Abstract
A novel aptamer-based competitive drug screening platform for osteoporosis was devised in which fluorescence-labeled, sclerostin-specific aptamers compete with compounds from selected chemical libraries for the binding of immobilized recombinant human sclerostin to achieve high-throughput screening for potential small-molecule sclerostin inhibitors and to facilitate drug repurposing and drug discovery. Of the 96 selected inhibitors and FDA-approved drugs, six were shown to result in a significant decrease in the fluorescence intensity of the aptamer, suggesting a higher affinity toward sclerostin compared with that of the aptamer. The targets of these potential sclerostin inhibitors were correlated to lipid or bone metabolism, and several of the compounds have already been shown to be potential osteogenic activators, indicating that the aptamer-based competitive drug screening assay offered a potentially reliable strategy for the discovery of target-specific new drugs. The six potential sclerostin inhibitors suppressed the level of both intracellular and/or extracellular sclerostin in mouse osteocyte IDG-SW3 and increased alkaline phosphatase activity in IDG-SW3 cells, human bone marrow-derived mesenchymal stem cells and human fetal osteoblasts hFOB1.19. Potential small-molecule drug candidates obtained in this study are expected to provide new therapeutics for osteoporosis as well as insights into the structure–activity relationship of sclerostin inhibitors for rational drug design.
Highlights
Osteoporosis is considered a major and global public health concern
We investigated whether fluorescence quenching was responsible for the reduction of fluorescence observed in Figure 3a,b.The aptamer-based competitive drug screening assay was performed as described previously but without the immobilized sclerostin
The results from this study indicate that the fluorescence of FAMlabeled sclerostin-specific aptamer and FITC-conjugated anti-sclerostin antibody was not affected in the presence of icariin, but the small-molecule inhibitor was reported to promote the osteogenic differentiation of rat BMSCs through the regulation of sclerostin [35]
Summary
Osteoporosis is considered a major and global public health concern. Because osteoporosis occurs primarily as a result of normal ageing and menopausal estrogen deficiency, the prevalence of osteoporotic fractures is expected to rise as the proportion of older populations increases worldwide [1]. Sclerostin was first recognized in patients with sclerosteosis, in which inactivating mutations of the sclerostin gene SOST were found to be associated with high bone mass [7] It is responsible for the inhibition of osteoblast differentiation as well as stimulation of RANKL from osteocytes to induce osteoclastogensis [8,9,10] and promotes the apoptosis of human mesenchymal stem cells [11]. Animal studies confirmed that sclerostin knockout mice displayed a high bone mass phenotype with increased bone formation and bone mineral density [12], while overexpression of sclerostin in mice resulted in osteopenia [13]. Romosozumab, a humanized anti-sclerostin monoclonal antibody designed for subcutaneous administration, was approved for the treatment of severe osteoporosis and postmenopausal women at high risk for osteoporotic fracture [14,15]. Romosozumab was demonstrated in clinical studies as a promising therapeutic for osteoporosis, increasing number of severe cardiovascular adverse events posed a potential risk to patients with cardiovascular diseases, and the safety and side effects of long-term treatment are unclear [19]
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