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.
Highlights
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
Inhibitors of MMP-9 activation were identified by highthroughput screening using the ThermoFluor platform to identify compounds that bound to MMP-9 and modified the protein’s thermal stability profile [34]
The resultant MMP-9 enzymatic activity was measured by monitoring cleavage of a fluorescent MMP-9 – specific substrate, DQ-gelatin, which was not cleaved to a great extent by catMMP-3 or proMMP-9 alone (Fig. 1A)
Summary
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. Of the ϳ50 other clinical trials conducted with active site MMP inhibitors, all have failed due to the onset of significant dose-limiting musculoskeletal toxicity or lack of efficacy [5] These compounds were based on hydroxamic acid or related chelator chemistry that acts to perturb the critical coordinating zinc in the catalytic domain, which results in loss of enzymatic activity. In growing efforts to identify inhibitor sites outside of the catalytic domain [15, 16], small peptides have been identified that can interfere with the MMP-2 interaction with collagen substrate [17] These new approaches hold promise due to enhanced selectivity, the clinical utility of these compounds and others employing alternative inhibitory strategies has not yet been determined. The specificity of JNJ0966 for inhibiting MMP-9 activation was explored, as was potential clinical utility in mouse experimental autoimmune encephalomyelitis (EAE), an in vivo model for human neuroinflammatory disorders such as multiple sclerosis
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