Fig 1.
Binding and inhibitory activity of engineered SPINK2-derived inhibitors.
(A) The binding activity to active MMP-9 was measured by ELISA. Various concentrations of engineered SPINK2-derived inhibitors or wild-type SPINK2 (1–1,000 nM) were added to the biotinylated active MMP-9_Cat-Avi (50 nM)-coated plate, and then inhibitors bound to active MMP-9 were detected by HRP-conjugated anti-S tag antibody. (B) The binding activities to pro- and active MMP-9 were measured by ELISA. Biotinylated pro-MMP-9_Cat-Avi or biotinylated active MMP-9_Cat-Avi (50 nM each) was coated on the plate, to which inhibitors (1 μM) were then added. The inhibitors bound to MMP-9 were detected by HRP-conjugated anti-S tag antibody. (C) MMP-9 inhibitory activity of M91005 was measured by enzymatic assay using peptide substrate. Active MMP-9_Cat-H6 (0.4 nM) was incubated with various concentrations of M91005 (0–25 nM) for 1 h at 37°C, and then peptide substrate 3226-v (10 μM) was added. MMP-9 activity was determined by monitoring the hydrolysis of the peptide substrate, and the activity in the absence of M91005 was taken as 100%. (D) MMP-9 inhibitory activity of engineered SPINK2-derived inhibitors was measured by enzymatic assay using the macromolecular substrate. Active MMP-9_Cat-H6 (0.6 nM) was incubated with various concentrations of inhibitors (0–100 nM) for 1 h at 37°C, and then DQ-gelatin (10 μg/ml) was added. MMP-9 activity was determined by monitoring the hydrolysis of DQ-gelatin and the activity in the absence of inhibitor was taken as 100%. Data are shown as the mean of duplicate experiments in (A) and the mean ± S.D. (n = 3) in (B–D). All curves were obtained by non-linear curve fitting as described in “Materials and methods”.
Table 1.
Binding affinity and inhibitory activity of inhibitors against active MMP-9.
Fig 2.
Cross-reactivity of engineered SPINK2-derived inhibitors against MMP-9.
The cross-reactivities against MMP-1, -2, -8, and -13 were measured by enzymatic assay using the peptide substrate. Each active MMP (MMP-1, 5 nM; MMP-2, 1.4 nM; MMP-8, 0.6 nM; MMP-13, 2 nM) was incubated with inhibitors (1 μM) for 10 min (for MMP-13) or 60 min (for MMP-1, -2, and -8) at 37°C, and then peptide substrate 3226-v (10 μM for MMP-1, -8, and -13, or 50 μM for MMP-2) was added. Enzymatic activity was determined by monitoring the hydrolysis of the peptide substrate, and each remaining enzymatic activity was normalized to the activity in the absence of inhibitors. Each bar represents the mean ± S.D. (n = 3).
Fig 3.
Design of MMP-9 mutants for analysis of interaction between MMP-9 and M91005.
(Top) The sequence alignment between MMP-9 and MMP-2 in the active-site cleft and exosite of MMP-9. The residues are numbered according to the generic MMP-9 nomenclature. Catalytic Glu-402 is indicated by an asterisk and shown in blue. Three histidine residues (His-401, His-405, and His-411) that chelate catalytic zinc ion are indicated by a “Z” and shown with a green background. Alanine substitution sites in the active-site cleft (for cleft mutants) and MMP-9/-2 chimeric mutation sites in the exosite (for exosite mutants) are highlighted in magenta and cyan, respectively. Three residues (Ala-189, His-190, and Ala-191) in the active-site cleft, the side chain of which is not exposed to solvent, are shown with an orange background. (Bottom) The structure of the catalytic domain of active MMP-9, colored in gray (Protein Data Bank code 4H3X). Each residue is shown with coloring as in the top panel.
Fig 4.
M91005 binds to the active site of MMP-9.
The binding activity to the catalytic Glu-402 residue of MMP-9 was evaluated by SEC. Active MMP-9_Cat-H6 or MMP-9_Cat-E402Q-H6 (25 μM each) was incubated with TIMP-1 or M91005 (75 μM each) in PBS for 1 h at 4°C, and then 10 μl of the reaction mixture was analyzed by monitoring the absorbance at 280 nm in SEC. Chromatograms are shown in panels as follows: active MMP-9_Cat-H6 (A), MMP-9_Cat_E402Q-H6 (B), and no MMP-9 (C). Dotted lines indicate the retention time (RT) of active MMP-9_Cat-H6 (7.0 min, A) and MMP-9_Cat_E402Q-H6 (7.1 min, B). Molecular weights (158, 44, and 17 kDa) of the gel filtration standard are indicated at the top of each panel. The RT of active MMP-9_Cat-H6 but not MMP-9_Cat-E402Q-H6 was shifted to a high molecular weight by incubation with M91005. mAU, milliabsorbance units.
Fig 5.
M91005 recognizes residues in the active-site cleft and the exosite of MMP-9.
MMP-9 inhibitory activities of M91005 towards the cleft mutants (A) and the exosite mutants (B) were measured by enzymatic assay using peptide substrate. Active MMP-9_Cat (WT) or the MMP-9 mutants activated by EK (1 nM each) were incubated with various concentrations of M91005 (0–330 nM or 0–1,000 nM) for 1 h at 37°C. Enzymatic activities were determined by monitoring the degradation of peptide substrate 3226-v (10 μM). Each enzymatic activity was normalized to the activity of the MMP-9 mutant without M91005. Data are shown as the mean ± S.D. (n = 3). All curves were obtained by non-linear curve fitting.
Table 2.
Inhibitory activity of M91005 against cleft mutants.
Fig 6.
Mapping of the possible interaction sites with M91005 onto the structure of active MMP-9.
In accordance with the results of binding and enzymatic assay using MMP-9 mutants, the residues identified as being involved in the interaction with M91005 were mapped onto the structure of the catalytic domain of active MMP-9 (gray, Protein Data Bank code 4H3X). The results of the binding assays (Fig 4) suggested that catalytic Glu-402 (blue) contributed to the interaction between M91005 and MMP-9. The results of enzyme inhibitory assays (Fig 5, Tables 2 and 3) suggested that M91005 recognized Phe-110, Tyr-179, Leu-187, Phe-192, Gln-199, Tyr-393, Tyr-420, Met-422, and Tyr-423 (magenta). The interaction with characteristic residues, Gln-199 and Met-422 (black dotted circle), is likely to contribute to the specific inhibition of M91005 toward MMP-9.
Table 3.
Inhibitory activity of M91005 against exosite mutants.