Biochemical characterization and structure determination of a potent, selective antibody inhibitor of human MMP9

Abstract

Matrix metalloproteinase 9 (MMP9) is a member of a large family of proteases that are secreted as inactive zymogens. It is a key regulator of the extracellular matrix, involved in the degradation of various extracellular matrix proteins. MMP9 plays a pathological role in a variety of inflammatory and oncology disorders and has long been considered an attractive therapeutic target. GS-5745, a potent, highly selective humanized monoclonal antibody inhibitor of MMP9, has shown promise in treating ulcerative colitis and gastric cancer. Here we describe the crystal structure of GS-5745·MMP9 complex and biochemical studies to elucidate the mechanism of inhibition of MMP9 by GS-5745. GS-5745 binds MMP9 distal to the active site, near the junction between the prodomain and catalytic domain, and inhibits MMP9 by two mechanisms. Binding to pro-MMP9 prevents MMP9 activation, whereas binding to active MMP9 allosterically inhibits activity.

Keywords: X-ray crystallography; allosteric regulation; antibody; enzyme inhibitor; matrix metalloproteinase (MMP).

Figure 1.

Inhibition of MMP9 by GS-5745. A, inhibition of activated MMP9 by GS-5745 was measured by incubating a fixed concentration of active MMP9 (0.5 nm MMP9-MYC-His_{6 APMA} (black circles), 5 nm MMP9-pro-cat_APMA (red squares), or 5 nm MMP9-cat (blue squares)) with various concentrations of GS-5745. DQ gelatin was added at 2 × K_m (30 nm for MMP9-MYC-His_{6 APMA} or 1500 nm for MMP9-pro-cat_APMA and MMP9-cat), and activity was monitored by measuring the increase in fluorescence of DQ gelatin upon cleavage by MMP9. IC₅₀ values were determined using a four-parameter curve fitting with GraphPad Prism 6. B and C, for competition studies, a fixed concentration of MMP9-MYC-His_{6 APMA} (0.2 nm) (B) or MMP9-cat (5 nm) (C) was mixed with varying concentrations of GS-5745 (0–2 nm for MMP9-MYC-His_{6 APMA} or 0–16 μm for MMP9-cat) and DQ gelatin (0–150 nm for MMP9-MYC-His_{6 APMA} or 0–2.3 μm for MMP9-cat). The rate of DQ gelatin cleavage was monitored, and the data were fit as described under “Experimental procedures.” Each data point represents the average of duplicate experiments, and error bars represent S.D. RFU, relative fluorescence units.

Figure 2.

Structure of GS-5745·MMP9-pro-cat and MMP9-pro-cat. A, the GS-5745·MMP9-pro-cat complex is represented as a ribbon diagram. The prodomain (pro) of MMP9 is colored yellow, and the catalytic domain (cat) is colored green. The heavy chain (Hc) of GS-5745 is colored pink, and the light chain (Lc) is colored cyan. Calcium atoms are colored gray, and zinc atoms are colored magenta. B, superposition of apo MMP9-pro-cat (gray) with MMP9-pro-cat bound to GS-5745 (yellow and green).

Figure 3.

Interactions between GS-5745 and MMP9. A, Arg-162 of MMP9 forms a salt bridge, hydrogen bonds, and hydrophobic interactions with GS-5745. B, Gln-108 forms hydrogen bonds with the heavy chain of GS-5745. The position of the cleavage site of trypsin/MMP3 between Arg-106 and Phe-107 is indicated. C, superposition of MMP9 bound to GS-5745 and active MMP9 (Protein Data Bank code 1GKD). Lc, light chain; Hc, heavy chain; Ab, antibody.

Figure 4.

GS-5745 prevents activation of pro-MMP9 by MMP3. A, activation of pro-MMP9 by MMP3 was measured by incubating a fixed concentration pro-MMP9 (2.5 ng/μl MMP9-pro-cat) with MMP3 (0.25 ng/μl) in the presence of GS-5745 (30 ng/μl) or control IgG4 (30 ng/μl). Reactions were incubated at 37 °C for the indicated time, and pro- and active MMP9 were visualized by immunoblotting analysis using an anti-MMP9 antibody. B, the relative intensity of pro-MMP9 in the presence of control IgG4 (○) or GS-5745 (●) in A, normalized to the control lane (0 h), was quantified by densitometry. C, the relative intensity of active MMP9-G100L in the presence of control IgG4 (□) or GS-5745 (■) in A, normalized to the control lane (0 h), was quantified by densitometry. Each data point represents the average of duplicate experiments, and error bars represent S.D.

Figure 5.

GS-5745 stabilizes pro-MMP9. A, HEK293 cells stably expressing either MMP9 or MMP9-G100L were treated with 30 μg/ml GS-5745 or 200 nm marimastat for 16 h. The medium was harvested and subjected to SDS-PAGE. Pro- and active MMP9 were visualized by immunoblotting analysis using an anti-MMP9 antibody. B, the stability of pro- and active MMP9-G100L was monitored by measuring the half-life of the protein in the absence or presence of GS-5745. HEK293 cells stably expressing MMP9-G100L were incubated with 50 μg/ml cycloheximide (CHX) and either 30 μg/ml GS-5745 or control IgG4. The medium was collected at various times, and pro- and active MMP9-G100L were detected as described previously. C, the relative intensity of pro-MMP9-G100L in the presence of control IgG4 (○) or GS-5745 (●) in B, normalized to the control lane (0 h), was quantified by densitometry. D, the relative intensity of active MMP9-G100L in the presence of control IgG4 (□) or GS-5745 (■) in B, normalized to the control lane (0 h), was quantified by densitometry. Each data point represents the average of duplicate experiments, and error bars represent S.D.

Figure 6.

GS-5745 prevents activation of pro-MMP9 by ulcerative colitis lysates. A, colon tissue was lysed, and MMP9 was visualized by immunoblotting. The upper band (molecular mass, ∼92 kDa) was detected in diseased and healthy colon lysate, whereas the lower band (∼82 kDa) was detected in only the ulcerative colitis lysate. B, endogenous MMP9 activity from each colon lysate was assessed. A standard curve of quantitatively activated MMP9 was used to determine the equivalents of active MMP9 in ng/ml. C, incubation of recombinant pro-MMP9 with ulcerative colitis lysates results in MMP9 activation. The activated MMP9 was detected by the neoepitope antibody (red), whereas both pro- and active MMP9 are detected by the total MMP9 antibody (green). MMP9 activation is inhibited by GS-5745 but not a human IgG4 isotype control antibody. D, the activity of MMP9 in C was measured with the Biotrak MMP9 assay kit. GS-5745, but not the isotype control IgG4, blocks this proteolysis. MMP9 was isolated from the lysate constituents prior to activity assessment. Each data point represents a single experiment, and error bars represent S.D.