Evolution of a matrix metalloproteinase assay to assess inhibitor potency and selectivity

Matrix metalloproteinases (MMPs) are a class of zinc-containing extracellular proteinases involved in extracellular matrix turnover and remodeling. As such, these enzymes are critical in maintaining the proper biochemical composition and physical characteristics of all tissues, but in particular those tissues whose composition is largely extracellular matrix, such as cartilage. Under normal physiological conditions, constitutive expression of the MMPs is low, as is their catalytic activity which is held in check by naturally occurring inhibitors termed TIMPs (tissue inhibitor of metalloproteinases). However, under pathologic conditions such as rheumatoid and osteoarthritis, MMP expression in cartilage is disregulated. MMP levels are high with enzymic activity exceeding the level of the natural inhibitors. This condition leads to a loss of proteoglycan and collagen from articular cartilage culminating in the ultimate destruction which characterizes the pathology of arthritic diseases. Based upon this scenario, intensive efforts are focused on the identification of inhibitors which will block the action of MMPs in arthritis. Predictive, high through-put enzymic assays are essential for inhibitor identification. Ideally, such assays must be convenient, kinetically predictive, and flexible to accommodate the numerous candidate MMPs potentially involved in cartilage degradation. Thus, we have evaluated several MMP assays for inhibitor assessment and these are detailed below.