Rhodamine B and 2-acetamido-1,3,6-tri-O-acetyl-4-deoxy-4-fluoro-d-glucopyranose (F-GlcNAc) inhibit chondroitin/dermatan and keratan sulphate synthesis by different mechanisms in bovine chondrocytes

Abstract

MPS disorders result from a deficiency or absence of glycosaminoglycan (GAG) degrading enzymes leading to an imbalance between the synthesis and degradation of GAGs and their subsequent accumulation in a range of cells. The inhibition of GAG synthesis using small chemical inhibitors has been proposed as a novel therapeutic approach to treatment. Several inhibitors have been shown to decrease heparan sulphate GAG synthesis and in this study we evaluated a novel fluorinated analog of N-acetylglucosamine (2-acetamido-1,3,6-tri-O-acetyl-4-deoxy-4-fluoro-d-glucopyranose (F-GlcNAc)) and rhodamine B for their ability to also inhibit the synthesis of chondroitin/dermatan and keratan sulphate GAGs present in bovine cartilage. Both inhibitors decreased GAG synthesis in chondrocyte monolayer culture and in cartilage chip explant culture in a dose dependent manner. Both inhibitors decreased the size of newly synthesised proteoglycans and in the case of F-GlcNAc this was due to a decrease in newly synthesised GAG chain size. Rhodamine B, however, did not affect GAG chain size, while both inhibitors decreased the amount of chondroitin/dermatan and keratan sulphate GAG equally. The expression of genes responsible for the initiation and elongation of chondroitin/dermatan sulphate and keratan sulphate GAGs were downregulated in the presence of rhodamine B but not in the presence of F-GlcNAc. Thus the 2 inhibitors appear to have differing effects on GAG synthesis, with F-GlcNAc inhibiting the epimerisation of UDP-GlcNAc to UDP-GalNAc thus decreasing the availability of monosaccharides for addition to the growing GAG chain, whereas rhodamine B is more likely to reduce the number of GAG chains. Together with previous data these 2 inhibitors are capable of non-specific inhibition of GAG synthesis, reducing the production of chondroitin/dermatan sulphate, keratan sulphate and heparan sulphate GAGs. As such they would be applicable to therapy in a range of MPS disorders.