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

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.

Diverse polyacetylenes from Atractylodes chinensis and their anti-osteoclastogenesis activity.

The phytochemical investigation on Atractylodes chinensis afforded 15 polyacetylenes 1-15 and one meroterpenoid 16. Of the 16 isolates, compounds 4 and 9 are new ones, and compounds 8 and 16 are first reported from nature. In addition, the relative configuration of 1 and the available NMR data of compounds 1, 8, and 16 were first provided. Their structures were elucidated by extensive analysis of MS, UV, IR, and NMR spectroscopic data. Besides, all isolated compounds were evaluated for their effects on RANKL-induced osteoclastogenesis in BMMs. Among them, polyacetylenes 12-14 showed potent inhibitory activity with IC50 values of 0.67 ± 0.05 µM, 0.72 ± 0.31 µM, and 2.40 ± 0.41 µM, respectively. The current work demonstrates the polyacetylenes are the main active constituents of A. chinensis against osteoclastogenesis.

Diverse Sesquiterpenoids and Polyacetylenes from Atractylodes lancea and Their Anti-Osteoclastogenesis Activity.

Twenty-two sesquiterpenoids (1-22) and 11 polyacetylenes (23-33) were obtained from the rhizomes of Atractylodes lancea. Among them, 11 compounds (1-5, 11, 12, 23, 24, 30, and 31) are new. The scaffolds represented by the isolates of sesquiterpenoids were found to be varied and included two rare rearranged spirovetivane sesquiterpenoids with a spiro [4,4] skeleton, eight spirovetivanes, three guaianes, eight eudesmanes, and one eremophilane. Their planar structures and relative configurations were elucidated by UV, IR, 1D and 2D NMR, and HRESIMS data analysis. The absolute configurations of the new sesquiterpenoids were determined using X-ray diffraction analysis and by comparison of the calculated and experimental electronic circular dichroism and optical rotation data, as well as chemical transformations. All the isolated compounds (1-33) were evaluated for their activity against RANKL-induced osteoclastogenesis in bone marrow macrophages. Two polyacetylene-type compounds, 25 and 32, showed potent activity with IC50 values of 1.3 and 0.64 µM, respectively. Rearranged spirovetivane sesquiterpenoids with a spiro [4,4] skeleton are reported herein from the genus Atractylodes for the first time. Polyacetylenes were demonstrated as the main active constituents of A. lancea with osteoclastogenesis inhibitory activity.

Chemical constituents of Ferula seravschanica.

Phytochemical investigation of Ferula seravschanica afforded seven new compounds, including three new bicyclic-type sesquiterpene coumarins (1-3), two new monocyclic-type sesquiterpene coumarins (16-17), two new phenylpropanoids (23-24) as well as twenty-two known compounds (4-15, 18-22, and 25-29). The structures of new compounds were determined by HRESIMS, NMR, ECD calculations, and X-ray single-crystal diffraction analysis. Furthermore, crude EtOAc extract and separated fractions (F1-F12) possessed cytotoxic activity against four human tumor cell lines (HT-29, DU145, HeLa, and Jurkat). Subsequently, we examined Jurkat inhibitory activity of isolated compounds. Compound 12 significantly inhibited the proliferation of the leukemia cells with IC50 value of 2.50 µM.

Diverse diterpenoids and sesquiterpenoids from Siegesbeckia pubescens and their activity against RANKL-induced osteoclastogenesis.

Phytochemical investigation of the aerial parts of Siegesbeckia pubescens led to seventeen diterpenoids (1-17) and twelve sesquiterpenoids (18-29). Their structures were varied including twelve ent-pimarane (1-12), three ent-kaurane (13-15), two acyclic diterpenoids (16-17), ten germacrene (18-27), one guaiane (28), and one caryolane (29) sesquiterpenoids. Eight of twenty-nine were new ones (1, 3, 4, 16-18, 23, and 28). Their structures were elucidated by extensive spectroscopic analysis. The absolute configurations of compounds 1 and 2 were identified using X-ray diffraction analysis, and of compounds 18, 23, and 28 were elucidated by the experimental and calculated electronic circular dichroism (ECD) spectra. All the isolated compounds (1-29) were assayed for their inhibition of RANKL-induced osteoclastogenesis in bone marrow macrophages (BMMs). Four sesquiterpenoids 18, 25, 26, and 27 exhibited potent inhibition of osteoclastogenesis with IC50 value of 0.51, 0.80, 0.50, and 0.83 µM, respectively. Here we demonstrated that S. pubescens may be a resource for discovery of anti-osteoporosis agents.

Discovery of diverse diterpenoid scaffolds from Euphorbia antiquorum and their activity against RANKL-induced osteoclastogenesis.

Seven new diterpenoids, euphorantones A-D (1, 3, 6, and 10), 8,12,13-epi-3,7,12-O-triacetyl-8-O-(2-methylbutanoyl)-ingol (9), 8,12,13-epi-3,12-O-diacetyl-7-O-benzoyl-8-methoxyingol (11), 2,3-epi-7,12-diacetate-8-benzoate-ingol (12), together with eighteen known compounds (2, 4-5, 7-8, and 13-25), were isolated from the aerial parts of Euphorbia antiquorum L.. The structures of new compounds 1, 3, 6, and 9-12 were elucidated by extensive spectroscopic analyses. The absolute configurations of new compounds were assigned using X-ray diffraction, Rh2(OCOCF3)4-induced CD spectrum, and confirmed through comparison of the calculated and experimental 13C NMR and electronic circular dichroism (ECD) data. Compounds 1-25 were evaluated for their inhibition of RANKL-induced osteoclastogenesis. Compound 1 showed the most potent inhibition of RANKL-induced osteoclastogenesis with IC50 value of 0.3 µM. It inhibited NFAT transcript activity and osteoclast related genes TRAcP, CTSK, and NFATc1 expression.

Cembrane-type diterpenoids from Macaranga pustulata.

Three new cembrane-type diterpenoids, deheiculatins M-O (1-3), together with five known analogues (4-8), were isolated from the twigs of Macaranga pustulata King ex Hook. The structures of new compounds 1-3 were elucidated by extensive spectroscopic analyses, modified Mosher's method, and the experimental and calculated electronic circular dichroism (ECD) experiments. All the isolates were evaluated for their cytotoxicity on three human cancer cell lines (CNE1, CNE2, and HCT 116), and all of them showed weak cytotoxicity (IC50 > 20 µM).