Cell lines

Bone marrow-derived macrophages

Preparation Method

Powder was dissolved in DMSO as 10 mM stock solutions and stored at -20!栵ntil use. BMM cells were plated at concentration of 160 x 103 cells/well in 24 wells osteo assay plates coated with bone biomimetic synthetic surface and cultured with rmM-CSF (30ng/ml) and rmRANKL (50ng/ml) for 3 days in absence or presence of different concentrations of HG-9-91-01.

Reaction Conditions

0, 0.1, 0.3, 0.5µM for 72 days

Applications

No significant toxicity was observed at the concentrations tested. Pre-treatment with 0.5 µM HG-9-91-01 significantly reduced the formation of resorption lacunae compared to RANKL alone.

Animal models

TNBS-induced colitis in female BALB/c mice, C57BL/6J mice

Preparation Method

The TNBS-challenged mice were treated with different doses of HG-9-91-01 (3, 10, 30 mg/kg/day intraperitoneally (i.p.), dissolved in 100 µL olive oil).

Dosage form

Intraperitoneal injection, 3, 10, 30 mg/kg/day for 8 days.

Applications

TNBS-treated mice given HG-9-91-01 at 3, 10, or 30 mg/kg rapidly recovered their lost body weight. The macroscopic inflammatory signs (gross bleeding, ulceration) and the shortening of colon length typically caused by TNBS were also improved by treatment with 3, 10, or 30 mg/kg HG-9-91-01

HG-9-91-01 is a salt-inducible kinases (SIK) inhibitor, inhibited SIK1, SIK2, SIK3 with IC50 values of 0.92nM, 6.5nM and 19.4nM, respectively^[1].

HG-9-91-01 potently inhibited the SIKs and did not inhibit any other member of the AMPK-related kinase subfamily. HG-9-91-01 increased LPS-stimulated IL-10 production and suppressed proinflammatory cytokine secretion^[2]. 0.5 µM HG-9-91-01 pretreated RAW264.7 for 30 min before RANKL stimulation resulted in reduction of multinucleated cell formation and of TRAP staining, and significantly reduced the mRNA of osteoclast differentiation markers in a concentration dependent manner. Pre-treatment with 0.5 µM HG-9-91-01 significantly reduced the formation of resorption lacunae compared to RANKL alone^[3].

HG-9-91-01 (3, 10, or 30 mg/kg) enhanced the expression of IL-10 whereas downregulated the levels of IL-12 and TNF-α in the colon tissue of the TNBS mice^[4].

References:
[1]. Sundberg T B, Choi H G, Song J H, et al. Small-molecule screening identifies inhibition of salt-inducible kinases as a therapeutic strategy to enhance immunoregulatory functions of dendritic cells[J]. Proceedings of the National Academy of Sciences, 2014, 111(34): 12468-12473.
[2]. Clark K, MacKenzie K F, Petkevicius K, et al. Phosphorylation of CRTC3 by the salt-inducible kinases controls the interconversion of classically activated and regulatory macrophages[J]. Proceedings of the National Academy of Sciences, 2012, 109(42): 16986-16991.
[3]. Lombardi M S, GilliÉron C, Berkelaar M, et al. Salt-inducible kinases (SIK) inhibition reduces RANKL-induced osteoclastogenesis[J]. PloS one, 2017, 12(10): e0185426.
[4]. Fu Y, Ma G, Zhang Y, et al. HG-9-91-01 Attenuates murine experimental colitis by promoting interleukin-10 production in colonic macrophages through the SIK/CRTC3 pathway[J]. Inflammatory Bowel Diseases, 2021, 27(11): 1821-1831.