Elsevier

Bioorganic & Medicinal Chemistry

Volume 23, Issue 21, 1 November 2015, Pages 7069-7078
Bioorganic & Medicinal Chemistry

5-Lipoxygenase inhibitors suppress RANKL-induced osteoclast formation via NFATc1 expression

https://doi.org/10.1016/j.bmc.2015.09.025Get rights and content

Abstract

5-Lipoxygenase synthesizes leukotrienes from arachidonic acid. We developed three novel 5-LO inhibitors having a benzoxazole scaffold as a potential anti-osteoclastogenics. They significantly suppressed RANKL-induced osteoclast formation in mouse bone marrow-derived macrophages. Furthermore, one compound, K7, inhibited the bone resorptive activity of osteoclasts. The anti-osteoclastogenic effect of K7 was mainly attributable to reduction in the expression of NFATc1, an essential transcription factor for osteoclast differentiation. K7 inhibited osteoclast formation via ERK and p38 MAPK, as well as NF-κB signaling pathways. K7 reduced lipopolysaccharide (LPS)-induced osteoclast formation in vivo, corroborating the in vitro data. Thus, K7 exerted an inhibitory effect on osteoclast formation in vitro and in vivo, properties that make it a potential candidate for the treatment of bone diseases associated with excessive bone resorption.

Introduction

Osteoclasts are multinucleated cells derived from the monocyte-macrophage lineage of hematopoietic precursors.1 Osteoclasts are bone-resorbing cells and increased numbers of osteoclasts can lead to the development of diseases characterized by bone loss, such as osteoporosis, rheumatoid arthritis, Paget’s disease, periodontal disease, osteosarcoma, and cancer bone metastasis.2, 3, 4, 5 Pharmaceutical inhibition of osteoclast differentiation is considered a promising therapeutic strategy for the prevention of bone loss-associated disorders and related fractures.

Osteoclast differentiation is primarily regulated by two cytokines: macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL). The binding of M-CSF to its receptor, M-CSFR on the surface of osteoclast precursor induces surface expression of RANK (a receptor of RANKL). The binding of RANKL to RANK triggers activation of signaling molecules, including mitogen-activated protein kinases (MAPKs) and NF-κB that then evoke downstream activation of c-Fos and nuclear factor of activated T cells c1 (NFATc1).6 c-Fos is an essential factor for induction of NFATc1, a master transcription factor of osteoclast-specific genes and therefore of osteoclast differentiation.7

5-Lipoxygenase (5-LO) is a crucial enzyme of the arachidonic acid (AA) cascade catalyzing the formation of bioactive leukotrienes (LTs). 5-LO catalyzes the conversion of AA to 5(S)-hydroperoxyeicosatetraenoic acid (5-HPETE) and leukotriene (LT)A4.8, 9 The intermediate LTA4 can be further converted to LTB4 by LTA4 hydrolase10 or to LTC4 by LTC4 synthase.11 5-LO-derived AA metabolites represent potent mediators of inflammatory reactions. In recent years, accumulating evidence indicates that the 5-LO pathway plays a role in the development of allergic diseases, such as asthma,12 and a variety of inflammatory disorders, such as rheumatoid arthritis.13 Furthermore, the 5-LO pathway also plays a key role in bone metabolism. Genetic loss of 5-LO activity alters bone morphology,14 and increases cortical bone thickness.15 Leukotrienes produced via 5-LO can stimulate osteoclast formation and activity, suggesting that the increased cortical thickness found in 5-LO knockout mice may be associated with reduced osteoclast number and activity.16, 17, 18 Thus 5-LO inhibitors are of therapeutic value for the treatment or prevention of bone related-diseases.

The 5-LO protein consists of a C-terminal catalytic domain and an N-terminal C2-like β-barrel domain.19 The catalytic domain contains a non-heme iron in the active site that acts as an electron acceptor or donor during catalysis.20 During enzyme activation, the iron is oxidized from the ferrous state (Fe2+) to the ferric (Fe3+) form, thus gaining entry in the catalytic cycle. The C2-like domain has a regulatory function. Most 5-LO inhibitors bind to the catalytic site of the enzyme and exhibit varying efficiencies of competitive inhibition.21 Zileuton, N-[1-(1-benzothien-2-yl)ethyl]-N-hydroxyurea, is the first and only approved 5-LO inhibitor to date. However, it only partially inhibits the synthesis of leukotrienes at clinical dose and the therapeutic index is limited by side-effects.22 Currently, there is no drug available that completely inhibits the actions of all the 5-LO-induced mediators. Therefore, there is a need for the development of new inhibitors with a pharmacological profile of high potency of enzyme inhibition.

Recently we reported the design and synthesis of a series of benzoxazoles and benzothiazoles as 5-LO inhibitors.23, 24 In our continuing effort, we synthesized the novel three compounds having a benzoxazole scaffold, investigated as a potential anti-osteoclastogenics using mouse bone marrow-derived macrophages (BMMs) in this study. Furthermore, we selected one compound K7, evaluated the pharmacological effect of K7 on osteoclast formation, in vitro and in vivo. Our findings suggested that K7 could be used as a new therapeutic agent against bone lytic diseases involving increased osteoclastogenesis.

Section snippets

Synthesis of (5-(4-(N′-hydroxycarbaimidoyl)-phenoxy)-N-(2-(4-substituted phenylamino)benzo[d]oxazole-5-yl)pentanamide (5ac) and their 5-LO inhibition

Chemical synthesis of benzoxazole derivatives is delineated in Scheme 1. The synthesis of starting compounds was reported in previous study.24 Briefly, 5-nitro-2-aminophenol was reacted with ethylphenyl isothiocyanate to give the corresponding thiourea, then benzoxazoles (1ac) were prepared by KO2 catalyzed ring closure. The nitro compounds 1ac were reduced to amino compounds 2ac by 5% Pd/C and hydrogen. Coupling of amino group and 5-bromovaleric acid by PyBOP resulted in the amides 3ac.

Discussion

5-LO converts AA into LTA4, which is used to synthesize LTB4 and the cysteinyl leukotrienes.39, 40 These metabolites of 5-LO are lipid signaling molecules that affect many physiological processes, including inflammation and allergy.41, 14 Recently 5-LO was suggested to be a direct regulator of bone metabolism. Furthermore, we have previously provided mechanistic insight by demonstrating that pharmacological blockade of 5-LO inhibits RANKL-induced osteoclastogenesis and bone loss.42 Thus 5-LO

Materials

Antibodies against ERK, phospho-ERK, IκB, β-actin, phospho-p38, p38, c-Fos and NFATc1 were purchased from Cell Signaling Technology (Beverly, MA, USA). All other reagents were from Sigma–Aldrich (St. Louis, MO, USA). ICR mice were obtained from Samtako Inc. (Seoul, Korea). Mice were maintained in the animal facility of the Sookmyung Women’s University on a 12:12-h light–dark cycle, and were allowed food and water ad libitum. All experiments were performed in accordance with institutional

Acknowledgments

This research was supported by Basic Science Research program through the National Research Foundation of Korea (NRF) – South Korea funded by the Ministry of Education (NRF-2013R1A1A2062631) (to M. Yim), and by Grant from National Leading Research Lab (NLRL) program (2011-0028885) funded by the Ministry of Science, ICT and Future Planning (MSIP) and the National Research Foundation of Korea (NRF) – South Korea (to S. Choi).

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