Discovery of a highly selective chemical inhibitor of matrix metalloproteinase-9 (MMP-9) that allosterically inhibits zymogen activation

Abstract

Aberrant activation of matrix metalloproteinases (MMPs) is a common feature of pathological cascades observed in diverse disorders, such as cancer, fibrosis, immune dysregulation, and neurodegenerative diseases. MMP-9, in particular, is highly dynamically regulated in several pathological processes. Development of MMP inhibitors has therefore been an attractive strategy for therapeutic intervention. However, a long history of failed clinical trials has demonstrated that broad-spectrum MMP inhibitors have limited clinical utility, which has spurred the development of inhibitors selective for individual MMPs. Attaining selectivity has been technically challenging because of sequence and structural conservation across the various MMPs. Here, through a biochemical and structural screening paradigm, we have identified JNJ0966, a highly selective compound that inhibited activation of MMP-9 zymogen and subsequent generation of catalytically active enzyme. JNJ0966 had no effect on MMP-1, MMP-2, MMP-3, MMP-9, or MMP-14 catalytic activity and did not inhibit activation of the highly related MMP-2 zymogen. The molecular basis for this activity was characterized as an interaction of JNJ0966 with a structural pocket in proximity to the MMP-9 zymogen cleavage site near Arg-106, which is distinct from the catalytic domain. JNJ0966 was efficacious in reducing disease severity in a mouse experimental autoimmune encephalomyelitis model, demonstrating the viability of this therapeutic approach. This discovery reveals an unprecedented pharmacological approach to MMP inhibition, providing an opportunity to improve selectivity of future clinical drug candidates. Targeting zymogen activation in this manner may also allow for pharmaceutical exploration of other enzymes previously viewed as intractable drug targets.

Keywords: allosteric regulation; drug action; drug discovery; enzyme inhibitor; enzyme processing; matrix metalloproteinase (MMP); pharmacology.

Figure 1.

JNJ0966 inhibited activation of proMMP-9 but exhibited no effect on catalytic activity or maturation of other MMPs. A, proMMP-9 activation assay utilizing the fluorescent substrate DQ-gelatin. proMMP-9, catMMP-3, or a combination of the two in the presence or absence of 10 μm JNJ0966 was incubated together then with DQ-gelatin. proMMP-9 activated by catMMP-3 exhibited significant increases in activity as compared with either enzyme alone, and this was significantly inhibited by JNJ0966 (n = 6). RFU, relative fluorescence units; ****, p < 0.0001, one-way ANOVA with Bonferroni multiple-comparison post-test. B, molecular structure of JNJ0966 and molecular weight (MW). C, proMMP-9 activation assay characterizing JNJ0966 concentration response with catMMP-3 activation (IC₅₀ = 440 nm). D and E, activity assay in which concentration responses of JNJ0966 and GM6001 were incubated with catMMP-3 (D) or catMMP-9 (E). GM6001 inhibited catMMP-3 (D, IC₅₀ = 7.2 nm) and catMMP-9 (E, IC₅₀ = 0.5 nm), but JNJ0966 had no effect at any concentration (n = 6). F, activation assay on proMMP-1, proMMP-2, proMMP-3, and proMMP-9 activated by trypsin or catMMP-14, as indicated, in the presence (black bars) or absence (white bars) of 10 μm JNJ0966. JNJ0966 only significantly inhibited the activation of proMMP-9 by trypsin. Data were normalized to 100% for each enzyme maximal activity (n = 6; ****, p < 0.001, two-tailed t test). G, proMMP-9 activation assay characterizing JNJ0966 concentration response with trypsin activation (IC₅₀ = 429 nm). H, HT1080 cellular invasion assay demonstrating the concentration-response curves of doxycycline (green squares; IC₅₀ = 21 μm), GM6001 (blue triangles; IC₅₀ = 1.4 μm), and JNJ0966 (red circle; IC₅₀ = 1.0 μm) inhibiting cellular transmigration across a Matrigel^TM layer (n = 4). I, representative images from the bottom side of transwells from the indicated treatments illustrate calcein AM–labeled cells that have migrated through Matrigel to the bottom filter insert layer. In all graphs (A, C, D, E, F, and G), data are presented as means ± S.D. (error bars). All curves are fit by nonlinear regression.

Figure 2.

JNJ0966 reduced proMMP-9 maturation through intermediate states to active fully processed enzyme. A–F, proMMP-9 samples were reacted with catMMP-3 (0, 20, 40, and 60 min) in the presence or absence of 10 μm JNJ0966. Sample aliquots from the same time points were loaded on replicate gels in each panel (A–E). Three arrowheads in each panel denote the migration of proMMP-9 at 92 kDa, intermediate MMP-9 at 86 kDa, and active MMP-9 at 82 kDa. A, gelatin zymograph demonstrating maturation of proMMP-9 through activation and the effects of reducing active MMP-9 in the presence of JNJ0966. B–E, replicate immunoblots probed with antibodies for total MMP-9 (B), proMMP-9 (C), intermediate MMP-9 (D), and fully processed MMP-9 (E). Combined, the immunoblots demonstrate the accumulation of fully processed MMP-9 in DMSO controls and the reduced conversion of MMP-9 to the mature species in the presence of JNJ0966. F, activity assay to monitor development of gelatinase activity in the same sample aliquots analyzed in A–E (n = 3 for each assay time point; data are represented as means ± S.D. (error bars); ****, p < 0.0001, two-tailed t test).

Figure 3.

Analysis of proMMP-9 maturation kinetics by gelatin zymography in the presence and absence of 10 μm JNJ0966. A, gelatin zymogram of proMMP-9 samples that have been reacted with catMMP-3 in the presence and absence of 10 μm JNJ0966 for 0, 10, 20, 30, 40, 50, and 60 min. Zymograms are duplicate pictures; the bottom panel is overlaid with graphical lines to illustrate the three different MMP-9 molecular species (92, 86, and 82 kDa). B–D, graphs of relative abundance of the individual MMP-9 forms as a percentage of total enzyme abundance in each condition as measured by densitometric quantitation of the zymogram. In all graphs, relative abundance of proMMP-9 species is depicted from DMSO control (red lines) and 10 μm JNJ0966 (blue lines) conditions. B, graph of the unprocessed 92-kDa proMMP-9 with (blue lines) and without (red line) JNJ0966. C, graph of the 86-kDa intermediately processed proMMP-9. D, graph of the 82-kDa fully processed MMP-9.

Figure 4.

Structural characterization of JNJ0966 and proMMP-9 complex. A, superposition of JNJ0966 structure on the proMMP-9 coordinates. A stick diagram of JNJ0966 (carbon backbone is represented in cyan, oxygen in red, nitrogen in blue, and sulfur in yellow) is superimposed on the ribbon diagram of uncomplexed proMMP-9 (green), demonstrating how JNJ09066 occupies space typically occupied by the side chains of Arg-106 and Phe-107 (green sticks). Residues 105–109 are shown in red on the proMMP-9 backbone. B, structure of JNJ0966 in a complex with proMMP9. In the ribbon diagram of proMMP9, residues near the interface with JNJ0966 are labeled in black (Val-101, Phe-110, and Tyr-179). The activation loop (residues 103–108) was disordered in the JNJ0966-MMP-9 structure. C, electron density of the proMMP9 JNJ0966 complex. The figure is in wall-eyed stereo. The 2|F_o| − |F_c| map (cyan) at the 1σ contour level shows the unambiguous binding mode of JNJ0966. D, activation assay comparing wild-type and amino acid substitution mutant versions of proMMP-9 activated by catMMP-3 in the presence or absence of 10 μm JNJ0966, as measured by DQ-gelatin cleavage. Basal activity (black bars), basal activity plus catMMP-3 activation (open bars), and basal activity plus catMMP-3 with 10 μm JNJ0966 (hatched bars) are shown for each MMP-9 mutant. Data are normalized to 100% for each MMP-9 mutant plus catMMP-3 levels. The R106A mutation in MMP-9 had little impact on the activity of JNJ0966, whereas V101A, F110A, and Y179A reduced the magnitude of the inhibitory effect. The triple mutant of the latter three residues abolished the effects of JNJ0966 in inhibiting activation. Data are represented as means ± S.D. (error bars), n = 4. *, p < 0.05; ***, p < 0.001; ****, p < 0.0001, two-tailed t test.

Figure 5.

JNJ0966 reduced motor symptoms in mouse EAE and penetrates into the brain. A, mice that had induced EAE were treated twice daily by oral gavage with vehicle (black squares), JNJ0966 at 10 mg/kg (green diamonds), or 30 mg/kg (blue circles) or dexamethasone (1 mg/kg; red triangles). Animals receiving either dose of JNJ0966 or dexamethasone evidenced delayed onset of motor disability and also had significantly reduced disease courses relative to the vehicle controls. Data were analyzed via repeated-measures ANOVA with Bonferroni multiple-comparison post-test to examine differences between the group (n = 7 for vehicle group, n = 5 for dexamethasone group, n = 9 for JNJ0966 10 mg/kg group, and n = 9 for JNJ0966 30 mg/kg group (*, p < 0.05; **, p < 0.01). Points, mean clinical scores; bars, S.E. B, cumulative clinical scores for individual animals plotted for vehicle, dexamethasone, JNJ0966 10 mg/kg, and JNJ0966 30 mg/kg. All treatment groups were significantly lower than vehicle (one-way ANOVA with Bonferroni multiple-comparison post-test; *, p < 0.05). C, plasma (gray circles) and brain (black squares) concentrations of JNJ0966 after 10- or 30-mg/kg dosing in five animals on the last day of dosing (day 17) in the EAE study. D, ratio of brain to plasma concentrations of JNJ0966 after 10-mg/kg (circles) or 30-mg/kg (squares) dosing for animal exposures shown in C. For C and D, individual values are plotted, with bars for means and S.D.