Inhibitory Effects of 2N1HIA (2-(3-(2-Fluoro-4-Methoxyphenyl)-6-Oxo-1(6H)-Pyridazinyl)-N-1H-Indol-5-Ylacetamide) on Osteoclast Differentiation via Suppressing Cathepsin K Expression

Abstract

Osteoclasts are large multinucleated cells which are induced by the regulation of the receptor activator of nuclear factor kappa-Β ligand (RANKL), which is important in bone resorption. Excessive osteoclast differentiation can cause pathologic bone loss and destruction. Numerous studies have targeted molecules inhibiting RANKL signaling or bone resorption activity. In this study, 11 compounds from commercial libraries were examined for their effect on RANKL-induced osteoclast differentiation. Of these compounds, only 2-(3-(2-fluoro-4-methoxyphenyl)-6-oxo-1(6H)-pyridazinyl)-N-1H-indol-5-ylacetamide (2N1HIA) caused a significant decrease in multinucleated tartrate-resistant acid phosphatase (TRAP)-positive cell formation in a dose-dependent manner, without inducing cytotoxicity. The 2N1HIA compound neither affected the expression of osteoclast-specific gene markers such as TRAF6, NFATc1, RANK, OC-STAMP, and DC-STAMP, nor the RANKL signaling pathways, including p38, ERK, JNK, and NF-κB. However, 2N1HIA exhibited a significant impact on the expression levels of CD47 and cathepsin K, the early fusion marker and critical protease for bone resorption, respectively. The activity of matrix metalloprotease-9 (MMP-9) decreased due to 2N1HIA treatment. Accordingly, bone resorption activity and actin ring formation decreased in the presence of 2N1HIA. Taken together, 2N1HIA acts as an inhibitor of osteoclast differentiation by attenuating bone resorption activity and may serve as a potential candidate in preventing and/or treating osteoporosis, or other bone diseases associated with excessive bone resorption.

PSTP-3,5-Me Inhibits Osteoclast Differentiation and Bone Resorption

“…Bone marrow macrophage cells (BMMs) were isolated from femurs and tibias of 8- to 10-week-old C57BL/6J females, as described previously [45]. Briefly, the cells were cultured in alpha minimal essential medium (a-MEM, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS containing 30 ng/mL M-CSF (PeproTech, NJ, USA) for three days.…”

“…Primary mouse osteoblasts were isolated from the calvaria of three-day-old C57BL/6J mice [45]. The cells were cultured in α-MEM with 10% FBS and differentiated into osteoblast by addition of 100 ng/mL BMP-2 (Sino Biological, Beijing, China).…”

“…Bone resorption activity was determined using bone resorption assay kit (CosMo Bio, Tokyo, Japan), as described previously [45]. Briefly, BMMs were cultured on a calcium phosphate-coated 48-well plate (2.5 × 10 4 cells/well) in the presence of M-CSF (30 ng/mL) and RANKL (100 ng/mL) with different concentrations of PSTP-3,5-Me (0, 3, and 10 µM) for six days.…”

N-[2-(4-Acetyl-1-Piperazinyl)Phenyl]-2-(3-Methylphenoxy)Acetamide (NAPMA) Inhibits Osteoclast Differentiation and Protects against Ovariectomy-Induced Osteoporosis

“…In recent years, we reported several compounds that target osteoclast inhibition [ 22 , 23 , 24 ]. In the present study, we selected a new chemical with a structure similar to that of previously reported chemicals that exhibit strong inhibitory effects on osteoclast differentiation in vitro, as well as an effect in vivo.…”

A novel regulatory role of TRAPPC9 in L‐plastin‐mediated osteoclast actin ring formation

“…OC expresse tartrate-resistant acid phosphatase (TRAP), calcitonin receptors, 1,2 and cathepsin-K. 3 OC differentiate from monocyte/macrophage precursors in response to two hematopoietic factors, the receptor activator of nuclear factor-κB ligand (RANKL) and macrophage colony-stimulating factor-1 (MCSF1). The main function of OC is to resorb bone.…”