The Journal of Immunology

Vibsanin B Preferentially Targets HSP90b, Inhibits Interstitial Leukocyte Migration, and Ameliorates Experimental Autoimmune Encephalomyelitis

Bai-Xin Ye,*,1 Xu Deng,†,1 Li-Dong Shao,† Ying Lu,* Run Xiao,* Yi-Jie Liu,* Yi Jin,* Yin-Yin Xie,* Yan Zhao,* Liu-Fei Luo,* Shun Ma,‡ Ming Gao,x Lian-Ru Zhang,‡ Juan He,† Wei-Na Zhang,* Yi Chen,* Cheng-Feng Xia,† Min Deng,{ Ting-Xi Liu,*,{ Qin-Shi Zhao,† Sai-Juan Chen,* and Zhu Chen*

Interstitial leukocyte migration plays a critical role in inflammation and offers a therapeutic target for treating inflammation- associated diseases such as multiple sclerosis. Identifying small molecules to inhibit undesired leukocyte migration provides promise for the treatment of these disorders. In this study, we identified vibsanin B, a novel macrocyclic diterpenoid isolated from Viburnum odoratissimum Ker-Gawl, that inhibited zebrafish interstitial leukocyte migration using a transgenic zebrafish line (TG:zlyz– enhanced GFP). We found that vibsanin B preferentially binds to heat shock protein (HSP)90b. At the molecular level, inacti- vation of HSP90 can mimic vibsanin B’s effect of inhibiting interstitial leukocyte migration. Furthermore, we demonstrated that vibsanin B ameliorates experimental autoimmune encephalomyelitis in mice with pathological manifestation of decreased leuko- cyte infiltration into their CNS. In summary, vibsanin B is a novel lead compound that preferentially targets HSP90b and inhibits interstitial leukocyte migration, offering a promising drug lead for treating inflammation-associated diseases. The Journal of Immunology, 2015, 194: 4489–4497.

eukocyte trafficking is a multistep process consisting of moattractant signals, plays an important role in immune cell de- transendothelial and interstitial migration of leukocytes, velopment, immunosurveillance, and effector functions. Leukocyte L and it is required for efficient immune response. Interstitial migration is tightly regulated and its deregulation has been im- leukocyte migration, an amoeboid migration mode characterized plicated in several human diseases (1, 2). Mounting evidence in- by polarized cell morphology and redistribution of signaling dicates that inhibiting interstitial leukocyte migration is a prom- molecules under the guidance of soluble and tissue-bound che- ising strategy for treating inflammation-associated diseases (3). For example, inhibition of monocyte/macrophage infiltration offers a potential therapeutic strategy for the treatment of multiple scle- *State Key Laboratory of Medical Genomics and Shanghai Institute of Hematology, rosis (MS), an autoimmune inflammation-mediated demyelinating RuiJin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China; †State Key Laboratory of Phytochemistry and Plant Resources in disease partially caused by an abnormal monocyte/macrophage in- West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming filtration into the CNS (4). 650201, China; ‡State Key Laboratory of Cellular Stress Biology, Xiamen University, Xiamen 361102, China; xDepartment of Immunology, School of Basic Medicine, Zebrafish is a widely used vertebrate animal model for disease Tongji Medical College, Huazhong University of Science and Technology, Wuhan modeling and phenotype-driven chemical screening because of its { 430030, China; and Institute of Health Sciences, Shanghai Institutes for Biological small size, transparent body, and morphological or physiological Sciences, Chinese Academy of Sciences and Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China similarity to mammals. This whole animal model could potentially 1B.-X.Y. and X.D. contributed equally to this work. benefit several steps of the drug development processes, including drug screening, lead discovery, target identification, as well as Received for publication November 17, 2014. Accepted for publication February 24, 2015. functional assays (5–7). Recently, the zebrafish model has been This work was supported by Chinese National Key Basic Research Project 973 Grant used to analyze the dynamic course of leukocyte chemotactic 2013CB966800, Chinese Ministry of Health Grant 201202003, Mega-Projects of migration in vivo, which facilitates novel insights into certain Scientific Research for the 12th Five-Year Plan Grant 2013ZX09303302, and by State Key Laboratories Project of Excellence Grant 81123005. inflammatory diseases that involve leukocytes. Thus, zebrafish is an ideal model organism to study the biological behavior of leu- Address correspondence and reprint requests to Dr. Zhu Chen, Dr. Sai-Juan Chen, or Dr. Qin-Shi Zhao, RuiJin Hospital, Shanghai Jiao Tong University School of Med- kocytes through live imaging techniques (8–11). We previously icine, 197 Rui Jin Road II, Shanghai 200025, China (Z.C. and S.-J.C.) or Kunming constructed a transgenic zebrafish line TG:zlyz–enhanced GFP Institute of Botany, Chinese Academy of Sciences, 132 Lanhei Road, Heilongtan, Kunming 650201, China (Q.-S.Z.). E-mail addresses: [email protected] (Z.C.), (EGFP) for tracking leukocyte migration in response to acute in- + [email protected] (S.-J.C.), or [email protected] (Q.-S.Z.) jury in vivo (9). In TG:zlyz-EGFP embryos, EGFP cells mainly The online version of this article contains supplemental material. represented primitive macrophages before 36 h postfertilization Abbreviations used in this article: Biotin-ViB, biotin-tagged vibsanin B; dHL60, (hpf), whereas in the later stage after 48 hpf of embryos, the differentiated HL60; dpf, days postfertilization; EAE, experimental autoimmune en- EGFP+ cells contained monocytes/macrophages and neutrophils, cephalomyelitis; EGFP, enhanced GFP; hpf, hours postfertilization; HSP, heat shock protein; MS, multiple sclerosis; siRNA, small interfering RNA; TRAP, TNFR- both of which are called “leukocytes” and play an important role + associated protein; ViB–18-F, vibsanin B derivative with conversion of the C-18 in innate immunity (9, 12). Thus, we refer these EGFP cells in hydroxyl group to fluorine; ViB–4-OH, vibsanin B derivative whose C4 carbonyl the TG:zlyz-EGFP embryos (at 3 d postfertilization [dpf]) as group was reduced to a hydroxyl group. leukocytes and use this zebrafish model to identify drug leads that Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 inhibit interstitial leukocyte migration (12, 13). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402798 4490 VIBSANIN B INHIBITS LEUKOCYTE MIGRATION AND AMELIORATES EAE

Heat shock protein (HSP)90 is a highly conserved molecular presented as chemotaxis indexes referring to the fold increase in the total chaperone that regulates diverse physiological and pathological number of migrating cells in response to stimuli over the spontaneous cell processes. It dynamically distributes in almost every cellular com- migration to the control medium. partment: the cytoplasmic HSP90, which is the dominant form in the Pull-down assay and mass spectrum analysis cell and is comprised of a and b isoforms; the endoplasmic retic- As described in previous work (25), THP-1 cell lysates were incubated with ulum–localized HSP90 protein glucose-regulated protein 94; and biotin or biotin-tagged vibsanin B (Biotin-ViB) in the absence or presence the mitochondrial HSP90, which includes the TNF receptor- of vibsanin B at 4˚C, followed by incubation with streptavidin-agarose associated protein (TRAP) family member TRAP1 (14). Recently, beads (Sigma-Aldrich, catalog no. S1638). The agarose bead–bound pro- it was shown that HSP90 regulates the migration of endothelial cells teins were pulled down, separated by SDS-PAGE, and stained by Coo- or tumor cells by influencing cell membrane polarization, which is massie brilliant blue R250. The indicated band in the gel was excised, followed by in-gel digestion and analysis by mass spectrometry (ABI mainly mediated by the PI3K-AKT pathway (1, 15–17). Given the 4700; Applied Biosystems, Foster City, CA). similarity between tumor cells and leukocytes with respect to their amoeboid migration (18), HSP90 could also be involved in inter- Western blot analysis stitial leukocyte migration; thus, targeting HSP90 could ameliorate The proteins separated by SDS-PAGE electrophoresis were transferred to inflammation-associated diseases. As has been reported, HSP90 is ECL nitrocellulose membranes and incubated with the primary Ab over- considered to be a promising druggable target for treating inflam- night at 4˚C, followed by incubation of HRP-linked secondary Ab (Cell matory and neurodegenerative diseases (19, 20). Therefore, the Signaling Technology) for 1 h at room temperature. Detection was per- formed by an Immobilon Western chemiluminescent HRP substrate kit development of novel HSP90 inhibitors is necessary for the HSP90- (Millipore, catalog no. WBKLS0100) according to the manufacturer’s based therapy of inflammation-associated diseases, including MS. instructions (LAS 4000, Fujifilm). Signal intensity of protein was nor- Vibsane-type diterpenoids are a class of structurally diverse and malized against ubiquitously expressed proteins GAPDH or actin using rarely occurring natural products exclusively found in a few Vi- Quantity One (Bio-Rad, Hercules, CA) software. The primary Abs in this burnum species. However, the biological profile and the underlying study are specific for HSP90a, HSP90b, HSP70, His-tag, p-AKT, AKT, p-p38, p38, p-ERK, and ERK (Cell signaling Technology). Extraction of mechanism of action of these diterpenoids have not yet been de- whole zebrafish embryo protein was described in our previous work (9). termined (21). In this study, we applied a transgenic zebrafish line TG:zlyz-EGFP to identify that vibsanin B, a novel macrocyclic Preparation of recombinant proteins diterpenoid from Viburnum odoratissimum Ker-Gawl, inhibits in- Both pET-28a(+)-HSP90a and pET-28a(+)-HSP90b plasmid vectors terstitial leukocyte migration. An activity-based protein profiling were transformed into Escherichia coli strain BL21. Then, the recombinant strategy revealed that vibsanin B preferentially targets HSP90b. proteins His-HSP90a and His-HSP90b expressed in BL21 bacteria were Furthermore, we demonstrated that vibsanin B ameliorates experi- purified according to the protocol in our previous work (26). mental autoimmune encephalomyelitis (EAE) in mice with histo- RNA interference and transfection logical evidence of inhibited leukocyte infiltration into the CNS. The RNA oligonucleotides were synthesized in Shanghai GenePharma: small interfering RNA (siRNA)-HSP90a,59-AACCCUGACCAUUCC- Materials and Methods AUUAUU-39; siRNA-HSP90b,59-CAAGAAUGAUAAGGCAGUUAA- Plasmid construction 39; and siRNA-negative control: 59-UUCUCCGAACGUGUCACGU-39. RNA oligonucleotides (50 ng) were transfected into THP-1 cells according Human HSP90a gene coding sequence was amplified from the plasmid to the manufacturer’s instructions for Lipofectamine LTX with Plus re- vector pcDNA3.1-Flag-HSP90a (Prof. Yong-zhang Luo’s Laboratory, agent (Invitrogen). Approximately 48 h after transfection, THP-1 cells Beijing, China) by PCR with the following primers: forward, 59-CT- were collected, followed by Western blot analysis or in vitro cell migration AGCTAGCCCTGAGGAAACCCAGACCCAAGAC-39, reverse, 59-AC- assays. GCGTCGACCTAATCGACTTCTTCCAT-39.HumanHSP90b gene coding sequence was cloned from the plasmid vector pcDNA3.1-Flag- Real-time quantitative PCR with reverse transcription HSP90b in our laboratory (22) by PCR with the following primers: for- ward, 59-GGAATTCCATATGCCTGAGGAAGTGCACCAT-39, reverse, Total RNA was isolated by a TRIzol kit (Ambion). RNA was reverse transcribed using random hexamers and oligo (deoxythymidine) primers 59-ACGCGTCGACCTAATCGACTTCTTCCAT-39. Both PCR products according to the manufacturer’s instructions (Invitrogen). Real-time were subcloned into a pET28a(+) vector containing a His6 tag sequence at quantitative PCR reaction for HSP70 and b-actin were performed with the N terminus to generate vectors pET-28a(+)-HSP90a and pET-28a SYBR Green PCR master mixture reagents (Toyobo) on the ABI Prism (+)-HSP90b, respectively. 7900HT sequence detector (Applied Biosystems). The relative expression Zebrafish based phenotype-driven chemical screen and natural values were normalized against the internal control b-actin. The specific product library primers used for these real-time PCRs were: forward, 59-CGA- GAGGGTGTCAGCCAAGA-39, reverse, 59-AGCCACGAGATGACCTC- As described in our previous reports (9, 12), transgenic zebrafish embryo TTGAC-39. (TG:zlyz-EGFP) with tail transection was applied to evaluate the effect of natural products on zebrafish leukocyte migration. The natural product Zebrafish morpholino knockdown library was supplied by the Kunming Institute of Botany, Chinese Acad- Zebrafish hsp90a and hsp90b morpholino antisense oligonucleotide (Gene emy of Sciences. Especially, among the library, vibsanin B was isolated Tools, Philomath, OR) injection was reported in the previous work (9, 27). from air-dried and powdered aerial parts of V. odoratissimum Ker-Gawl. The sequences are shown as: MO-hsp90a,59-TCTTTGTTGAAT- Transwell assay TATTCGCTGTATT-39; MO-hsp90b,59-TCGTTGATTTTTGATGTTT- TAATCG-39; and MO-control,59-CCTCTTACCTCAGTTACAATTTATA- To obtain differentiated HL60 (dHL60) cells, HL60 cells were conditioned 39. in culture medium containing 1.3% DMSO for 5–6 d. THP-1 or dHL60 cell chemotaxic migration ability was measured using a Transwell assay in EAE induction and treatment a 96-well plate (ChemoTx system; Neuro Probe, Gaithersburg, MD, cat- Female C57BL/6 mice (6–8 wk of age) were obtained from the Shanghai alog no. 101-8) as described previously (23, 24). Before the Transwell Laboratory Animal Center, Chinese Academy of Sciences, Shanghai. The assay, THP-1 or dHL60 cells were pretreated with chemicals for 2–3 h. animals were housed in specific pathogen-free conditions. Experiments Then, THP-1 or dHL60 cells were incubated with chemicals in the upper were carried out according to the National Institutes of Health Guide for chamber of the ChemoTx system for 1 h with the stimulation of MCP-1 Care and Use of Laboratory Animals. The mice EAE model establishment, (Roche) or fMLF (Gene Operation) in the lower chamber, respectively. drug administration, and clinical score evaluation method were described Finally, the total number of cells were counted, including the ones fixed on in our previous work (28). Briefly, mouse (∼20 g) immunization was in- the filter and those that transversed into the lower chamber. These data are duced by mixing 300 mg of myelin oligodendrocyte glycoprotein 35–55 The Journal of Immunology 4491 peptide (MEVGWYRSPFSRVVHLYRNGK) with .95% (w/w) purity model (TG:zlyz-EGFP) (Fig. 1A, 1B) (9, 12). The transgenic (GL Biochem) in CFA containing 5 mg/ml Mycobacterium tuberculosis zebrafish embryos (3 dpf) were treated with 68 natural products, H37Ra (Difco Laboratories). Pertussis toxin (200 ng; List Biological leading to the identification of the hit compound vibsanin B Laboratories) in PBS was administered i.v. on days 0 and 2 post- immunization. The clinical severity of EAE was typically scored using (Fig. 1C), which showed a significant inhibitory effect on zebra- a grading scale of 0–5 according to Stromnes and Goverman (29). The fish leukocyte migration in a dose-dependent manner (Fig. 1D, natural product vibsanin B was dissolved in vehicle (2.5% DMSO, 2% 1E). Vibsanin B showed no effect on the overall number of leu- Tween 80, saline water) to the indicated concentration and then was ad- kocytes at zebrafish trunk and tail without tail transection ministrated into EAE-treated mice. (Supplemental Fig. 1A, 1B). Phosphorylation of histone H3 (PH3 Histopathology and quantification of inflammatory cells in Supplemental Fig. 1C, left) and TUNEL staining (Supplemental On days 15–18 postimmunization, mice spinal cords were obtained and Fig. 1C, right) assays also indicated that vibsanin B does not in- fixed with 4% (w/v) paraformaldehyde. Paraffin-embedded 3-mm trans- duce the proliferation or apoptosis of the EGFP-labeled leuko- verse sections were obtained from lumbar spinal cord, stained with Luxol cytes, suggesting that the decreased number of leukocytes that 3 fast blue or H&E, and then photographed by light microscopy ( 200). migrated to the injury site is attributed to the impaired leukocyte Inflammatory cells were quantified by cell count on an average of three spinal cord sections with H&E staining (five random 3400 photomicro- migration. graphs for each section) selected from each group in a blinded fashion. THP-1 cells (a human acute monocyte leukemia cell line) and Chemokine detection dHL60 cells (neutrophil-like cells derived from human promye- locytic leukemia-like cell line HL60) are simple model systems to Total proteins lysates were obtained from mice spinal cords and prepared study leukocyte migration in vitro (23, 24). To further validate the according to the previous report (30). The level of chemokines was detected by BioTNT (Shanghai, China) using a Quantibody mouse chemokine array effect of vibsanin B on human leukocyte migration in vitro, we (RayBiotech, catalog no. QAM-CHE-1) that was processed according to performed a Transwell assay to determine THP-1 or dHL60 cell the manufacturer’s instructions. chemotaxis upon vibsanin B treatment at indicated concentrations Statistical analysis without inducing cell apoptosis (Supplemental Fig. 1D, 1E) or proliferation (Supplemental Fig. 1F). The result showed that the A Student t test was used to analyze the differences between the groups in chemotaxis index for both of THP-1 or dHL60 cells was signifi- GraphPad software. A p value , 0.05 was considered statistically significant. cantly inhibited by vibsanin B in a dose-dependent manner (Fig. 1E, Supplemental Fig. 1G), suggesting that the inhibition of in- Results flammatory migration by vibsanin B is an intrinsic cellular Identification of vibsanin B that inhibits leukocyte migration property. via its Michael acceptors Vibsanin B contains a,b-unsaturated moieties, namely Michael To identify effective compounds that regulate interstitial leukocyte acceptors (25), which potentially react with positionally suited migration, we performed a chemical screening using a zebrafish nucleophiles, such as hydrosulfuryl groups and amino groups.

FIGURE 1. Identification of vibsanin B that inhibits interstitial leukocyte migration in vivo and in vitro. (A)TG:zlyz-EGPF embryos (at 3dpf) were subjected with tail transection for generating zebrafish leukocyte migration model. The red line means the incision of tail transection, and the square region means the injury site. (B) Chemical screening strategy. (C) Chemical structure of vibsanin B. (D and E)TG:zlyz-EGFP embryos (3 dpf) with tail transection were treated with 30 or 50 mM vibsanin B, respectively. Leukocytes that migrated to the highly inflamed regions [white boxes in (D)] at 6 h after tail transection were quantitatively analyzed (E). (F) Effects of ViB on the THP-1 cell chemotaxis index induced by 100 ng/ml MCP-1, as measured by Transwell assay. Cells in blank group were not treated with MCP-1 or chemicals. Data represent mean 6 SD. The p values were obtained from a two-tailed t test. 4492 VIBSANIN B INHIBITS LEUKOCYTE MIGRATION AND AMELIORATES EAE

This reaction could mediate the formation of adducts between further mechanistic study. We incubated Biotin-ViB or free biotin vibsanin B and its target protein. Consistently, ViB–4-OH, a vib- (Supplemental Fig. 2C) with THP-1 cell lysate. Subsequently, the sanin B derivative whose C4 carbonyl group was reduced to a mixture was precipitated and the complexes were purified using hydroxyl group (Supplemental Fig. 2A), lost its inhibitory effect on streptavidin-coated agarose beads followed by gel electrophoresis the chemotactic migration of zebrafish leukocytes (Supplemental and Coomassie blue staining (Fig. 2A). We found that a protein Fig. 2E) and THP-1 cells (Supplemental Fig. 2F). In contrast, the band with a molecular mass of ∼90 kDa was precipitated by vibsanin B derivative with conversion of the C-18 hydroxyl group to Biotin-ViB but not by free biotin. Notably, this band was competed fluorine (ViB–18-F; Supplemental Fig. 2B) did not disrupt its activity away in the presence of free vibsanin B at higher concentrations in zebrafish and the THP-1 cell model (Supplemental Fig. 2E, 2F). (Fig. 2A, lane 3), indicating that vibsanin B and Biotin-ViB bind to These results suggest that the Michael acceptor moieties are likely the the same protein at the same site. key pharmacophores of vibsanin B that are responsible for its Next, we excised the protein band of interest, and mass spectrum inhibitory effect on leukocyte migration. analysis indicated that the vibsanin B–bound protein was HSP90b (Fig. 2A, Supplemental Fig. 3A, 3B). Notably, HSP90a and other b Vibsanin B preferentially targets HSP90 proteins were not readily detected (Supplemental Fig. 3A). To To identify the direct targets of vibsanin B in leukocytes, we determine whether the binding of vibsanin B is isotype specific, performed a pull-down assay using Biotin-ViB (Supplemental Fig. we used HSP90b- or HSP90a-specific Abs to blot the precipitates 2D), followed by mass spectrometry analysis (25). Biotin-ViB bound by Biotin-ViB. The result indicated that both HSP90a or partially retained its inhibitory effect on leukocyte migration HSP90b precipitates can be detected (Fig. 2B). However, the af- (Supplemental Fig. 2G, 2H), but its mechanism of action requires finity of vibsanin B binding with HSP90b was much stronger than

FIGURE 2. Vibsanin B preferentially targets HSP90b.(A and B) THP-1 cell lysates were incubated with Biotin-ViB or biotin in the absence or presence of a 10-fold excess of unlabeled vibsanin B, followed by pull-down assay with streptavidin-agarose. The precipitates were resolved by SDS-PAGE. The gel was stained with Coomassie brilliant blue R250 (A) or subjected to Western blot analysis with HSP90a and b-specific Abs (B). The black arrowhead refers to the protein that binds to ViB. MW, molecular mass. (C) Recombinant His-HSP90b protein was incubated with Biotin-ViB, vibsanin B, ViB–4-OH, or ViB–18-F for 2 h and the mixtures were blotted with streptavidin-HRP or HSP90b Abs. (D) Recombinant His-HSP90a or His-HSP90b was incubated with Biotin-ViB for the indicated time course, and the mixture was analyzed by Western blot assay with streptavidin-HRP and His Ab. (E and F) Quantitative real-time PCR (E) and Western blot analysis (F) showed that vibsanin B and 17AAG induce HSP70 expression in THP-1 cells in a dose-dependent manner. The Journal of Immunology 4493 with HSP90a, and this interaction could also be blocked by leukocyte migration upon treatment with HSP90 inhibitors. As higher concentrations of free vibsanin B (Fig. 2B). Additionally, shown in Fig. 3A and 3B, zebrafish leukocyte inflammatory mi- the pull-down assay showed that vibsanin B does not bind the gration was impaired by 17AAG, which phenocopied the effect other HSP90 homologs, such as glucose-regulated protein 94 and of vibsanin B. Consistently, morpholino-mediated knockdown of TRAP1 (Supplemental Fig. 3C), supporting the hypothesis that zebrafish hsp90a or hsp90b resulted in the significantly decreased vibsanin B preferentially binds to HSP90b. migration of leukocytes to the site of injury (Fig. 3C, 3D). Fur- To investigate whether vibsanin B binds to HSP90b directly, we thermore, knockdown of HSP90a or HSP90b in the THP-1 cells performed an in vitro binding assay using purified recombinant by specific siRNAs (Fig. 3E, 3F), as well as 17AAG treatment His-HSP90b protein. It was revealed that Biotin-ViB efficiently (Fig. 3G), led to a decreased chemotaxis index. In addition to binds to recombinant His-HSP90b. Moreover, the binding THP-1 cells, 17AAG at indicated concentrations could also inhibit could be blocked by excessive vibsanin B and its active the dHL60 cell chemotaxis index significantly (Supplemental derivative ViB–18-F, but not by the inactive derivative ViB–4-OH Fig. 4F), without inducing THP-1 or dHL60 cell prolifer- (Fig. 2C). To further verify the preferential binding of vibsanin B to ation (Supplemental Fig. 4A) or apoptosis (Supplemental Fig. HSP90b instead of HSP90a, we incubated Biotin-ViB with re- 4B, 4C) during drug treatment. Thus, our findings suggested combinant His-HSP90a or His-HSP90b for different time that HSP90 is a potential endogenous target for inhibiting courses (Fig. 2D). The Western blot analysis indicated that Biotin- leukocyte migration. ViB preferentially binds to His-HSP90b in a time-dependent To further determine the effect of vibsanin B on leukocyte manner. migration upon HSP90 inactivation, we combined vibsanin B and Moreover, evidence has been accumulated to show that most 17AAG to treat THP-1 or dHL60 cells. The results showed no existing HSP90 inhibitors can induce HSP70 expression (31). obvious additive effect of two chemicals on cell chemotaxis Consistent with these data, vibsanin B upregulated HSP70 ex- (Supplemental Fig. 4D, 4E), suggesting that vibsanin B exerts no pression in THP-1 cells at the mRNA and protein levels, which additive effect on HSP90 inhibition by 17AAG, and thus HSP90b was less strong than the effect of 17AAG (Fig. 2E, 2F), suggesting might be a functional target of vibsanin B for impaired cell that vibsanin B can inhibit HSP90b activity. chemotaxis. However, in the zebrafish embryo, combination of 17AAG and vibsanin B could take an additive inhibitory effect on Inactivation of HSP90 abrogates leukocyte migration leukocyte migration (Supplemental Fig. 4F). This inconsistency HSP90 was reported to regulate tumor cell or endothelial cell suggested that, except in a direct manner at the cell level, vibsanin migration (15, 16). To explore whether HSP90 regulates leukocyte B might have additional mechanisms to influence leukocyte mi- migration, we performed an in vivo chemotaxis assay to evaluate gration at the whole animal level.

FIGURE 3. Inactivation of HSP90 abrogates leukocyte migration. (A–D) Zebrafish TG:zlyz-EGFP (3 dpf) leukocytes migrating to the injury site at 6 h after transection were quantitatively analyzed after treatment with 17AAG (50 mM) (A and B) or morpholino-control (Ctrl-MO), morpholino-hsp90a (hsp90a-MO, 0.4 mM), and morpholino-hsp90b (hsp90b-MO, 0.8 mM) injection at the one-cell stage egg (C and D). (E–G) Transwell assay showing MCP- 1–induced THP-1 chemotaxis index in the treatment of Si-NC (siRNA of negative control), Si-HSP90a (siRNA of HSP90a), or Si-HSP90b (siRNA of HSP90b)(E and F) and HSP90 inhibitor 17AAG (G). The p values are annotated as obtained from a two-tailed t test. 4494 VIBSANIN B INHIBITS LEUKOCYTE MIGRATION AND AMELIORATES EAE

Vibsanin B downregulates Akt-mediated pathways that are phorylated Akt was correlated with the extent of its inhibitory responsible for leukocyte migration effect on zebrafish leukocyte migration. Our findings indicated Next, we wanted to clarify which signaling pathways are re- that the impaired leukocyte recruitment is coupled with the vib- sponsible for vibsanin B’s inhibitory effect on leukocyte migra- sanin B–induced downregulation of phosphorylated Akt level in tion. Ample evidence has indicated that the PI3K-Akt pathway a dose- or time-dependent manner (Fig. 4D–F). Thus, the Akt- controls the directionality of leukocyte chemotactic migration (1). mediated pathway could be responsible for vibsanin B’s inhibitory In zebrafish embryos, the PI3Kg-Akt pathway promotes Rac- effect on leukocyte migration. mediated actin polymerization around the leading edge, which guides leukocyte migration in vivo (32). To determine which Vibsanin B ameliorates mice EAE by inhibiting leukocyte pathways are involved in vibsanin B’s inhibitory effect on leu- infiltration into the CNS kocyte migration, we analyzed the alterations of selected path- MS is an autoimmune inflammation-mediated demyelinating ways, including the PI3K-Akt pathway in THP-1 cells and disease in the CNS that is pathologically characterized by the zebrafish embryos upon vibsanin B treatment. Western blot anal- degeneration of the myelin sheath and axon loss in the white matter. ysis showed that the levels of phosphorylated Akt and p38 were EAE serves as an ideal animal model for MS and for evaluating the markedly downregulated. In contrast, other signaling components efficiency of anti-inflammatory drugs (28, 29). The HSP90 in- such as pERK showed no obvious change (Fig. 4A, 4B). Fur- hibitor 17AAG has been reported to ameliorate mice EAE (33). thermore, we determined that the PI3K inhibitor LY294002 and Additionally, previous work indicated that leukocyte infiltration the Akt inhibitor MK2206 could mimic vibsanin B’s effect, but into the CNS is involved in the progression of murine EAE. Thus, the p38 inhibitor SB202190 could not (Fig. 4C). These data we hypothesized that vibsanin B could be effective for MS suggested that the PI3K-Akt pathway, but not the p38-mediated treatment. We used the murine EAE model to evaluate the clinical pathway, could be responsible for the inhibitory effect of vibsanin implication of vibsanin B in MS. In the prophylactic treatment B on zebrafish leukocyte migration. To further validate this point, regimen, we treated mice daily, starting from the day of EAE we tested whether vibsanin B–mediated downregulation of phos- induction, with vibsanin B (25 mg/kg, n = 11) or vehicle (n = 11)

FIGURE 4. Downregulation of phosphorylated Akt level by vibsanin B associates with leukocyte migration inhibition. (A and B) Western blot analysis showing the effect of vibsanin B on selected signaling pathway components. THP-1 cells (A) in the induction of MCP-1 or zebrafish embryos (B) with tail transection were treated with vibsanin B at the indicated concentration. Then, the lysates of THP-1 cells or whole zebrafish embryo were subjected to Western blot analysis. (C) Zebrafish embryo (TG:zlyz-EGFP, 3 dpf) leukocytes migrating to the injury site at 6 hours after transaction were analyzed quantitatively upon the treatment of ViB (50 mM), Ly294002 (PI3Kg inhibitor, 60 mM), MK2206(AKT inhibitor, 100 mM), or SB202190 (p38 inhibitor, 100 mM). (D–F) TG:zlyz-EGFP embryos (3 dpf) with tail transection were treated with vibsanin B at the indicated concentration (0, 10, 30, 50 mM) for 6 h or for the indicated time (0, 2, 4, 6 h after tail transection [hptt]). The whole zebrafish embryo lysate was analyzed by Western blot assay to evaluate the level of phosphorylated Akt (D and E), and zebrafish leukocytes migrating to the injury site were analyzed quantitatively (F). The p values were obtained from a two-tailed t test. ***p , 0.0001. The Journal of Immunology 4495 as a control. As shown in Fig. 5A and 5B, vibsanin B adminis- possess extraordinary biological specificity and potency compared tration led to a significantly delayed disease onset (Fig. 5C) and with artificially designed molecules because of evolutionary se- decreased disease severity (Fig. 5D) compared with the vehicle lection (35), have promoted the development of most new drugs group. Histological analysis of spinal cord tissue sections indi- (36). They not only are fantastic chemical probes to fish novel cated that whereas the control mice had intact myelin sheaths insights into biological processes, but also serve as drug leads for without inflammatory foci (Fig. 5E, left), the EAE mice exhibited the development of therapeutic agents against a variety of diseases characteristic inflammation and demyelination (Fig. 5E, center). (25). Vibsanin B administration attenuated the CNS inflammation and Although zebrafish-based phenotypic screening of natural demyelination in EAE mice (Fig. 5E, right). Additionally, quan- products followed by target identification is challenging, it is ex- tification of inflammatory cells in spinal cord with H&E staining tremely informative because of the huge benefits at many steps in showed that the number of inflammatory cells was significantly the drug development process (5–7). In the present study, we decreased upon vibsanin B treatment (Fig. 5F), suggesting that applied a transgenic zebrafish (TG:zlyz-EGFP) to screen a natural vibsanin B could inhibit leukocyte infiltration into the CNS of product-based library, leading to the identification of a novel mice. Furthermore, we also found that chemokine CCL22 (a natural lead compound, vibsanin B, which is a vibsane-type macrophage-derived chemokine) in EAE mouse spinal cord, diterpenoid containing an 11-membered macrocyclic ring core which can induce macrophage or T cell infiltration into the CNS isolated from the leaves of V. odoratissimum Ker-Gawl (37). Al- and is involved in EAE progression (34), was significantly though vibsanin B has been shown to inhibit plant growth and the downregulated in the vibsanin B–treated mouse group (Fig. 5G), neurite outgrowth activity of nerve growth factor–mediated PC12 suggesting that vibsanin B might have additional effects other than cells (38, 39), its clinical implication and underlying mechanism direct impairment of leukocyte migration in the EAE model. in inflammation-associated diseases have not been documented. Our study disclosed that vibsanin B, together with its C-18 fluo- Discussion ride analog ViB–18-F, can inhibit leukocyte migration and ame- Hyperactive leukocyte response in tissues plays a critical role in liorate the treatment of murine EAE, implying a therapeutic the pathology of inflammation-associated diseases. The develop- potential of this class of compounds for the treatment of inflam- ment of effective therapeutic agents to control undesired leukocyte matory disease. migration is an effective strategy for the prevention and treatment Identifying the direct targets of natural products is critical for of inflammation-associated disease (2). Natural products, which understanding their inherent molecular mechanisms and acceler-

FIGURE 5. Vibsanin B ameliorates mice EAE via inhibiting leukocyte migration to the CNS. (A–D) Mice were injected i.p. daily with vehicle (d; n = 11) and vibsanin B (25 mg/kg, O; n = 11) on the day of EAE induction. The clinical score (A) and weight (B) of mice are represented. The average day of EAE onset (C) and cumulative clinical score (D) of each group are also shown. (E) Spinal cords from normal or EAE mice treated with vehicle or ViB were obtained at 15–18 d postimmunization and stained by H&E (top) or Luxol fast blue (LFB) (bottom). Original magnification 3200. (F) Quantification of inflammatory cells in spinal cord sections with H&E staining were analyzed by cell count under microscopy. (G) Chemokine CCL22 level in the spinal cord of mice EAE (n = 3) at peak disease in the treatment with vehicle (filled bar) or vibsanin B (open bar). The p values were obtained from a two-tailed t test. 4496 VIBSANIN B INHIBITS LEUKOCYTE MIGRATION AND AMELIORATES EAE ating the progress of drug discovery, but it is a rather challenging that vibsanin B might influence leukocyte migration through ex- process (25). In this study, we revealed that vibsanin B prefer- tracellular mechanisms. Thus, the detailed mechanism of vibsanin entially targets HSP90b and has a very weak binding with B in leukocyte migration needs further investigation. HSP90a through activity-based protein profiling method (40), Patients with MS usually have multiple disabling symptoms that indicating that inhibition of HSP90b, but not HSP90a, would be lead to a substantial social and economic burden on society. Be- mainly responsible for the inhibitory effect of vibsanin B on cause existing therapies are only partially effective for slowing leukocyte migration. Because of high conservation in sequence, disease progression and alleviating associated symptoms, MS the function of HSP90a and HSP90b is very similar (41), al- patients require more effective management (45, 46). Our results though previous work reported that HSP90a, but not HSP90b, indicated that vibsanin B could ameliorate mouse EAE severity exerts an essential extracellular role in cancer cell invasiveness and suppress leukocyte infiltration into the CNS of mice, implying (42). At the level of structural biology, the HSP90 monomer that vibsanin B might serve as a novel natural lead compound for contains three separate domains: the N-terminal domain, middle the development of therapeutic agents in treating inflammation- domain, and carboxyl-terminal domain, all of which bind different associated diseases, including MS. Additionally, we also showed molecules to form functional HSP90 chaperone machinery (41). that vibsanin B could exert an inhibitory effect on leukocyte mi- Among these domains, the HSP90 carboxyl-terminal domain has gration in mice, zebrafish, and the human cell model system, many differences in sequence between HSP90a and HSP90b (43). suggesting that the biological function of vibsanin B is evolu- Given that vibsanin B has much stronger affinity with HSP90b tionarily conserved. Thus, vibsanin B as a novel chemical struc- than with HSP90a, we expect vibsanin B might bind with some ture preferentially targeting HSP90b is a potential drug lead that special pockets in HSP90 carboxyl-terminal domain, which merits further development for the treatment of leukocyte migra- remained to be precisely determined. tion–mediated inflammatory diseases such as MS. In similarity with other HSP90 inhibitors such as 17AAG, vibsanin B induced HSP70 upregulation at the mRNA or protein Acknowledgments expression level, suggesting that vibsanin B might inhibit HSP90 We commemorate Dr. Ting-xi Liu, a young mentor who used to guide this function in cells. Our results showed that inactivation of HSP90 work and unfortunately died in 2011. His golden thought shines like a star in phenocopied the inhibitory effect of vibsanin B on leukocyte the sky. We thank Dr. Guang-biao Zhou and Dr. Yong-zhang Luo for the migration in THP-1 cells, dHL60 cells, and zebrafish embryos. plasmids pcDNA3.1-Flag-HSP90b and pcDNA3.1-Flag-HSP90a, respec- Moreover, vibsanin B could not exert obvious additive negative tively. We thank Dr. Rui-bao Ren, Xiao-jian Sun, and Xiao-wei Zhang for effects on THP-1 or dHL60 cell chemotaxis upon HSP90 inhibition revision in writing. We thank Dr. Fang Zheng, Huo-ying Chen, Hao Wu, by 17AAG, suggesting that HSP90 might be a functional target of Yan Sun, and Rui-hong Zhang for help with the EAE experiment. vibsanin B for inhibiting leukocyte migration in the cell model system. Mounting evidence indicated that the polarized PI3K-Akt Disclosures pathway components distributed around the leading edge can The authors have no financial conflicts of interest. regulate the directionality of migrating leukocytes (1). In our study, we showed that vibsanin B could downregulate phosphorylated References Akt/total Akt level. Additionally, the PI3Kg inhibitor LY294002 1. Friedl, P., and B. Weigelin. 2008. Interstitial leukocyte migration and immune or Akt inhibitor MK2206, which can downregulate the level of function. Nat. Immunol. 9: 960–969. Akt phosphorylation, phenocopies vibsanin B’s inhibitory effect 2. Nathan, C., and A. Ding. 2010. Nonresolving inflammation. Cell 140: 871–882. 3. Mackay, C. R. 2008. 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