In vitro anti-osteoclastogenic activity of p38 inhibitor doramapimod via inhibiting migration of pre-osteoclasts and NFATc1 activity

Abstract

The mitogen activated protein kinase p38 plays a role in the receptor activator of NF-ĸB ligand (RANKL)-induced osteoclast differentiation. In this study, we investigated the effect of p38 inhibitor doramapimod on the osteoclast differentiation. Doramapimod significantly inhibited the osteoclastogenesis of bone marrow macrophages (BMMs) via attenuating the activation of p38 induced by M-CSF and RANKL. Importantly, doramapimod blocked the migration and fusion in pre-osteoclasts via the down-regulating NFATc1. The inhibitory effect of doramapimod on the migration/fusion of pre-osteoclasts via inhibiting NFATc1 activity were confirmed by measuring NFATc1 luciferase activity and evaluating the mRNA expression of NFATc1-responsive genes related to the osteoclastic migration/fusion. These results suggested anti-osteoclastogenic activity of doramapimod via inhibiting migration/fusion of pre-osteoclasts and NFATc1 activity.

A High-Throughput Screening-Compatible Strategy for the Identification of Inositol Pyrophosphate Kinase Inhibitors

“…The 16-fold lower hit rate for the same library screened against PPIP5K may, in part, reflect its lack of homology with protein kinases (see the Introduction ). On the other hand, among the 15 initial hits for PPIP5K, we are only aware of one (UNC10225354) that has previously been reported to inhibit any other kinase (p38 MAPK [ 44 ]). That information suggests the 5K library may have yielded leads with some specificity for an inositol phosphate kinase.…”

“…The data obtained from UNC10225354 ( S5 Fig ) could not be fitted to a thermogram with acceptable curvature for the nonlinear regression analysis required to obtain an accurate value for Ki [ 48 ], potentially because of the compound’s poor solubility in aqueous buffer. Moreover, UNC10225354 (also known as Doramapimod), is a potent inhibitor of p38 MAPK [ 44 ], whereas UNC10225498 and UNC10112646 are not known to interact with any other cellular targets. Thus, the interactions of the latter two inhibitors with PPIP5K were characterized in more detail.…”

Antibacterial, anti-inflammatory, and anti-osteoclastogenic activities of <i>Colocasia antiquorum</i> var. <i>esculenta</i>: Potential applications in preventing and treating periodontal diseases

“…TRAP is considered an established cytochemical marker for recognizing polynucleated and mononucleated osteoclasts 32) . The study of TRAP + cell formation and activity is a well-known method of determining osteoclast formation and function 24,33) . The differentiation of osteoclasts by mediating inflammatory cytokines is directly promoted and activated by LPS itself, which can ultimately lead to bone resorption 7,25,34) .…”

“…Therefore, for the complete formation of TRAP + -MNCs, BMMs (1×10 4 cells/well in a 96-well plate or 3×10 5 cells/well in a 6-well plate) were cultured with M-CSF, RANKL, and 0, 3, 10, and 30 μg/mL CA for 4 days. For TRAP staining and activity assay, mature osteoclasts were visualized by staining for TRAP, a biomarker of osteoclast differentiation, as described previously 24) . Briefly, BMM-derived MNCs were fixed with 3.7% formaldehyde for 5 min, permeabilized with 0.1% Triton X-100 for 5 min, and stained with the Leukocyte Acid Phosphatase Kit 387-A (Sigma-Aldrich).…”

“…Briefly, BMM-derived MNCs were fixed with 3.7% formaldehyde for 5 min, permeabilized with 0.1% Triton X-100 for 5 min, and stained with the Leukocyte Acid Phosphatase Kit 387-A (Sigma-Aldrich). To measure TRAP activity, the permeabilized cells were incubated with the TRAP buffer (100 mM sodium citrate [pH 5.0] and 50 mM sodium tartrate) including 3 mM p-nitrophenyl phosphate (Sigma-Aldrich) at 37°C for 5 min, as described previously 24) . The reaction mixtures were transferred onto a new plate containing an equal volume of 0.1 N sodium hydroxide, and optical density values were determined at 405 nm by using a Wallac EnVision microplate reader (PerkinElmer, Finland).…”