A novel small molecule screening assay using normal human chondrocytes toward osteoarthritis drug discovery

Osteoarthritis (OA) is the most common form of arthritis and a leading cause of pain and disability in adults. A central feature is progressive cartilage degradation and matrix fragment formation driven by the excessive production of matrix metalloproteinases (MMPs), such as MMP-13, by articular chondrocytes. Inflammatory factors, including interleukin 6 (IL-6), are secreted into the joint by synovial fibroblasts, and can contribute to pain and inflammation. No therapeutic exists that addresses the underlying loss of joint tissue in OA. To address this, we developed and utilized a cell-based high-throughput OA drug discovery platform using normal human chondrocytes treated with a recombinant fragment of the matrix protein fibronectin (FN-f) as a catabolic stimulus relevant to OA pathogenesis and a readout using a fluorescent MMP-13 responsive probe. The goal was to test this screening platform by identifying compounds that inhibited FN-f-induced MMP-13 production and determine if these compounds also inhibited catabolic signaling in OA chondrocytes and synovial fibroblasts. Two pilot screens of 1344 small molecules revealed five "hits" that strongly inhibited FN-f induced MMP-13 production with low cytotoxicity. These included RO-3306 (CDK1 inhibitor (i)), staurosporine (PKCi), trametinib (MEK1 and MEK2i), GSK-626616 (DYRK3i), and edicotinib (CSF-1Ri). Secondary testing using immunoblots and cells derived from OA joint tissues confirmed the ability of selected compounds to inhibit chondrocyte MMP-13 production and FN-f stimulated IL-6 production by synovial fibroblasts. These findings support the use of this high throughput screening assay for discovery of disease-modifying osteoarthritis drugs.