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Identification of a binding site on soluble RANKL that can be targeted to inhibit soluble RANK-RANKL interactions and treat osteoporosis.

ABSTRACT: One of the major challenges for discovering protein-protein interaction inhibitors is identifying selective and druggable binding sites at the protein surface. Here, we report an approach to identify a small molecular binding site to selectively inhibit the interaction of soluble RANKL and RANK for designing anti-osteoporosis drugs without undesirable immunosuppressive effects. Through molecular dynamic simulations, we discovered a binding site that allows a small molecule to selectively interrupt soluble RANKL-RANK interaction and without interfering with the membrane RANKL-RANK interaction. We describe a highly potent inhibitor, S3-15, and demonstrate its specificity to inhibit the soluble RANKL-RANK interaction with in vitro and in vivo studies. S3-15 exhibits anti-osteoporotic effects without causing immunosuppression. Through in silico and in vitro experiments we further confirm the binding model of S3-15 and soluble RANKL. This work might inspire structure-based drug discovery for targeting protein-protein interactions.

SUBMITTER: Huang D

PROVIDER: S-EPMC9468151 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

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Identification of a binding site on soluble RANKL that can be targeted to inhibit soluble RANK-RANKL interactions and treat osteoporosis.

Huang Dane D Zhao Chao C Li Ruyue R Chen Bingyi B Zhang Yuting Y Sun Zhejun Z Wei Junkang J Zhou Huihao H Gu Qiong Q Xu Jun J

Nature communications 20220912 1

One of the major challenges for discovering protein-protein interaction inhibitors is identifying selective and druggable binding sites at the protein surface. Here, we report an approach to identify a small molecular binding site to selectively inhibit the interaction of soluble RANKL and RANK for designing anti-osteoporosis drugs without undesirable immunosuppressive effects. Through molecular dynamic simulations, we discovered a binding site that allows a small molecule to selectively interru ...[more]

PMID: 36097003

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Project description:Background and purposeOsteoporosis is characterized by excessive bone resorption due to enhanced osteoclast activation. Stimulation of nuclear factor of activated T cells 1 (NFATc1) and accumulation of reactive oxygen species (ROS) are important mechanisms underlying osteoclastogenesis. Robinin (Rob) is a flavonoid glycoside that has shown anti-inflammatory and antioxidative effects in previous studies, but little is known about its effects on bone homeostasis. The purpose of our research was to investigate whether Rob could prevent bone resorption in ovariectomized (OVX) mice by suppressing osteoclast production through its underlying mechanisms.MethodsThe docking pose of Rob and RANKL was identified by protein-ligand molecular docking. Rob was added to bone marrow macrophages (BMMs) stimulated by nuclear factor-κB (NF-κB) ligand (RANKL). The effects of Rob on osteoclastic activity were evaluated by positive tartrate resistant acid phosphatase (TRAcP) staining kit and hydroxyapatite resorption assay. RANKL-induced ROS generation in osteoclasts was detected by H2 DCFDA and MitoSox Red staining. The classic molecular cascades triggered by RANKL, such as NF-κB, ROS, calcium oscillations, and NFATc1-mediated signaling pathways, were investigated using Fluo4 staining, western blot, and quantitative real-time polymerase chain reaction. In addition, an OVX mouse model mimicking estrogen-deficient osteoporosis was created to evaluate the therapeutic effects of Rob in vivo.ResultsComputational docking results showed that Rob could bind specifically to RANKL's predicted binding sites. In vitro, Rob inhibited RANKL-mediated osteoclastogenesis dose-dependently without obvious cytotoxicity at low concentrations. We also found that Rob attenuated RANKL-induced mitochondrial ROS production or enhanced activities of ROS-scavenging enzymes, and ultimately reduced intracellular ROS levels. Rob abrogated the RANKL-induced mitogen-activated protein kinase (MAPK) and NF-κB signaling pathways, and subsequently blocked NFATc1 signaling and TRAcP expression. In addition, Rob inhibited osteoclast proliferation by downregulating the expression of osteoclast target genes (Acp5, Cathepsin K, Atp6v0d2, Nfact1, c-Fos, and Mmp9) and reducing Ca2+ oscillations. Our in vivo results showed that Rob reduced bone resorption in OVX animal model by repressing osteoclast activity and function.ConclusionsRob inhibits the activation of osteoclasts by targeting RANKL and is therefore a potential osteoporosis drug.

Dataset Information

Identification of a binding site on soluble RANKL that can be targeted to inhibit soluble RANK-RANKL interactions and treat osteoporosis.

Publications

Identification of a binding site on soluble RANKL that can be targeted to inhibit soluble RANK-RANKL interactions and treat osteoporosis.

Similar Datasets

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