Small-molecule anticancer compounds selectively target the hemopexin domain of matrix metalloproteinase-9

Abstract

Lack of target specificity by existing matrix metalloproteinase (MMP) inhibitors has hindered antimetastatic cancer drug discovery. Inhibitors that bind to noncatalytic sites of MMPs and disrupt protease signaling function have the potential to be more specific and selective. In this work, compounds that target the hemopexin (PEX) domain of MMP-9 were identified using an in silico docking approach and evaluated using biochemical and biological approaches. Two of the selected compounds interfere with MMP-9-mediated cancer cell migration and proliferation in cells expressing exogenous or endogenous MMP-9. Furthermore, these inhibitors do not modulate MMP-9 catalytic activity. The lead compound, N-[4-(difluoromethoxy)phenyl]-2-[(4-oxo-6-propyl-1H-pyrimidin-2-yl)sulfanyl]-acetamide, specifically binds to the PEX domain of MMP-9, but not other MMPs. This interaction between the compound and the PEX domain results in the abrogation of MMP-9 homodimerization and leads to blockage of a downstream signaling pathway required for MMP-9-mediated cell migration. In a tumor xenograft model, this pyrimidinone retarded MDA-MB-435 tumor growth and inhibited lung metastasis. Thus, we have shown for the first time that a novel small-molecule interacts specifically with the PEX domain of MMP-9 and inhibits tumor growth and metastasis by reducing cell migration and proliferation.

Figure 1. Clinical relevance of MMP-9 in patients with breast cancer

DNA microarray data mining of van de Vijver (21) and Stockholm (22) cohorts was performed using Kaplan-Meier survival analysis for correlation of MMP-9 expression with breast cancer survival rate (A & B) and recurrence (C). Levels of MMP-9 RNA were dichotomized at mean. n=cases.

Figure 2. Identification of small molecular weight compounds bound to the MMP-9 PEX domain

A) Ribbon model of the PEX domain of MMP-9 with the compounds 1, 2, 4 and 5 docked. The sulfate ions are shown for references, but were not included as part of the docking receptor. B) The same docked structure as shown in (A) rotated 90° about the X-axis with a solvent-accessible surface on the protein. C) Compounds 1, 2, 4, and 5 overlaid in their docked conformations. Atom colors: Carbon (grey), oxygen (red), nitrogen (blue), sulfur (yellow), and fluorine (green). The images and the solvent-accessible surface of MMP-9 PEX monomer were generated in UCSF Chimera (42). D) Structures of the best five docked molecules.

Figure 3. Inhibition of migration of cells expressing MMP-9 by the selected compounds

A–E) Cell migration assay: Transfected COS-1 cells were pre-incubated with the compounds at different concentration for 30 minutes followed by a Transwell chamber migration assay. Each concentration was assayed in triplicate and the experiments were repeated three times. F) Cell cytotoxic assay: COS-1 cells were incubated with the five compounds (100 μM) for 24 hours followed by a cell viability assay. DMEM media alone and media containing thapsigargin (1 μM) were included as negative and positive controls, respectively.

Figure 4. Specific inhibition of MMP-9-induced cell migration and invasion by the selected compounds

A) Specific inhibition of MMP-9-induced cell migration: COS-1 cells transfected with cDNAs as indicated were incubated with compound 1 or 2 (100 μM) for 30 minutes followed by a Transwell chamber migration assay. B–C) Dose-dependent inhibition of cancer cell migration by the selected compounds. HT-1080 cells and MDA-MB-435 cells were incubated with 1% DMSO, or different concentrations of compound 1 or 2 for 30 minutes followed by a Transwell chamber migration assay. D–E) Reduction of HT-1080 cell invasion by the selected compounds. HT-1080 cells (1 × 10⁴) were pre-treated with DMSO (1%), compound 1 or 2 (100 μM) for 30 minutes followed by 3D type I collagen invasion assay in the presence or absence of the compounds for additional 18 hours. Invading cells at the cell-collagen interface were microscopically counted.

Figure 5. Interaction of compound 2 with the PEX domain of MMP-9

A) No effect of the compounds on MMP-9 expression: Cell lysate of compound-treated HT1080 cells was examined by Western blotting (WB) using antibodies against MMP-9 and α/β tubulin, respectively. B) No inhibition of proteolytic activity of MMP-9 by the compounds: Latent and APMA-activated MMP-9 were incubated with DMSO control or the selected compounds 1 and 2 (100 μM) for 3 hours at 37°C followed by a fluorogenic substrate assay. C–D) Compound 2 binds selectively to the PEX domain of MMP-9: The λ_max of tryptophan fluorescence emission was monitored upon titration with compound 2 or buffer only as a control with (C) purified recombinant MMP-9 (50 nM) and (D) MMP-9/MMP-2_PEX chimera 9 (50 nM). E) Compound 2 interferes with MMP-9 homodimerization. COS-1 cells transfected with MMP-9-Myc and MMP-9-HA in the presence or absence of compounds 2 and 4 (100 μM) were pulled down with anti-HA antibody followed by Western blotting with anti-Myc antibody. The aliquots of total cell lysates serving as input were examined by Western blotting using anti-Myc antibodies. Reciprocal co-immunoprecipitation was also performed. F) Compound 2 decreased MMP-9-mediated ERK1/2 activation: COS-1 cells transiently transfected with vector control and MMP-9 cDNAs were serum-starved for 18 hours in the presence or absence of compounds 2 and 4 (100 μM) followed by Western blotting using anti-pERK1/2 and total ERK1/2 antibodies.

Figure 6. Inhibition of cell proliferation by compound 2

The effect of compound 2 on cell proliferation was examined in MMP-9 cDNA transfected COS-1 cells (A) or cancer cells expressing endogenous MMP-9 (HT-1080 and MDA-MB-435) (C & D), as well as GFP cDNA transfected COS-1 cells (control) (B) in the presence or absence of the compounds 2 and 4 (10 μM) for 9 days.

Figure 7. Retarded tumor growth and metastasis by compound 2 in mice bearing MDA-MB-435/GFP tumor xenografts

A–B) Effect of compound 2 on tumor growth: Mice bearing MDA-MB-435/GFP cells were administered compounds or vehicle control and tumor was measured. Arrows indicate tumor mass. C–E) Inhibition of metastasis by compound 2: Lung sections ~3 mm thick were examined by fluorescent microscopy. Incidence of metastasis was determined (C–D) and the average area of tumor in the lungs was determined using ImageJ software (E).