WP1066, a small molecule inhibitor of the JAK/STAT3 pathway, inhibits ceramide glucosyltransferase activity

https://doi.org/10.1016/j.bbrc.2017.07.115Get rights and content

Highlights

  • WP1066 is a novel inhibitor of ceramide glucosyltransferase.

  • Modes of inhibition of ceramide glucosyltransferase by WP1066 were uncompetitive.

  • WP1066 increases intracellular ceramide levels in B16 melanoma cells.

  • WP1066 may induce cell death by the ceramide accumulation.

Abstract

WP1066 is a well-known inhibitor of the JAK/STAT3 signaling pathway. By a screen of known small molecule inhibitors of various enzymes and protein factors, we identified WP1066 as a ceramide glucosyltransferase inhibitor. Ceramide glucosyltransferase catalyzes the first glycosylation step during glycosphingolipid synthesis. We found that WP1066 inhibited the activity of ceramide glucosyltransferase with an IC50 of 7.2 μM, and that its action was independent of JAK/STAT3 pathway blockade. Moreover, the modes of inhibition of ceramide glucosyltransferase were uncompetitive with respect to both C6-NBD-cermide and UDP-glucose.

Introduction

Ceramide glucosyltransferase (GlcT-1. EC 2.4.1.80) catalyzes the transfer of glucose from UDP-glucose to ceramide (Cer), generating the simplest glycosphingolipid (GSL), namely glucosylceramide (GlcCer) [1], [2]. GlcCer represents the core structure of >300 GSL species that play important roles in various physiological processes, such as cellular differentiation, adhesion, and proliferation [2]. In addition, the lipid substrate of GlcT-1, namely Cer, serves as a second messenger of various cellular processes, including cell death [3].

GlcT-1 regulates levels of Cer and total GSL in cells [2], [4]. Recent studies have demonstrated that increased GSL levels are associated with insulin resistance [5]. Moreover, enhanced GlcT-1 activity in cancer cells is a known cause of multi-drug resistance, as GlcT-1 metabolizes and detoxifies Cer. Inhibition of GlcT-1 has been shown to revert the sensitivities of cancer cells to anti-cancer drugs [6].

Gaucher's disease is a disorder caused by a deficiency of glucocerebrosidase, an enzyme that degrades GlcCer in lysosomes. Patients with Gaucher's disease accumulate excess GlcCer in cells, and develop symptoms such as spleen and liver enlargement, anemia, bone pain, and fatigue [7]. In addition, various neurological symptoms are present in certain types of Gaucher's disease (type 2 and type 3) [7].

Thus, GlcT-1 inhibitors represent a class of drug candidates for the treatment of Gaucher's disease, as they can reduce GlcCer content by inhibiting its synthesis [7].

As noted above, GlcT-1 inhibitors exhibit the potential for therapeutic use in a number of diseases, and many GlcT-1 inhibitors have been developed to date. Among them, the Cer analog PDMP [8] and an imino sugar inhibitor, N-butyl deoxynojirimycin (NB-DNJ, Miglustat) [9], are extensively used in basic studies of both GSL and GlcT-1 function. Two small molecule GlcT-1 inhibitors, Miglustat and the Cer analog Eliglustat, are now available for the treatment of Gaucher's disease without neurological symptoms (type 1) [7], [9].

Signal transducer and activator of transcription 3 (STAT3) is a protein that regulates various cellular processes, such as proliferation and survival. STAT3 is phosphorylated by the action of Janus kinases (JAKs) in response to cytokines or growth hormones. Phosphorylated STAT3 in turn dimerizes and is subsequently transferred from the cytosol to the nucleus, where it acts as a transcription factor to regulate various genes [10]. The JAK/STAT3 pathway is activated by various cancer types, including glioma, and blockade of the pathway induces cell death in cancer cells. Thus, the JAK/STAT3 pathway is a target for cancer therapy [11]. To date, many small molecule compounds targeting JAKs have been developed. In particular, WP1066 is an analog derived from the JAK2 inhibitor AG490 [12]. WP1066 induces degradation of JAK2 and inhibition of its phosphorylation [13]. WP1066 has been shown to exhibit more potent anti-cancer activity than AG490 toward various cancer types, including malignant glioma [12]. A Phase I trial investigating WP1066 is ongoing.

Herein, we report that WP1066, but not AG490, exhibits the ability to inhibit GlcT-1 activity. WP1066 inhibition of GlcT-1 activity is shown to be independent of JAK/STAT3 pathway blockade.

Section snippets

Materials

(S, E)-3-(6-bromopyridin-2-yl)-2-cyano-N-(1-phenylethyl)acrylamide (WP1066) and N-(1ʹ,2-dihydroxy-1,2ʹ-binaphthalen-4ʹ-yl)-4-methoxybenzenesulfonamide (STAT3 inhibitor XIII) were obtained from Merck (Darmstadt, Germany). 2-cyano-3-(3,4-dihydroxyphenyl)-N-(phenylmethyl)-2-propenamide (AG490 or Tyrphostin) was obtained from CosmoBio Co., Ltd, (Tokyo, Japan). 6-{((N-7-nitrobenz-2-oxa-1,3-diazol-4yl)amino)caproyl}sphingosine (C6-NBD-Cer) was purchased from Invitrogen (CA, USA). The SCADS Inhibitor

Inhibitory effect of WP1066 against GlcT-1

To search for GlcT-1 inhibitors, we screened a library of 325 bioactive small molecules with known activities. WP1066, a well-known inhibitor of the JAK/STAT3 pathway [11], [12], [13], was one of the compounds identified as exhibiting inhibitory activity against GlcT-1. WP1066 blocks the JAK/STAT3 pathway by inhibiting JAK2 [12]. As shown in Fig. 1A (a & b), WP1066 inhibited mouse GlcT-1 activity in a dose-dependent manner, with an IC50 of 7.2 μM. In addition to WP1066, we also evaluated the

Discussion

In the present study, we identified WP1066 as a GlcT-1 inhibitor with a novel structure. WP1066 degraded JAK2 and inhibited its phosphorylation at concentrations of 3 μM [12], while inhibiting intracellular GlcT-1 activity at a concentration of 10 μM (Fig. 3A). Although WP1066 is more effective at inhibiting JAK2 than GlcT1, the effective concentrations toward each target are not very different. Thus, special care needs to be taken when investigating the JAK/STAT3 pathway using WP1066. However,

Conflict of interest

None.

Acknowledgements

We wish to thank the Screening Committee of Anticancer Drugs for providing the SCADS Inhibitor Kit. The Screening Committee of Anticancer Drugs is supported by a Grant-in-Aid for Scientific Research on Innovative Areas, Scientific Support Programs for Cancer Research, from The Ministry of Education, Culture, Sports, Science and Technology, Japan.

This study was supported by a grant from the Strategic Research Foundation Grant-aided Project for Private Universities from The Ministry of Education,

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