration and proliferation in cancer cells expressing either exogenous or endogenous MMP-9. Furthermore, these inhibitors did not affect the catalytic activity of MMP-9. The lead compound, N-[4-(difluoromethoxy)phenyl]-2-[(4-oxo-6-propyl-1H-pyrimidin-2-yl)thio]-acetamide, specifically binds to the PEX domain of MMP-9 without binding to other MMPs. This interaction with the PEX domain inhibits MMP-9 homodimerization, thereby blocking the downstream signaling pathways required for MMP-9-mediated cell migration. In a tumor xenograft model, this pyrimidinone compound inhibited the growth of MDA-MB-435 tumors and suppressed lung metastasis. Therefore, we demonstrate for the first time that a novel small molecule can specifically interact with the PEX domain of MMP-9 and inhibit tumor growth and metastasis by reducing cell migration and proliferation. [1]

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The lack of target specificity has greatly hampered the development of inhibitors against matrix metalloproteinases (MMPs) for the treatment of various cancers. The catalytic domains of MMPs are highly conserved, while the heme-binding-like domains of MMPs vary among family members. The heme-binding-like domain of MMP-9 enhances cancer cell migration through self-interactions and heterologous interactions with cell surface proteins, including CD44 and α4β1 integrins. These interactions activate the EGFR-MAP kinase-dependent signaling pathway, leading to cell migration. In this study, we constructed a library of compounds based on the lead compound N-[4-(difluoromethoxy)phenyl]-2-[(4-oxo-6-propyl-1H-pyrimidin-2-yl)thio]-acetamide that targets the heme-binding protein-like domain of MMP-9. We identified N-(4-fluorophenyl)-4-(4-oxo-3,4,5,6,7,8-hexahydroquinazolin-2-ylthio)butyramide (compound 3c) as a highly potent lead compound (Kd = 320 nM) that specifically binds to the heme-binding protein-like domain of proMMP-9. We demonstrated that compound 3c disrupts the homodimerization of MMP-9, thereby preventing the binding of proMMP-9 to α4β1 integrin and CD44, and leading to the dissociation of EGFR. This interference resulted in reduced phosphorylation levels of Src and its downstream targets, focal adhesion kinase (FAK) and paxillin (PAX), which are closely associated with promoting tumor cell growth, migration, and invasion. In vivo experiments using chicken embryo chorioallantoic membranes demonstrated that 500 nM of 3c can block cancer cell invasion of the basement membrane and reduce angiogenesis. In summary, we proposed the mechanism of action of 3C: targeting the heme-binding domain can simultaneously interfere with the α4β1 integrin and EGFR signaling pathways, thereby blocking signal bypass and reducing cancer cell migration. Targeting the heme-binding-like domain is an effective strategy for developing anti-metastatic drugs. [2]

C16H17N3O3F2S

369.386

369.095

C, 52.03; H, 4.64; F, 10.29; N, 11.38; O, 12.99; S, 8.68

502887-71-0

502887-71-0

135415473

White to off-white solid powder

3.2

2

7

8

25

547

0

CCCC1=CC(=O)NC(=N1)SCC(=O)NC2=CC=C(C=C2)OC(F)F

OLTRRVUORWPRGF-UHFFFAOYSA-N

InChI=1S/C16H17F2N3O3S/c1-2-3-11-8-13(22)21-16(20-11)25-9-14(23)19-10-4-6-12(7-5-10)24-15(17)18/h4-8,15H,2-3,9H2,1H3,(H,19,23)(H,20,21,22)

N-[4-(difluoromethoxy)phenyl]-2-[(6-oxo-4-propyl-1H-pyrimidin-2-yl)sulfanyl]acetamide

MMP9-IN-1; MMP-9-IN-1; MMP-9-IN1; OUN87710; OUN 87710; OUN-87710

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 66.7~74 mg/mL (180.5~200.3 mM)

Solubility in Formulation 1: 5 mg/mL (13.54 mM) in 10% DMSO + 40% PEG300 50% PBS (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (5.63 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)