Osteoclasts, bone-resorptive multinucleated cells derived from hematopoietic stem cells, are associated with many bone-related diseases, such as osteoporosis. Osteoclast-targeting small-molecule inhibitors are valuable tools for studying osteoclast biology and for developing antiresorptive agents. Here, we have discovered that methyl-gerfelin (M-GFN), the methyl ester of the natural product gerfelin, suppresses osteoclastogenesis. By using M-GFNimmobilized beads, glyoxalase I (GLO1) was identified as an M-GFN-binding protein. GLO1 knockdown and treatment with an established GLO1 inhibitor in osteoclast progenitor cells interfered with osteoclast generation, suggesting that GLO1 activity is required for osteoclastogenesis. In cells, GLO1 plays a critical role in the detoxification of 2-oxoaldehydes, such as methylglyoxal. M-GFN inhibited the enzymatic activity of GLO1 in vitro and in situ. Furthermore, the cocrystal structure of the GLO1/M-GFN complex revealed the binding mode of M-GFN at the active site of GLO1. These results suggest that M-GFN targets GLO1, resulting in the inhibition of osteoclastogenesis.osteoclast ͉ small molecule ͉ crystal structure
Mature bone-resorbing osteoclasts (OCs) mediate excessive bone loss seen in several bone disorders, including osteoporosis. Here, we showed that reveromycin A (RM-A), a small natural product with three carboxylic groups in its structure, induced apoptosis specifically in OCs, but not in OC progenitors, nonfunctional osteoclasts, or osteoblasts. RM-A inhibited protein synthesis in OCs by selectively blocking enzymatic activity of isoleucyl-tRNA synthetase. The proapoptotic effect of RM-A was inhibited by neutralization or disruption of the acidic microenvironment, a prominent characteristic of OCs. RM-A was incorporated in OCs but not in nonfunctional osteoclasts and OC progenitors in neutral culture medium. Effects of RM-A on OC apoptosis increased under acidic culture conditions. RM-A not only was incorporated, but also induced apoptosis in OC progenitors in acidic culture medium. RM-A inhibited osteoclastic pit formation, decreased prelabeled 45 Ca release in organ cultures, and antagonized increased bone resorption in ovariectomized mice. These results suggested that preventive effects of RM-A on bone resorption in vitro and in vivo were caused by apoptosis through inhibition of isoleucyl-tRNA synthetase in OCs and that specific sensitivity of OCs to RM-A was due to the acidic microenvironment, which increased cell permeability of RM-A by suppressing dissociation of protons from carboxylic acid moieties, making them less polar. This unique mechanism suggested that RM-A might represent a type of therapeutic agent for treating bone disorders associated with increased bone loss.isoleucyl-tRNA synthetase ͉ inhibitor ͉ antiresorptive agent M ature osteoclasts resorb bone and mediate excessive bone loss seen in several bone disorders, including osteoporosis, arthritis, periodontitis, bone metastasis, and corticosteroidinduced bone loss (1-4). Mature bone-resorbing osteoclasts (OCs) exhibit highly specialized morphological structures, such as actin rings, clear zones, and ruffled borders, which are functional markers of OCs (2-5). These structures permit OCs to establish an isolated acidic microenvironment between themselves and the bone surface. The acidic microenvironment formed by V-ATPase on the ruffled borders of OCs allows dissolution of bone minerals and degradation of the bone matrix by proteases (6, 7). Therefore, these specialized features in OCs represent major potential targets for reducing OC activity and, consequently, could be useful for treatment of bone disorders. In addition, recent studies showed that receptor activator of NF-B ligand (RANKL), which is an essential factor for OC differentiation and activation, was one of the major targets of antiresorptive agents (2).Two types of antiresorptive drugs targeting OCs, i.e., bisphosphonates (BPPs) and calcitonin (CT), are used for clinical treatment of osteoporosis. BPPs, the most effective class of antiresorptive drugs available, inhibit bone resorption by suppressing functions of OCs and by shortening their lifespan (8 -10). Because of targeting of ...
Library on a glass slide: A variety of small molecules, including complex natural products, are introduced to glass slides using a photoaffinity reaction (see picture). The small molecules retain the inherent ability to interact with the relevant binding proteins. Importantly, this immobilization procedure does not require a specific functional group, and can be applied for use with a diverse small‐molecule library.
Identification of small-molecule ligands for a protein of interest can facilitate the analysis of the protein's functions in biological systems. Small-molecule microarrays have allowed for rapid detection of such ligand-protein interactions in a high-throughput manner, although a label on a protein is needed to observe these interactions. By combining SPR imaging technology with our recently developed photo-cross-linked small-molecule array platform, we developed a novel platform that allows in situ observation of interactions between photo-cross-linked small molecules on gold surfaces and nonlabeled proteins in solution. Interactions of estrogenic and androgenic substances with estrogen receptor alpha were observed using this platform.
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