Screening small molecule ihibitors against proteoglycan degradation to control tissue degeneration

Abstract

Defective biosynthesis or function of proteoglycans leads various pathological conditions and defects in stem/progenitor activities. A high-throughput platform is an important asset to understand the complex and yet unclear metabolic network of proteoglycan. By using chondrocytes, known to be active in proteoglycan production, as a model system we tested the feasibility of a spectrophotometry-based platform in search for small molecules potent in regulating proteoglycan metabolism. We developed a miniaturized format of dimethylmethylene blue (DMMB) assay with optimized detection range to measure levels of glycosaminoglycan, the major constituent of proteoglycan. We demonstrated that such assay exhibits rigorous and unbiased performance in large batch processing. A pilot screening of 960 small molecules in primary porcine chondrocytes suggested the platform may favor identification of both positive and negative regulators. Screening a chemical library of over 50,000 compounds identified a lead panel that can regulate sulfated glycosaminoglycan (GAG) levels without changing global metabolic activity. An alginate 3D culture system of bovine and human nucleus pulposus (NP) cells, which have a dominant function of high-level proteoglycan production, showed dosage-dependent positive response to the lead compounds in chondroitin-4-sulfate GAG production in the optimized DMMB and FACE (fluorescence-assisted carbohydrate electrophoresis) assays. Interestingly, degenerative human NP cells exhibited a remarkable response to compound stimulation. Two lead compounds showed capacity in inhibiting interleukin-1 (IL-1) induced loss of proteoglycans, as well as reducing the MMP-2 and -9 enzyme activity and nitric oxide production. By local delivery in a rat model, we verified that the leads could inhibit the loss of proteoglycan in intervertebral disc undergoing injury-induced degeneration. Overall, our study demonstrated the effectiveness of a high-throughput library screen platform in identifying proteoglycan regulators, and implicated the potential of small molecules in regulating chondroitin sulfate expression and alleviating cartilaginous tissue degeneration. Our findings warrant the future use of the DMMB assay-based screening platform to discover agents that regulate proteoglycans for specific cell types or disease models. This proof of concept study presents a new ground to facilitate drug discovery, in particular targeting conditions associated with proteoglycan degradation, which is a major and common catabolic consequence in the IL-1 as well as other pro-inflammatory cascades.