Synthesis and Pharmacological Evaluation of σ2 Receptor Ligands Based on a 3-Alkoxyisoxazole Scaffold: Potential Antitumor Effects against Osteosarcoma.

Since its initial discovery as the basis for nicotinic acetylcholine receptor ligands, the 3-alkoxyisoxazole scaffold has been shown to be a versatile platform for the development of potent σ1 and σ2 receptor ligands. Herein we report a further SAR exploration of the 3-alkoxyisoxazole scaffold with the aim of obtaining potent σ2 receptor ligands. Various substitutions on the benzene ring and at the basic amino regions resulted in a total of 21 compounds that were tested for their binding affinities for the σ2 receptor. In particular, compound 51 [(2S)-1-(4-ammoniobutyl)-2-(((5-((3,4-dichlorophenoxy)methyl)isoxazol-3-yl)oxy)methyl)pyrrolidin-1-ium chloride] was identified as one of the most potent σ2 ligands within the series, with a Ki value of 7.9 nM. It demonstrated potent antiproliferative effects on both osteosarcoma cell lines 143B and MOS-J (IC50 values of 0.89 and 0.71 µM, respectively), relative to siramesine (IC50 values of 1.81 and 2.01 µM). Moreover, compound 51 inhibited clonal formation of osteosarcoma 143B cells at 1 µM, corresponding to half the dose required of siramesine for similar effects. The general cytotoxicity profile of compound 51 was assessed in a number of normal cell lines, including HaCaT, HAF, and LO2 cells. Furthermore, FACS analysis showed that compound 51 likely inhibits osteosarcoma cell growth by disruption of the cell cycle and promotion of apoptosis.

Regulation of body length and bone mass by Gpr126/Adgrg6.

Adhesion G protein-coupled receptor G6 (Adgrg6; also named GPR126) single-nucleotide polymorphisms are associated with human height in multiple populations. However, whether and how GPR126 regulates body height is unknown. In this study, we found that mouse body length was specifically decreased in Osx-Cre;Gpr126fl/fl mice. Deletion of Gpr126 in osteoblasts resulted in a remarkable delay in osteoblast differentiation and mineralization during embryonic bone formation. Postnatal bone formation, bone mass, and bone strength were also significantly affected in Gpr126 osteoblast deletion mice because of defects in osteoblast proliferation, differentiation, and ossification. Furthermore, type IV collagen functioned as an activating ligand of Gpr126 to regulate osteoblast differentiation and function by stimulating cAMP signaling. Moreover,the cAMP activator PTH(1-34), could partially restore the inhibition of osteoblast differentiation and the body length phenotype induced by Gpr126 deletion.Together, our results demonstrated that COLIV-Gpr126 regulated body length and bone mass through cAMP-CREB signaling pathway.

Loss of Lgr4 inhibits differentiation, migration and apoptosis, and promotes proliferation in bone mesenchymal stem cells.

The key signaling networks regulating bone marrow mesenchymal stem cells (BMSCs) are poorly defined. Lgr4, which belongs to the leucine-rich repeat-containing G protein-coupled receptor (LGR) family, is widely expressed in multiple tissues from early embryogenesis to adulthood. We investigated whether Lgr4 functions in BMSCs and in osteogenesis, adipogenesis, and skeletal myoblasts, using mice with a ß-geo gene trap inserted into the Lgr4 gene. Abundant Lgr4 expression was detected in skeletal, adipose and muscular tissue of Lgr4+/- mice at E16.5 by ß-gal staining, and Lgr4-deficiency promoted BMSC proliferation (16 ± 4 in wild-type [WT] and 28 ± 2 in Lgr4-/- ) using colony forming units-fibroblast assay, while suppressing BMSC migration (from 103 ± 18 in WT to 57 ± 10 in Lgr4-/- ) by transwell migration assay and apoptosis ratio (from 0.0720 ± 0.0123 to 0.0189 ± 0.0051) by annexin V staining assay. Deletion of Lgr4 decreased bone mass (BV/TV from 19.16 ± 2.14 in WT mice to 10.36 ± 1.96 in KO) and fat mass through inhibiting BMSC differentiation to osteoblasts or adipocytes. Furthermore, LGR4-regulated osteogenic, adipogenic, and myogenic gene expression. Importantly, our data showed that loss of Lgr4-inhibited fracture healing by suppressing osteoblast differentiation. Moreover, deletion of Lgr4 in BMSCs-delayed fracture healing following stem cell therapy by BMSC transplantation. Together, our results demonstrated that LGR4 is essential for mesoderm-derived tissue development and BMSC differentiation, demonstrating that LGR4 could be a promising drug target for related diseases and a critical protein for stem cell therapy.

A betulinic acid derivative SH479 inhibits collagen-induced arthritis by modulating T cell differentiation and cytokine balance.

The ideal therapeutic drug for rheumatoid arthritis (RA) should not only inhibit inflammation, but also prevent articular joint damage and particularly inhibit osteoclastogenesis. Betulinic acid (BA) is a natural pentacyclic triterpene that has displayed moderate anti-inflammatory and anti-osteoclastogenesis activities in various experimental systems, suggesting that BA or its derivatives could have an inhibitory effect on RA. In this study, we screened BA derivatives and found a heterocyclic ring-fused BA derivative, SH479, which had greater inhibitory effect than BA on Th17 differentiation. Moreover, we investigated the immune regulatory activity and potential therapeutic effects of SH479 in an experimental model of rheumatoid arthritis, the collagen-induced arthritis (CIA) mouse model. SH479 significantly inhibited Th1 and Th17 polarization, antigen-specific T cell proliferation and splenic lymphocyte-induced osteoclastogenesis. Furthermore, it diminished arthritis scores as well as bone destruction and cartilage depletion in the CIA mouse model. The protective effect of SH479 was accompanied by decreased levels of pro-inflammatory cytokines IL-17 and IFN-γ, together with enhanced anti-inflammatory cytokine expression including IL-10 and IL-4, as well as elevated CD4+ Foxp3+ cell number. At the molecular level, our results indicated that SH479 alleviated CIA through regulation of CD4+ T cell subtypes by JAK-STAT pathways. In conclusion, this study demonstrates that SH479 has therapeutic potential for rheumatoid arthritis through an anti-inflammatory effect by shifting a pathogenic Th17/Th1 response to a Th2/Treg phenotype, and also through an additional articular bone protection effect.