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. Author manuscript; available in PMC: 2018 Apr 20.
Published in final edited form as: Bioorg Med Chem Lett. 2017 Oct 9;27(22):4975–4978. doi: 10.1016/j.bmcl.2017.10.017

Enantioselective inhibitory abilities of enantiomers of notoamides against RANKL-induced formation of multinuclear osteoclasts

Hikaru Kato a, Aika Kai a, Tetsuro Kawabata a, James D Sunderhaus b, Timothy J McAfoos b, Jennifer M Finefield b, Yukihiko Sugimoto c, Robert M Williams b,d, Sachiko Tsukamoto a,*
PMCID: PMC5909812  NIHMSID: NIHMS958118  PMID: 29037945

Abstract

The marine-derived Aspergillus protuberus MF297-2 and the terrestrial A. amoenus NRRL 35600 produce enantiomeric prenylated indole alkaloids. Investigation of biological activities of the natural and synthetic derivatives revealed that (−)-enantiomers of notoamides A and B, 6-epi-notoamide T, and stephacidin A inhibited receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)–induced osteoclastogenic differentiation of murine RAW264 cells more strongly than their respective (+)-enantiomers. Among them, (−)-6-epi-notoamide T was the most potent inhibitor with an IC50 value of 1.7 μM.

Keywords: Notoamide, Enantiomer, Osteoclastogenesis, Aspergillus, Fungus

Notoamides A–D were isolated from the fungus Aspergillus protuberus MF297-2,1 and successively fifteen new notoamides were obtained from the same strain.2–6 During the biosynthetic studies of notoamides, two antipodes, (−)-stephacidin A and (+)-notoamide B, were isolated from A. amoenus (formerly A. versicolor) NRRL 35600 along with (+)-versicolamide B, a diastereomer of (+)-notoamide B,7 and recently five antipodal congeners were isolated.8 We have been interested in the question if the antipodes showed enantiomerically-specific biological activities, and we tested notoamides of natural as well as synthetic origin in our in-house screening. We here report that the (−)-enantiomers of notoamides A and B, 6-epi-notoamide T,9 and stephacidin A inhibited receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)–induced osteoclastogenic differentiation of murine RAW264 cells more strongly than their respective (+)-enantiomers. Among them, (−)-6-epi-notoamide T was the most potent inhibitor with an IC50 value of 1.7 μM.

Osteoporosis is associated with the deregulation of osteoclast function, and therefore agents that affect osteoclastogenesis have been attracted much attentions.10,11 Bone homeostasis is maintained by the balance between bone formation by osteoblasts and bone resorption by osteoclasts.12 Stimulation of a monocyte/macrophage lineage by RANKL leads to its differentiation into multinuclear osteoclasts.12–14 RANKL stimulus activates downstream signaling pathways including the NF-κB and MAPK signaling pathways, which up-regulate the expression of osteoclast-specific genes, such as those encoding tartrate-resistant acid phosphatase (TRAP) and enzymes involved in cell fusion.

Notoamide derivatives, used in this study, were isolated from A. protuberus,1 A. amoenus,7 and A. taichungensis IBT 1940415 or synthesized9 (Figure 1). In order to test the inhibition by notoamides of RANKL-induced formation of multinuclear osteoclasts, the murine RAW264 cells were incubated with RANKL in the presence of samples at a concentration of 10 μg/mL.16 With respect to three pairs of natural enantiomers, notoamides A and B and stephacidin A, the (−)-enantiomers inhibited the osteoclastogenesis, detected by TRAP assay,16 more strongly than their respective (+)-enantiomers (Figure 2). Although synthetic (±)-notoamide T showed no inhibition at 10 μg/mL, whereas the C6-epimers, (±)-6-epi-notoamide T, completely inhibited at the same concentrations. We then separated the racemate, (±)-6-epi-notoamide T, into enantiomers by HPLC with a chiral-phase column17 and found that the separated (+)- and (−)-enantiomers showed inhibitory activities with IC50 values of 4.4 and 1.7 μM, respectively, also indicating enantio-specific inhibitory activity. Next, we examined the inhibitory effect of (−)-6-epi-notoamide T against the differentiation of RAW264 cells induced by treatment with RANKL, detected by tartrate-resistant acid phosphatase (TRAP) staining.16 In an experimental control, the cells differentiated into multinuclear osteoclasts by stimulus of RANKL, but (−)-6-epi-notoamide T at 5 μM inhibited the differentiation completely (Figure 3).

Figure 1.

Figure 1

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Structures of notoamide derivatives isolated from (a) A. protuberus, (b) A. amoenus, and (c) A. taichungensis, and (d) synthesized notoamides.

Figure 2.

Figure 2

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Inhibitory effects of notoamide congeners on RANKL-induced TRAP activity. RAW264 cells were treated with RANKL (250 ng/mL) in the presence or absence of notoamide derivatives (10 μg/mL) or quercetin (a positive control; 3.1 μg/mL) and allowed to differentiate for four days. TRAP activity was measured as absorbance at 405 nm. Quadruplicate experiments were carried out and the error bars represent the standard deviation.

Figure 3.

Figure 3

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Inhibitory effect of (−)-6-epi-notoamide T on RANKL-induced multinuclear osteoclastogenesis. RAW264 cells were allowed to differentiate by treatment with RANKL (250 ng/mL) in the presence or absence of (−)-6-epi-notoamide T (5 μM) for four days and were then stained with TRAP-staining solution. TRAP-positive cells stained red.

RANKL-induced osteoclastogenic differentiation is associated with up-regulation of specific genes. In order to examine if the inhibitory effect of (−)-6-epi-notoamide T relates to the expression of osteoclastogenic-specific genes, total RNA was prepared and analyzed by real-time RT-PCR.18 Although RANKL (50 ng/mL) induced the expression of Nfatc1 (Nuclear factor of activated T cells c1; NFATc1) in RAW264 cells as well as osteoclastgenesis-specific genes including Acp5 (tartrate-resistant acid phosphatase 5; TRAP), Ctsk (cathepsin K; CTSK), Atp6v0d2 (ATPase, H+ transporting, lysosomal V0 subunit D2), Dcstamp (dendrocyte expressed seven transmembrane protein; DC-STAMP), and Ocstamp (Osteoclast stimulatory transmembrane protein; OC-STAMP), (−)-6-epi-notoamide T (5 μM) suppressed the mRNA expression levels of these genes by 20–50% (Figure 4).

Figure 4.

Figure 4

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Inhibitory effect of (−)-6-epi-notoamide T on the expression levels of osteoclast marker genes. RAW264 cells were treated with RANKL (50 ng/mL) and (−)-6-epi-notoamide T (5 μM) for five days. Gene expression levels were evaluated by real-time RT-PCR and calculated from the data of duplicate experiments.

Regarding other enantiomeric compounds containing the bicyclo[2.2.2]diazaoctane ring system, (−)-versicolamides B and C have never been isolated from fungi and only a trace amount of (−)-6-epi-stephacidin A has been isolated from A. amoenus. Therefore, we could not compare their effects with (+)-enantiomers. Notoamides C and D, containing a dioxopiperazine ring, scarcely inhibited RANKL-induced osteoclastogenesis (see Figure 2).

To date, we have been studying the structures,1–8,15 syntheses,9,19–24 and biosyntheses6,9,23,25,26 of prenylated indole alkaloids from three fungi, A. protuberus, A. amoenus, and A. taichungensis. We have, for a long time, been interested in the subject if the enantiomers of notoamide derivatives showed the enantiomerically distinct biological activities. This is the first report describing the enantioselective biological activities of notoamide enantiomers. Interestingly, among the natural and synthetic notoamide congeners tested in this study, the (−)-enantiomers of notoamides A and B, 6-epi-notoamide T, and stephacidin A showed more potent inhibition of RANKL–induced osteoclastgenesis than their respective (+)-enantiomers. Among them, (−)-6-epi-notoamide T was the most potent inhibitor (IC50, 1.7 μM). Expression of osteoclast-specific genes, encoding TRAP and other enzymes involved in cell fusion, is up-regulated by the NF-κB and MAPK signaling pathways, which are activated by RANKL stimulus. Efforts to clarify the inhibitory mechanisms of notoamides are under investigation in our laboratory.

Acknowledgments

This work was financially supported in part by Grants-in-Aid for Scientific Research (No. 17H0399400 to ST) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Financial support from the National Institutes of Health (Grant CA 070375 to RMW) is gratefully acknowledged.

References and notes

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