Selective small-molecule inhibitors as chemical tools to define the roles of matrix metalloproteinases in disease.

The focus of this article is to highlight novel inhibitors and current examples where the use of selective small-molecule inhibitors has been critical in defining the roles of matrix metalloproteinases (MMPs) in disease. Selective small-molecule inhibitors are surgical chemical tools that can inhibit the targeted enzyme; they are the method of choice to ascertain the roles of MMPs and complement studies with knockout animals. This strategy can identify targets for therapeutic development as exemplified by the use of selective small-molecule MMP inhibitors in diabetic wound healing, spinal cord injury, stroke, traumatic brain injury, cancer metastasis, and viral infection. This article is part of a Special Issue entitled: Matrix Metalloproteinases edited by Rafael Fridman.

Acceleration of diabetic wound healing using a novel protease-anti-protease combination therapy.

Nonhealing chronic wounds are major complications of diabetes resulting in >70,000 annual lower-limb amputations in the United States alone. The reasons the diabetic wound is recalcitrant to healing are not fully understood, and there are limited therapeutic agents that could accelerate or facilitate its repair. We previously identified two active forms of matrix metalloproteinases (MMPs), MMP-8 and MMP-9, in the wounds of db/db mice. We argued that the former might play a role in the body's response to wound healing and that the latter is the pathological consequence of the disease with detrimental effects. Here we demonstrate that the use of compound ND-336, a novel highly selective inhibitor of gelatinases (MMP-2 and MMP-9) and MMP-14, accelerates diabetic wound healing by lowering inflammation and by enhancing angiogenesis and re-epithelialization of the wound, thereby reversing the pathological condition. The detrimental role of MMP-9 in the pathology of diabetic wounds was confirmed further by the study of diabetic MMP-9-knockout mice, which exhibited wounds more prone to healing. Furthermore, topical administration of active recombinant MMP-8 also accelerated diabetic wound healing as a consequence of complete re-epithelialization, diminished inflammation, and enhanced angiogenesis. The combined topical application of ND-336 (a small molecule) and the active recombinant MMP-8 (an enzyme) enhanced healing even more, in a strategy that holds considerable promise in healing of diabetic wounds.

Restructuring of the extracellular matrix in diabetic wounds and healing: A perspective.

Diabetic foot ulcers are a complication of diabetes for which treatment options are limited and not effective, resulting in 73,000 lower-limb amputations in the United States every year. Wound healing is a complex process with a highly orchestrated cascade of events, in which the extracellular matrix (ECM) interacts with growth factors and cells. Matrix metalloproteinases (MMPs) are involved in all wound healing events, in particular MMP-8 and MMP-9, whose physiological functions are to degrade damaged collagen type I and to facilitate keratinocyte migration and re-epithelialization, respectively. MMP substrate redundancy permits another MMP to substitute for MMP-9 during normal wound healing. Under the hypoxic and inflammatory environment of diabetic wounds, increased reactive oxygen species (ROS) and upregulation of MMP-9 results in wounds that are recalcitrant to healing. We have determined that MMP-8 plays a role in the body's response to wound healing and that MMP-9 is the pathological consequence of the disease with detrimental effects. Thus, selective inhibition of MMP-9, while leaving MMP-8 activity unaffected, is desirable. ND-336 has such inhibitory profile and is a promising strategy for treatment of diabetic foot ulcers.

Targeting extracellular matrix remodeling sensitizes glioblastoma to ionizing radiation.

Background: The median survival of Glioblastoma multiforme (GBM) patients is 14+ months due to poor responses to surgery and chemoradiation. Means to counteract radiation resistance are therefore highly desirable. We demonstrate the membrane bound matrix metalloproteinase MT1-MMP promotes resistance of GBM to radiation, and that using a selective and brain permeable MT1-MMP inhibitor, (R)-ND336, improved tumor control can be achieved in preclinical studies. Methods: Public microarray and RNA-sequencing data were used to determine MT1-MMP relevance in GBM patient survival. Glioma stem-like neurospheres (GSCs) were used for both in vitro and in vivo assays. An affinity resin coupled with proteomics was used to quantify active MT1-MMP in brain tissue of GBM patients. Short hairpin RNA (shRNA)-mediated knockdown of MT1-MMP and inhibition via the MT1-MMP inhibitor (R)-ND336, were used to assess the role of MT1-MMP in radio-resistance. Results: MT1-MMP expression inversely correlated with patient survival. Active MT1-MMP was present in brain tissue of GBM patients but not in normal brain. shRNA- or (R)-ND336-mediated inhibition of MT1-MMP sensitized GSCs to radiation leading to a significant increase in survival of tumor-bearing animals. MT1-MMP depletion reduced invasion via the effector protease MMP2; and increased the cytotoxic response to radiation via induction of replication fork stress and accumulation of double strand breaks (DSBs), making cells more susceptible to genotoxic insult. Conclusions: MT1-MMP is pivotal in maintaining replication fork stability. Disruption of MT1-MMP sensitizes cells to radiation and can counteract invasion. (R)-ND336, which efficiently penetrates the brain, is therefore a novel radio-sensitizer in GBM.