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. MMPs2 are a family of structurally related zinc-binding proteolytic enzymes that digest extracellular matrix proteins and participate in tissue remodeling and signaling events (1). Currently, ϳ23 MMPs have been identified, comprising secreted and membrane-bound forms, and different family members share some common structural and functional domains and have varying degrees of substrate specificity. Abnormal expression and activation of MMPs has been implicated in the pathogenesis and pathological progression of several different human diseases that are centered in many different tissues in the periphery and central nervous system (2, 3). Initial clinical exploration of synthetic MMP inhibitors was focused on oncology indications, as preventing the breakdown of tissue matrices and barriers was viewed as a potential mechanism to limit tumor metastasis.Despite intensive efforts over many years to develop synthetic MMP inhibitors, only a single MMP inhibitor, Periostat, a tetracycline derivative used in periodontal disease, has progressed into regular clinical use (4). 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 ...

Merkel Cell Polyomavirus Small Tumor Antigen Activates Matrix Metallopeptidase-9 Gene Expression for Cell Migration and Invasion

“…Although results from early animal studies utilizing broad spectrum inhibitors were compelling, it was determined that inhibiting such a broad scale inhibition of MMPs interfered with normal tissue function, which could support disease progression (66, 67). Moreover, MMP-9 active site inhibitors were prone to off target binding which induced unexpected malignancies including musculoskeletal toxicity (68).…”

Proteases: Pivot Points in Functional Proteomics

“…None of the 50some clinical trials with MMP inhibitors were successful, due to off-target toxicity or absence of efficacy. A recent study reports effective allosteric prevention of pro-MMP-9 activation in a mouse neuroinflammatory model by a small, highly selective heterocyclic chemical inhibitor [204]. This orally administered compound does not prevent activation of the structurally related pro-MMP-2, and does not inhibit catalytically active MMP-1, −2, −3, −9 or −14.…”

“…Additional allosteric, exosite and effector-binding site interactions are expected to contribute significantly to exclusive selection of the target protease. Structural conservation of the specificity site throughout a protease family with diverse catalytic properties and biological targets may pose a problem in designing specific drugs, and a promising alternative was developed by targeting zymogen activation rather than the active protease [204]. A highly selective compound that allosterically inhibits MMP-9 activation by binding to a pocket near the zymogen cleavage site may be a first viable drug candidate.…”

Third time lucky? Getting a grip on matrix metalloproteinases

“…C, the chemical structure of compound JNJ0966. D, a novel approach by Scannevin et al (9), in which the MMPI (green) targets the final activation point of the zymogen and prevents prodomain (blue) removal. This MMPI does not inhibit the mature MMP.…”

“…As one role of MMPs in cancer is associated with excessive activity, blocking zymogen activation could provide a fairly targeted mechanism to disrupt cancer development. The conceptually simple but elegant working hypothesis, then, of Scannevin et al (9), is that repressing activation of the zymogen could be an alternate approach to preventing activity. Using high-throughput screening employing the ThermoFluor approach, which reports on protein stability variations due to ligand binding, the authors identified compound JNJ0966 (Fig.…”