A Small Molecule Binding HMGB1 and HMGB2 Inhibits Microglia-Mediated Neuroinflammation

article published online: 12 October 2014 | doi: 10.1038/nchembio.1669

A small molecule binding HMGB1 and HMGB2 inhibits microglia-mediated neuroinflammation

Sanghee Lee1, Youngpyo Nam2, Ja Young Koo1, Donghyun Lim3, Jongmin Park1, Jiyeon Ock2, Jaehong Kim2, Kyoungho Suk2* & Seung Bum Park1,3*

Because of the critical role of neuroinflammation in various neurological diseases, there are continuous efforts to identify new therapeutic targets as well as new therapeutic agents to treat neuroinflammatory diseases. Here we report the discovery of inflachromene (ICM), a microglial inhibitor with anti-inflammatory effects. Using the convergent strategy of phenotypic screening with early stage target identification, we show that the direct binding target of ICM is the high mobility group box (HMGB) proteins. Mode-of-action studies demonstrate that ICM blocks the sequential processes of cytoplasmic localization and extracellular release of HMGBs by perturbing its post-translational modification. In addition, ICM effectively downregulates proinflammatory functions of HMGB and reduces neuronal damage in vivo. Our study reveals that ICM suppresses microglia-mediated inflammation and exerts a neuroprotective effect, demonstrating the therapeutic potential of ICM in neuroinflammatory diseases.

euroinflammation, considered mainly as innate immune Furthermore, we demonstrate the therapeutic potentials of ICM in responses in the central nervous system (CNS), is triggered CNS inflammatory disease. in response to diverse inflammatory signals such as patho- N 1,2 gen infection, injury or trauma, which may result in neurotoxicity . RESULTS An inflammatory response in the brain is strongly associated with Discovery of the anti-neuroinflammatory agent ICM the activation of microglia and is characterized by various indica- Microglia, a type of immune cell in the brain, serve as the first line tions, including the synthesis and secretion of proinflammatory of defense in the CNS by recognizing external pathogens, and they cytokines and chemokines and the breakdown of the blood-brain contribute to innate and adaptive immunity by continuously survey- barrier1,2. Although there is some debate about the cause-and-effect ing the microenvironment and protecting the brain. Once micro- relationship, it is generally believed that chronic and excessive glia are stimulated by immunogens or bacterial endotoxins, such microglial activation results in neuronal damage and death, which as lipopolysaccharide (LPS), activated microglia have a central role contribute to neurodegenerative diseases, including Alzheimer’s in neuroinflammation and secrete various neurotoxic factors, such

disease, Parkinson’s disease, multiple sclerosis and amyotrophic as IL-1β, TNF-α, PGE2, nitric oxide (NO) and superoxide anions lateral sclerosis1–3. Therefore, there is a growing need for the dis- (O −)2. To identify anti-neuroinflammatory agents, we used cell- Nature America, Inc. All rights reserved. America, Inc. © 201 4 Nature 2 covery of new therapeutic agents targeting microglial activation based phenotypic screening with LPS-induced nitrite release in the and neuroinflammation. To find a better treatment for devastating BV-2 mouse microglial cell line as a readout. Using high-throughput neuroinflammatory diseases of the CNS, the drug discovery pro- screening of an approximately 3,500-member in-house library con- npg cesses should be accompanied by the identification of potential structed by the pDOS strategy10, we identified a new benzopyran- therapeutic targets. embedded tetracyclic compound named ICM (Fig. 1a). ICM In the post-genomic era, cell-based phenotypic screening has efficiently blocked LPS-induced nitrite release in a dose-dependent emerged as a promising approach for the discovery of new chemical manner without any toxicity in BV-2 microglial cells (Fig. 1b and entities (NCEs) as candidates for first-in-class drugs4. This para- Supplementary Results, Supplementary Fig. 1a). We also con- digm shift has enabled innovation in the functional network of firmed that ICM inhibited nitrite release in a broad range of cell bioactive small molecules through the identification of new mecha- lines, including rat microglia (HAPI), mouse macrophages (RAW nisms of action5. However, this attractive approach is quite limited 264.7) and mouse primary microglial cultures (Supplementary in the actual drug discovery process because of the lengthy process Fig. 1b). However, ICM exhibited less potency in culture of primary of target identification or deconvolution6,7. In addition, further astrocytes, another major glial cell type in the CNS, than in micro- development and clinical applications of NCEs can be extremely glial cells, indicating an excellent selectivity of ICM toward microglia difficult without elucidation of their molecular targets, even though (Supplementary Fig. 1b). To confirm the anti-inflammatory effect the NCEs show prominent therapeutic effects in vivo8. Therefore, of ICM, we examined the expression or production of other pro efficient target identification has become an indispensable element inflammatory mediators in murine microglial cell lines and primary in phenotype-based drug discovery. microglial cells. The increased levels of inflammation-related genes, Here we report a new anti-neuroinflammatory agent, ICM (1), such as Il6, Il1b, Nos2 and Tnf, after LPS stimulation were markedly discovered by cell-based phenotypic screening and the subsequent suppressed by treatment with ICM (Fig. 1c and Supplementary identification of its molecular target using fluorescence difference Figs. 3a and 4a), and LPS-induced secretion of the proinflamma- in two-dimensional (2D) gel electrophoresis (FITGE) technology9. tory cytokine TNF-α was also reduced by ICM treatment (Fig. 1d

1Department of Chemistry, Seoul National University, Seoul, Korea. 2Department of Pharmacology, Brain Science and Engineering Institute, BK21 Plus KNU Biomedical Convergence Program, Kyungpook National University School of Medicine, Daegu, Korea. 3Department of Biophysics and Chemical Biology/N-Bio Institute, Seoul National University, Seoul, Korea *e-mail: [email protected] or [email protected]

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25 LPS (–) a b c LPS –+–+++ On the basis of these results, we confirmed LPS (+) 20 ICM (μM) 110–– 510 that ICM as a newly identified microglial O N Il6 inhibitor has both an anti-inflammatory and a M]

μ 15 O N Il1b neuroprotective effect. N 10 H Nos2 Nitrite [ Potential ICM targets 5 Tnf HO O We recently reported a cell-based target iden- 0 9 Gapdh tification method, FITGE . To overcome the Inﬂachromene, ICM (1) 0.01 0.1110 100 [ICM] (μM) limitation of weak binding affinity or low abundance of target proteins, we introduced d DMSO e DMSO f DMSO g DMSO ICM ICM ICM ICM covalent anchoring of a bioactive small mol- 6,000 100 150 NS 150 NS ecule to cellular target proteins in live cells 80 ** *** using a photocrosslinker. The resolution of the 4,000 100 100 in-gel protein analysis was clearly enhanced by 60 *** (pg/ml) 2D gel electrophoresis with dual-color labeling α 40 2,000 ** of p65 (%) 50 50 to differentiate specific target proteins from TNF- 20 9

Nuclear translocation nonspecific protein binders . On the basis of eGFP-positive cell (%) 0 0 0 CMFDA-positive cell (%) 0 a structure-activity relationship study for ICM Control LPS Control LPS Control LPS Control LPS analogs (Supplementary Fig. 7), we designed Figure 1 | Discovery of ICM as an anti-inflammatory agent in LPS-induced microglial activation. and synthesized the chemical probe ICM-BP (2) (a) Chemical structure of ICM. (b) Dose-dependent effect of ICM on BV-2 cells in the absence or (Fig. 2a). Benzophenone was embedded in presence of LPS stimulation. The level of nitrite in the extracellular milieu was measured by Griess the phenyl substituent of triazole as a photo assay. (c) RT-PCR analysis revealing the inhibitory effect of ICM on the expression levels of activatable crosslinking moiety, and an LPS-induced inflammation-related genes in HAPI cells (full-length blots are shown in alkyne group was incorporated at the end of Supplementary Fig. 29). Data are representative of triplicate experiments. (d) LPS-induced benzophenone as a functional tag for a bio- orthogonal click reaction with azide-linked TNF-α secretion in BV-2 cells was measured by ELISA with ICM (5 μM) treatment. (e) The fluorescent dyes or biotin. Before the appli- nuclear translocation of p65 (an NF-κB subunit) was investigated by immunofluorescence cation of ICM-BP for target identification, analysis with ICM (5 μM) treatment in HAPI cells. (f) The effect of ICM (10 μM) on microglial neurotoxicity as measured by coculture of LPS-stimulated HAPI microglial cells and B35-eGFP we evaluated its inhibitory activity toward LPS-induced nitrite release and confirmed that neuroblastoma cells. (g) The effect of ICM (10 μM) on coculture of LPS-stimulated primary microglial cells and CMFDA-labeled primary cortical neurons. The eGFP-positive cells and the ICM-BP exhibited inhibitory activity at a level CMFDA-positive cells for viability assessment are presented as the percentage of nontreated comparable to that of the original compound, cells in f and g. Cells were treated with ICM for 30 min before LPS treatment (100 ng/ml). ICM (Fig. 2b). All data represent the mean and s.d. in triplicate. *P < 0.05, **P < 0.001, ***P < 0.0001 compared With the optimized probe, we sought to to DMSO, as calculated by Student’s t-test. NS, not significant compared to DMSO. identify the target protein of ICM in live cells using competitive labeling, which facilitated an effective exclusion of nonspecific binding and Supplementary Fig. 3b). We previously reported that a series proteins. Once the cells were treated with ICM-BP in the absence of ICM analogs inhibited RANKL-induced osteoclastogenesis by or presence of ICM as a soluble competitor, they were irradiated Nature America, Inc. All rights reserved. America, Inc. © 201 4 Nature perturbing the NF-κB and MAPK signaling pathways in bone mar- with UV light to photocrosslink ICM-BP to cellular target proteins row monocytes and macrophages11. To confirm that these signaling and subjected to the click reaction with an azide-linked fluorescent pathways were influenced by the anti-neuroinflammatory effect of dye. The resulting proteomes were analyzed by gel electrophoresis npg ICM, NF-κB and MAPK signaling were investigated in microglia. and gel-fluorescence scanning. As determined by one-dimensional ICM substantially suppressed the nuclear translocation of NF-κB (1D) gel analysis, a direct comparison of the labeling patterns of (Fig. 1e and Supplementary Fig. 3c) and the degradation of IκB ICM-BP with and without ICM competition showed two distinct (Supplementary Fig. 5a) in LPS-stimulated microglia. In addition, bands at 29 kDa and 45 kDa (Fig. 2c) regardless of LPS stimulation. ICM treatment inhibited LPS-induced phosphorylation of ERK, We selected the protein in the 29-kDa band, as it competed out at a JNK and p38 MAPK in microglia (Supplementary Fig. 5b). lower concentration of ICM more effectively than the 45-kDa band Microglia release various neurotoxic factors after activation. in a dose-dependent competitive assay (Supplementary Fig. 9b). Microglia-mediated neurotoxicity is thought to be responsible for Even though the probe labeling pattern in cell lysates was substan- cellular damage to neighboring neurons; the damaged neurons lead tially different from that in live cells, we failed to observe any spe- to subsequent reactivation of microglia, a response known as micro- cific binding events of ICM-BP or competitive binding with ICM gliosis2. The perpetual accumulation of neuronal damage and micro- in the cell lysates (Supplementary Fig. 9c). On the basis of these glial activation eventually lead to the death of adjacent neurons in results, the bands were excised from 1D gels of live cell labeling, and the brain and to various neurodegenerative diseases. Therefore, the the proteins of the 29-kDa band were explored by LC/MS/MS anal- inhibition of microglial activation might confer a protective effect ysis. According to MS analysis, however, numerous proteins were against microglia-mediated neurotoxicity. To evaluate the neuro- implicated as potential targets by 1D gel analysis (Supplementary protective effect of ICM in vitro, coculture assays of LPS-treated Fig. 10a). To narrow down the number of candidates, we performed microglia were performed with either enhanced GFP (eGFP)- a 2D gel analysis to improve the resolution of the in-gel analysis. labeled neuroblastoma cells or 5-chloromethylfluorescein diacetate ICM-BP–labeled proteomes with and without ICM competition (CMFDA)-labeled primary cortical neurons. ICM completely pre- were treated with Cy5-azide (red for specific binding) and Cy3-azide vented the death of cocultured neuroblastoma and primary neuronal (green for nonspecific binding), respectively. Two samples labeled cells by inhibiting microglia-mediated neurotoxicity (Fig. 1f,g and with different fluorescent dyes were mixed and analyzed together Supplementary Fig. 4c). Furthermore, ICM itself had no significant by 2D gel electrophoresis, which reduced the gel-to-gel variation. effect on the viability of neurons (Supplementary Fig. 6; P values of In-gel fluorescence scanning of 2D gels effectively revealed char- 0.3225, 0.9908 and 0.6763 for 1 μM, 5 μM and 10 μM ICM treatment). acteristic spots around 29 kDa and 45 kDa, corresponding to the

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abDMSO c ICM 0.5 μM LPS – –++ –– ++ effect of ICM for both HMGB1 and HMGB2. ICM 5 μM ICM-BP ++++ ++ ++ 12 Transfection of short interfering RNA (siRNA) O ICM-BP 0.5 μM ICM––+ + –+–+ HN ICM-BP 5 μM for HMGB1 and HMGB2 individually

O induced a reduction in the protein expression 8 O M] μ N * levels of HMGB1 and HMGB2, respectively. O N *** N In addition, the dual knockdown of both H 4 Nitrite [ *** *** HMGB1 and HMGB2 decreased in their *** HO O expression levels simultaneously (Fig. 3a). 0 ICM-BP (2) Control LPS The loss of function resulting from individual In-gel ﬂuorescenceSilver knockdown of HMGB1 and HMGB2 mark- d pH 3 10 edly inhibited LPS-induced nitrite release in e BV-2 microglial cells, and dual knockdown

ICM-BP + + synergistically enhanced the inhibitory effect ICM– + (Fig. 3b). These results indicate a crucial role * Input of both HMGB1 and HMGB2 in microglial activation. We then tested whether HMGB PD knockdown affected the anti-inflammatory IB: HMGB2 effect of ICM. Transfection with siRNA for the two HMGBs negated the inhibitory effect of ICM, as it elevated LPS-induced nitrite release compared to mock and scramble siRNA trans- Figure 2 | Target identification of ICM by 1D and 2D gel analysis in live cells. (a) The chemical fections. In simultaneous knockdown of both structure of ICM-BP. Blue represents a photoaffinity group. Red represents a tag moiety for the HMGB1 and HMGB2, ICM treatment resulted click reaction. (b) The inhibitory activity of ICM-BP on nitrite release compared to that of ICM in in even higher nitrite release than after indi- BV-2 cells. The data represent the mean and s.d. in triplicate. ***P < 0.0001 compared to DMSO, vidual knockdown of either after LPS stimula- as calculated by Student’s t-test. (c) After light-induced labeling of ICM-BP (5 μM) to target the tion (Fig. 3c). The attenuated inhibitory effect proteome in live cells, ICM-BP–labeled proteomes with or without ICM competition (80 μM) were of ICM after knockdown of the HMGBs might analyzed by 1D gel electrophoresis in the absence or presence of LPS (100 ng/ml) stimulation. be due to the elimination of its target protein, The arrow and asterisk designate the major bands with significant differences after ICM which confirmed an HMGB-dependent anti- competition. Blots in c and e were carried out as individual experiments, and several dose and time inflammatory effect of ICM. Overall these ranges were tested to find the optimal conditions. (d) Target identification with ICM-BP (20 μM) results support that HMGBs as the target of ICM using 2D gel analysis. ICM-BP–labeled proteomes with and without ICM competition (20 μM) have a critical role in the anti-inflammatory were treated with Cy3-azide (green) and Cy5-azide (red), respectively. The merged fluorescence effect of ICM. images of the whole gel (left) and the expanded image of the dotted box (right). The arrow and asterisk designate the major red spots in the 2D gel showing specific binding. Scale bars, 2 cm. ICM as a PTM modulator (e) Pulldown (PD) assay performed with biotin-labeled proteome and streptavidin beads. ICM-BP In mammals, HMGBs are known as multifunc- (5 μM) with or without ICM competition (20 μM) was subjected to PD assay and immunoblotting tional proteins14. Originally, the HMGBs were (IB) for HMGB2 (full-length blots are shown in Supplementary Fig. 30). known as DNA-binding nuclear proteins12–15. Since the unexpected role of HMGB1 as a late Nature America, Inc. All rights reserved. America, Inc. © 201 4 Nature bands in the 1D gel analysis (Fig. 2d). After MS/MS analysis, we mediator of inflammation was reported16, numerous studies have obtained a shorter list of proteins detected from the 29-kDa spot elucidated interesting features of the HMGBs, especially HMGB1, than in 1D gel analysis, and we selected a number of potential can- in the regulation of inflammatory signaling12–14,17. HMGB1 shuttles npg didates for target proteins that were identified in both the 1D and between the nucleus and the cytosol, and cytoplasmic HMGB1 can 2D gel analyses (Supplementary Fig. 10). be released into the extracellular space through the secretory lyso- Among those candidate proteins, we focused on the HMGB2 somal pathway or by passive diffusion in necrotic cells18–20. Once protein as a potential target, as there are a number of reports in the released, extracellular HMGB1, similar to proinflammatory cyto literature indicating that the HMGBs have crucial roles in inflam- kines, interacts consecutively with various receptors to activate the mation12–14. To assess the specific binding event of ICM-BP with inflammatory signaling pathway12,13. Other HMGBs are expected to HMGB2, we performed an affinity pulldown assay using avidin- have similar roles based on their structural similarity to HMGB1 based enrichment of an ICM-BP–treated proteome after the click (ref. 14). Therefore, we hypothesized that ICM regulates HMGB2- reaction for labeling with biotin-azide. HMGB2 efficiently bound mediated inflammatory signaling. with ICM-BP, whereas the excess ICM as a soluble competitor To test this hypothesis, we first monitored the intracellular or markedly lowered the level of binding for HMGB2 (Fig. 2e), which extracellular translocation of HMGB2 after ICM treatment. LPS confirmed the specific interaction between ICM-BP and HMGB2. stimulation increased the cytoplasmic accumulation of HMGB2, as well as the secretion of HMGB2 into the extracellular milieu, but Functional validation of HMGB as a target of ICM ICM completely suppressed LPS-induced cytoplasmic transloca- Next we elucidated the HMGB-dependent anti-inflammatory activ- tion or extracellular release of HMGB2 (Fig. 4a). Because of these ity of ICM with a loss-of-function study to determine whether the observations, we then focused on the post-translational modifica- interaction between ICM and HMGB functionally influenced the tion (PTM) of HMGB2. HMGBs have diverse PTM sites and are desired inhibitory effect of ICM. There are four isoforms of HMGB, modified extensively by acetylation, phosphorylation, methyla- HMGB1–HMGB4, with a shared structural motif. Among them, tion, glycosylation and poly(ADP) ribosylation21. In particular, HMGB1 and HMGB2 share 80% sequence homology, contain- several PTMs might be closely related to the cellular transloca- ing two tandem HMG box domains (box A and box B) and a long tion of HMGB1 in monocytes or macrophages22,23, as acetylation acidic tail at the C terminus14. Because of the structural and func- and phosphorylation were observed at lysine and serine residues, tional similarity of the two proteins, we speculated that ICM might respectively, near two nuclear localization sequences (NLSs) in the also interact with HMGB1, and so we examined the inhibitory HMGBs. Therefore, we sought to determine the potential effect of

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ab c animals showed limp tails (grade 1) (Fig. 5b). 30 LPS (–) 0.4 ** *** ICM markedly delayed the onset of disease LPS (+) NS and decreased disease severity by reducing M] siRNA Mock sc si-HMGB1 si-HMGB2 si-HMGB1 + si-HMGB2 20 *** μ 0.3 the maximal clinical score and degree of ** HMGB1 ** hindlimb paralysis compared to vehicle treat- 10 * Nitrite [ 0.2 * ment (Supplementary Fig. 17). To examine HMGB2 *** E ect of ICM on NS

0 (normalized to DMSO) the activation of microglia and the inflamma- Actin siRNA LPS-induced nitrite release siRNA sc sc tory response, we euthanized mice at the peak Mock Mock of disease and performed histological analysis. si-HMGB1 si-HMGB2si-HMGB1 si-HMGB1si-HMGB2si-HMGB1 Microglial activation in the lumbar spinal + si-HMGB2 + si-HMGB2 cords of EAE mice was clearly suppressed, and Figure 3 | Functional validation of HMGB2 as a direct binding target of ICM. (a) Representative inflammatory lesions in EAE spinal cords were western blot results in triplicate confirming siRNA-mediated knockdown of HMGB1 and HMGB2 substantially attenuated by ICM treatment in BV-2 cells (mock, control transfection; sc, scrambled siRNA; si-HMGB1, siRNA for HMGB1; (Fig. 5c,d). In addition, microglial cells iso- si-HMGB2, siRNA for HMGB2) (full-length blots are shown in Supplementary Fig. 30). (b) The lated from the spinal cords of EAE mice were loss-of-function effect by siRNA knockdown with either si-HMGB1 or si-HMGB2 and double subjected to analysis of the mRNA levels of knockdown with both si-HMGB1 and si-HMGB2 confirmed by LPS-induced nitrite release in proinflammatory cytokines and chemokines, BV-2 cells. (c) The HMGB-dependent effect of ICM. LPS-induced nitrite release after siRNA which confirmed that ICM treatment sup- transfection. ICM (5 μM) treatment was normalized to the individual DMSO treatments. All data pressed the production of inflammatory represent the mean and s.d. in triplicate. *P < 0.05, **P < 0.001, ***P < 0.0001 compared to mock markers, such as IL-1β, TNF-α, CXCL10, CCL2 transfection, as calculated by Student’s t-test NS, not significant compared to mock transfection. and IL-6, in microglia in vivo (Fig. 5e). The Asterisks above the lines indicate significance between single and dual knockdown. colocalization of HMGBs and microglia in EAE mice supported a microglia-specific inhibitory effect of ICM (Supplementary Fig. 18). The ICM on LPS-induced phosphorylation and acetylation of HMGBs in effects of ICM on the proliferation of neuronal progenitor cells in the microglia. LPS treatment increased the phosphorylation and acetyla- subventricular zone (SVZ) and the inflammatory activation of devel- tion of HMGB2 in microglia, and these PTMs were effectively inhib- oping glial cells and neuronal progenitor cells were next assessed. ited by treatment with ICM (Fig. 4b). Identical inhibitory effects Injection of ICM did not significantly influence the proliferation of on the PTMs for HMGB1 were also observed after ICM treatment neuronal progenitor cells, as determined by counting BrdU-positive (Supplementary Fig. 14). Considering that PTMs of HMGB1 regu- proliferating cells in SVZ (Supplementary Fig. 19; P values of late the nuclear export of HMGB1, this ICM-induced PTM inhibition 0.0736 and 0.0717 for 10 nM and 50 nM ICM treatment). Similarly, can explain the ICM-induced suppression of HMGB2 transloca- ICM treatment did not have significant effects on NO or TNF-α tion. In other words, LPS-induced phosphorylation and acetylation production in glial cells or neuronal progenitor cells derived from lead to relocalization of HMGB2 to the cytoplasm, but ICM treat- striatum at embryonic day (E) 15 (Supplementary Fig. 20), further ment inhibits the PTMs and suppresses the cytoplasmic accumula- supporting the microglia selectivity of ICM. Moreover, we clearly tion of HMGB2. Therefore, we confirmed that ICM disrupts PTMs observed that ICM ameliorated neuroinflammation, consequently and sequentially blocks intracellular translocation and extracellular attenuating neurite damages and spinal cord demyelination in EAE secretion of HMGB2, thereby inhibiting the role of HMGB2 as a spinal cords, as determined by immunostaining of microtubule- proinflammatory cytokine. associated protein-2 (MAP-2) and myelin basic protein (MBP) Nature America, Inc. All rights reserved. America, Inc. © 201 4 Nature

The therapeutic effect of ICM a LPS – ++ b LPS – ++ Considering the impact of microglia-mediated neurotoxicity in ICM – –+ ICM – –+ npg neuroinflammatory disease, we envisioned the therapeutic poten- hnRNP Input tial of ICM and tested the effect of ICM on microglial activation Nu in vivo using an LPS-induced mouse neuroinflammation model. HMGB2 IP: pSer

Briefly, ICM was injected intraperitoneally daily for 4 days, then IB: HMGB2 the mice were euthanized, and the levels of Iba-1 as a marker of Actin microglial activation were analyzed (Supplementary Fig. 15a). Cyt HMGB2 Input Quantitative analysis of different regions of the mouse brain, including the cortex, hippocampus and substantia nigra, revealed that IP: AcLys ICM effectively blocked LPS-mediated microglial activation, even HMGB2 CM IB: HMGB2 at a low dose (2 mg per kg body weight of the mouse) (Fig. 5a and Supplementary Fig. 16). Figure 4 | Mode-of-action study of ICM for perturbing the inflammatory To evaluate the protective role of ICM in the pathogenesis of function of HMGB2. (a) The effect of ICM on the translocation of HMGB2 neuroinflammatory disease, we used a mouse experimental auto- from the nucleus (Nu) to the cytoplasm (Cyt) and extracellular milieu in immune encephalitis (EAE) model, which is an animal model of conditioned medium (CM). (b) Anti-phosphoserine (pSer) and acetylated multiple sclerosis, a prototype organ-specific autoimmune and lysine (AcLys) immunoprecipitation (IP) followed by immunoblotting inflammatory disease in the CNS. After myelin oligodendrocyte (IB) for HMGB2 indicates the effect of ICM on the post-translational glycoprotein (MOG) peptide immunization, EAE developed by day modification of HMGB2. BV-2 cells were treated with ICM (10 μM) 12 and reached a maximal score on day 17. To evaluate the thera- before LPS (200 ng/ml) and analyzed by subcellular fractionation or peutic effect of ICM, we administered 10 mg/kg of ICM daily after IP assay. Heterogeneous nuclear ribonucleoprotein (hnRNP) and actin MOG immunization (Supplementary Fig. 15b). We found that were detected as housekeeping proteins in the nucleus and cytoplasm, ICM administration significantly reduced the progression of dis- respectively. Input represents 5% of the total lysate used in IP (full-length ease, as determined by EAE clinical score (Fig. 5b). When the EAE blots are shown in Supplementary Fig. 30). Blots were carried out as phenotype reached its peak, most of the mice treated with vehicle individual experiments, and several dose and time ranges were tested to showed hindlimb paralysis (grade 3), but most of the ICM-treated find the optimal conditions.

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Vehicle a 600 Cortex SN Hippocampus b 3 have been focused on HMGB1 (refs. 12,13,17), ICM 10 mg/kg and the role of other HMGB isoforms remains unclear, especially in neuroinflammation. In * 2 400 this study, we found that HMGB2 has a criti- *** ** *** cal role in microglia-mediated neuroinflam- cell number

+ ** *** ** ** *** 1 *** ** * 200 ** ** Clinical score *** *** mation through the discovery of ICM and the Iba-1 *** subsequent identification of HMGB2 as the ** ** target of ICM. 0 0 LPS –––+++–––+++ –––+++ 0510 15 20 25 30 Because of their important roles as late medi- ICM (mg/kg) –210 –210 –210 –210 –210 –210 Time after immunization (d) ators of inflammation, HMGBs have received c Naive EAE d Naive EAE increasing attention as therapeutic targets for Vehicle Vehicle ICM Vehicle VehicleICM sepsis, cancer, arthritis and other inflammatory conditions, including hepatitis and pan- creatitis, as well as neuroinflammation14,25. horn Ventral In fact, the dual inhibitory activity of ICM Fluoromyelin toward both HMGB1 and HMGB2 ensures the therapeutic potential of ICM because of Iba-1

Ventral column the significant roles of both proteins in neuro inflammation (Fig. 3b). The excellent thera- 50 25 peutic efficacy of ICM in vivo, even at low efNaive f Naive 40 EAE + vehicle 20 EAE + vehicle doses (Fig. 5a), compared to its efficacy in the EAE + ICM EAE + ICM in vitro system might be due to the synergistic 30 15 inhibitory activity of ICM for both HMGB2 20 10 *** and HMGB1. ICM effectively suppressed

(fold change) ** *

10 HMGB2 (ng/ml) 5 mRNA expression ** microglial activation, inhibited neuroinflam- ** 0 ** 0 mation and mediated a neuroprotective effect Il1b Tnf Cxcl10 Ccl2 Il6 CSF Serum in the EAE spinal cord. According to the time- course experiment, ICM exhibited a lasting Figure 5 | The therapeutic effect of ICM in vivo. (a) Suppression of glial activation by ICM anti-inflammatory effect and not just a sim- administration was analyzed in the cortex, substantia nigra (SN) and hippocampus regions of ple delay of inflammation (Supplementary LPS-injected mouse brains. Quantification for immunofluorescence staining of Iba-1. (b) EAE Fig. 23). Interestingly, ICM retained its inhibi- was induced in C57BL/6 mice by immunization of the MOG35–55 peptide and pertussis toxin. tory activity when administered after LPS The animals were administered daily with either vehicle or ICM for 30 d. Data are presented stimulation as well as when given before LPS as a clinical score and represent the mean and s.e.m. (n = 7). (c) Histological analysis of ICM’s treatment (Supplementary Fig. 24), suggest- effects on the EAE model compared with preimmunized mice (naive). Frozen sections of lumbar ing the therapeutic potential of ICM in the spinal cords were stained with FluoroMyelin for myelin (top) and Iba-1–specific antibody for clinical setting. Therefore, ICM might provide microglial activation (bottom). (d) The frozen sections in c were subjected to hematoxylin and an important breakthrough for the treatment of eosin staining to show inflammatory lesions. Scale bars (c,d), 200 μm. (e) The effect of ICM on HMGB-mediated neuroinflammation as well as the mRNA expression of proinflammatory cytokines and chemokines in microglia isolated from neurodegenerative diseases. Nature America, Inc. All rights reserved. America, Inc. © 201 4 Nature the spinal cords of naive or EAE mice. (f) The levels of HMGB2 in cerebrospinal fluid and serum of HMGB-targeted anti-inflammatory ther- naive or EAE mice were measured by ELISA. ICM (10 mg per kg body weight) was administrated apy was previously focused on a scaveng- in the EAE experiment, and samples were prepared at the disease peak time (Supplementary ing strategy using HMGB1-specific antibody npg Fig. 15). The data in a, e and f represent the mean and s.d. in triplicate. *P < 0.05, **P < 0.001, and A-box mimic peptides or on a receptor- ***P < 0.0001 compared to vehicle, as calculated by Student’s t-test. blocking strategy with RAGE-specific antibody. However, both strategies suffer from (Supplementary Figs. 21 and 22). Because ICM blocked the extra- limited cellular uptake or metabolic instability14,16,26,27. A small cellular release of HMGBs as an inflammatory cytokine, we mea- molecule–based strategy can effectively overcome these limitations. sured HMGB2 levels in the serum and cerebrospinal fluid of EAE Recently, glycyrrhizin, a natural product isolated from the root of mice. MOG immunization caused an increase in HMGB2 plasma Glycyrrhiza glabra, was reported to be the first HMGB1-binding levels, whereas ICM treatment effectively suppressed the release molecule28. However, the anti-inflammatory efficacy of glycyr- of HMGB2 into body fluids after EAE induction (Fig. 5f). These rhizin is very low in LPS-activated microglia, with an approximately results strongly support the therapeutic potential of ICM as an anti- 600-fold lower potency than that of ICM (Supplementary Fig. 25), neuroinflammatory and neuroprotective agent in vivo. and its interaction with other HMGBs has not been explored. In contrast, using functional studies and additional mechanistic stud- DISCUSSION ies, we demonstrated that ICM regulated an inflammatory function Despite the deteriorating role of neuroinflammation in many of the HMGBs through direct binding. neurological diseases, the number of existing anti-inflammatory ICM exhibits a unique mode of action as a PTM modulator, drugs is quite limited because of insufficient efficacy or undesired regulating the secretion of HMGBs (Fig. 4). A computational study side effects24. Therefore, there is a growing need for the develop- of the binding site of ICM to HMGB1 led to a structural insight for ment of NCEs with new modes of action for the treatment of neuro the ICM-induced nuclear translocation of HMGBs. On the basis of inflammatory disease. To address these unmet needs, we used the docking simulation, the binding site of ICM was predicted to be high-throughput phenotypic screening and discovered ICM as a the DNA-binding domain in box A of HMGB, which is located adja- new anti-microglial and anti-inflammatory agent. Furthermore, we cent to the NLS (Supplementary Figs. 26 and 27). This result sup- identified HMGBs as a direct protein target of ICM using FITGE ports the idea that ICM binding perturbed the PTM near the NLS technology. Although there are reports in the literature regarding site and then inhibited the nuclear translocation of HMGBs. In fact, the role of HMGBs as inflammatory cytokines, most of the studies the HMGBs were originally identified as DNA-binding proteins that

nature CHEMICAL BIOLOGY | vol 10 | december 2014 | www.nature.com/naturechemicalbiology 1059 article Nature chemical biology doi: 10.1038/nchembio.1669

regulated transcription, replication or DNA repair15. If ICM univer- 10. Oh, S. & Park, S.B. A design strategy for drug-like polyheterocycles with sally blocked the functions of HMGBs, it would clearly cause severe privileged substructures for discovery of specific small-molecule modulators. side effects. In spite of the various properties of the HMGBs, how- Chem. Commun. (Camb.) 47, 12754–12761 (2011). 11. Zhu, M. et al. Discovery of novel benzopyranyl tetracycles that act as ever, ICM inhibited the inflammatory function of HMGBs without inhibitors of osteoclastogenesis induced by receptor activator of NF-κB affecting the interaction of HMGBs with DNA (Supplementary ligand. J. Med. Chem. 53, 8760–8764 (2010). Fig. 28), thereby exhibiting no severe adverse effects or cytotoxic- 12. Lotze, M.T. & Tracey, K.J. High-mobility group box 1 protein (HMGB1): ity (Supplementary Figs. 1c and 6). In addition, further evaluation nuclear weapon in the immune arsenal. Nat. Rev. Immunol. 5, 331–342 (2005). with neuronal progenitor cells in SVZ using BrdU-injected animals 13. Sims, G.P., Rowe, D.C., Rietdijk, S.T., Herbst, R. & Coyle, A.J. HMGB1 and RAGE in inflammation and cancer. Annu. Rev. Immunol. 28, 367–388 (2010). confirmed no statistically significant effect of ICM on neurogenesis 14. Yang, H. & Tracey, K.J. Targeting HMGB1 in inflammation. Biochim. Biophys. in vivo (Supplementary Fig. 19), supporting the specific regulation Acta 1799, 149–156 (2010). of ICM on microglia-mediated neuroinflammation. 15. Štros, M. HMGB proteins: interactions with DNA and chromatin. Biochim. In our previous study, we observed that ICM analogs suppressed Biophys. Acta 1799, 101–113 (2010). κ 16. Wang, H. et al. HMG-1 as a late mediator of endotoxin lethality in mice. osteoclastogenesis by inhibiting the NF- B and MAPK signaling Science 285, 248–251 (1999). 10 pathways in mouse macrophages and bone marrow monocytes . 17. Ulloa, L. & Messmer, D. High-mobility group box 1 (HMGB1) protein: friend The NF-κB and MAPK signaling pathways are the key regulators and foe. Cytokine Growth Factor Rev. 17, 189–201 (2006). of inflammation induced by HMGBs17, and HMGB1 signaling has a 18. Gardella, S The nuclear protein HMGB1 is secreted by monocytes via a crucial role in arthritis as well as in the activation of macrophages29. non-classical, vesicle-mediated secretory pathway. EMBO Rep. 3, 995–1001 κ (2002). In this study, we found that ICM also inhibited the NF- B and 19. Rendon-Mitchell, B. et al. IFN-γ induces high mobility group box 1 protein MAPK signaling pathways in microglia, resulting in an anti- release partly through a TNF-dependent mechanism. J. Immunol. 170, inflammatory effect in macrophages (Fig. 1e and Supplementary 3890–3897 (2003). Figs. 2a and 5). The inhibitory activity of ICM in these signaling 20. Scaffidi, P., Misteli, T. & Bianchi, M.E. Release of chromatin protein HMGB1 cascades provides strong support for the molecular target of ICM by necrotic cells triggers inflammation. Nature 418, 191–195 (2002). 21. Zhang, Q. & Wang, Y. High mobility group proteins and their post- and explains the observed phenotype and mechanism of action of translational modifications. Biochim. Biophys. Acta 1784, 1159–1166 (2008). ICM in activated microglia. The identification of the target of ICM 22. Bonaldi, T. et al. Monocytic cells hyperacetylate chromatin protein HMGB1 allowed the discovery of the ‘missing link’ for the integration of pre- to redirect it towards secretion. EMBO J. 22, 5551–5560 (2003). vious observations into a coherent explanation. 23. Youn, J.H. & Shin, J.-S. Nucleocytoplasmic shuttling of HMGB1 is regulated In summary, the integration of phenotype-based screening with by phosphorylation that redirects it toward secretion. J. Immunol. 177, 7889–7897 (2006). FITGE-based target identification led to the discovery of a new 24. Craft, J.M., Watterson, D.M. & Eldik, L.J.V. Neuroinflammation: a potential chemical entity, ICM. This convergent strategy and subsequent therapeutic target. Expert Opin. Ther. Targets 9, 887–900 (2005). mechanistic studies revealed that a new small molecule, a PTM 25. Ellerman, J.E., Brown, C.K. & Vera, M. Masquerader: high mobility group modulator of HMGBs, is an inhibitor of neuroinflammation with a box-1 and cancer. Clin. Cancer Res. 13, 2836–2848 (2007). broad window of therapeutic possibilities for various neuroinflam- 26. Yang, H. et al. Reversing established sepsis with antagonists of endogenous high-mobility group box 1. Proc. Natl. Acad. Sci. USA 101, 296–301 (2004). matory diseases. 27. Fiuza, C. et al. Inflammation-promoting activity of HMGB1 on human microvascular endothelial cells. Blood 101, 2652–2660 (2003). Received 19 March 2014; accepted 18 September 2014; 28. Mollica, L. et al. Glycyrrhizin binds to high-mobility group box 1 protein and published online 12 October 2014 inhibits its cytokine activities. Chem. Biol. 14, 431–441 (2007). 29. Kokkola, R. et al. Successful treatment of collagen-induced arthritis in mice and rats by targeting extracellular high mobility group box chromosomal Methods protein 1 activity. Arthritis Rheum. 48, 2052–2058 (2003). Methods and any associated references are available in the online Nature America, Inc. All rights reserved. America, Inc. © 201 4 Nature version of the paper. Acknowledgments This work was supported by a Creative Research Initiative grant (2014R1A3A2030423), References the Bio & Medical Technology Development Program (2012M3A9C4048780) and the npg 1. Streit, W.J., Mrak, R.E. & Griffin, W.S.T. Microglia and neuroinflammation: a Basic Research Laboratory (2010-0019766) funded by the National Research Foundation pathological perspective. J. Neuroinflammation 1, 14 (2004). of Korea (NRF). K.S. was supported by a NRF grant funded by the Korean government 2. Block, M.L., Zecca, L. & Hong, J.-S. Microglia-mediated neurotoxicity: (MSIP) (2008-0062282) and a grant of the Korea Healthcare Technology R&D Project, uncovering the molecular mechanisms. Nat. Rev. Neurosci. 8, 57–69 (2007). Korean Ministry of Health and Welfare (A111345). S.L., Y.N., J.Y.K., D.L., J.P. and J.K. are 3. Block, M.L. & Hong, J.-S. Microglia and inflammation-mediated grateful for a BK21 Scholarship. neurodegeneration: multiple triggers with a common mechanism. Prog. Neurobiol. 76, 77–98 (2005). Author contributions 4. Swinney, D.C. & Anthony, J. How were new medicines discovered? Nat. Rev. S.L. performed the biochemical assays, analyzed the data and prepared the manuscript. Drug Discov. 10, 507–519 (2011). Y.N. and J.K. performed the biochemical assays and in vivo experiments and analyzed the 5. Kotz, J. Phenotypic screening, take two. SciBX. doi:10.1038/scibx.2012.380 data. J.Y.K. synthesized the compounds and conducted the computational study. D.L. (2012). and J.O. performed experiments for target validation. J.P. contributed to the target 6. Terstappen, G.C., Schlüpen, C., Raggiaschi, R. & Gaviraghi, G. Target identification. K.S. and S.B.P. directed the study and were involved in all aspects of the deconvolution strategies in drug discovery. Nat. Rev. Drug Discov. 6, 891–903 experimental design, data analysis and manuscript preparation. All authors critically (2007). reviewed the text and figures. 7. Schenone, M., Dančík, V., Wagner, B.K. & Clemons, P.A. Target identification and mechanism of action in chemical biology and drug discovery. Nat. Chem. Competing financial interests Biol. 9, 232–240 (2013). The authors declare no competing financial interests. 8. Andreux, P.A., Houtkooper, R.H. & Auwerx, J. Pharmacological approaches to restore mitochondrial function. Nat. Rev. Drug Discov. 12, 465–483 (2013). Additional information 9. Park, J., Oh, S. & Park, S.B. Discovery and target identification of an Supplementary information, chemical compound and chemical probe information is antiproliferative agent in live cells using fluorescence difference in two- available in the online version of the paper. Reprints and permissions information is dimensional gel electrophoresis. Angew. Chem. Int. Ed. Engl. 51, 5447–5451 available online at http://www.nature.com/reprints/index.html. Correspondence and (2012). requests for materials should be addressed to S.B.P. or K.S.

1060 nature chemical biology | vol 10 | december 2014 | www.nature.com/naturechemicalbiology ONLINE METHODS RT-PCR. For the analysis of gene expression, cells were incubated with ICM Reagents. Chemicals, including LPS (L2880, Sigma) and glycyrrhizin (G0150, for 6 h in the absence or presence of LPS. Total RNA was extracted from cells TCI), were purchased from commercial venders. Cell culture reagents, using TRIzol reagent (Invitrogen) according to the manufacturer’s instruc- including FBS, media and antibiotic-antimycotic solution, were purchased tions. Reverse transcription was conducted using Superscript II (Invitrogen) from Gibco, Invitrogen. All antibodies for immunoblotting analysis were and oligo(dT) primers. PCR amplification was conducted using a DNA Engine purchased from R&D systems, Santa Cruz, Abcam, Cell Signaling and Wako. Tetrad Peltier Thermal Cycler and a C1000 Touch Thermal cycler (Bio-Rad) at The purified HMGB1 (ab73658) and HMGB2 (ab91926) proteins were an annealing temperature of 55–60 °C for 20–30 cycles using specific primer purchased from Abcam. Fluorescent-labeled ODN and native ODN were sets (Supplementary Table 4). To analyze PCR products, each sample was purchased from InvivoGen. electrophoresed on a 1% agarose gel and detected under UV light. Gapdh was used as an internal control. Cell culture. The BV-2 and HAPI microglial cell lines were obtained from α α American Type Culture Collection and cultured in DMEM supplemented ELISA TNF- secretion was measured using a TNF- ELISA kit (DY410, with 1% (v/v) antibiotic-antimycotic solution and heat-inactivated 5% or 10% R&D Systems). BV-2 cells were treated with LPS in the absence or presence of α (v/v) FBS, respectively. RAW 264.7 cells were obtained from American Type ICM. After a 24-h incubation, the levels of TNF- in the culture medium were α Culture Collection and cultured in RPMI 1640 medium supplemented with measured with rat monoclonal anti-mouse TNF- antibody (1:180 dilution, heat-inactivated 10% (v/v) FBS and 1% (v/v) antibiotic-antimycotic solution. 840143, R&D Systems) as the capture antibody and goat biotinylated poly α B35-eGFP neuroblastoma cells were obtained by stably transfecting an eGFP clonal anti-mouse TNF- antibody (1:180 dilution, 840144, R&D Systems) as α construct into B35 rat neuroblastoma cells. Cells were maintained in a humidi- the detection antibody. The biotinylated anti–TNF- antibody was detected by sequential incubation with streptavidin–horseradish peroxidase (HRP) conju- fied atmosphere of a 5% CO2 incubator at 37 °C and cultured in 100-mm cell culture dishes. For primary astrocytes and microglial cultures, the whole brains gate (1:120 dilution, 890803, R&D Systems) and TMB substrates (DY999, R&D of 3-day-old mice were chopped and mechanically disrupted using a nylon Systems). After incubation for 20 min, the color development was stopped by mesh. The mixed glial cells obtained were seeded in culture flasks and grown at adding 2 N H2SO4. The absorbance was then read at 450 nm and 540 nm using a microplate reader (Molecular devices). The level of HMGB2 in body fluids 37 °C in a 5% CO2 atmosphere in DMEM supplemented with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin. Culture media were changed initially was measured by a HMGB2-ELISA kit (CSB-EL010560MO, CUSABIO Life after 5 d and then changed every 3 d. After 14 d in culture, primary astrocytes Science) according to the manufacturing guidelines. and microglia were obtained using shaking or a mild trypsinization method Immunofluorescence staining. Cells were pretreated with compounds, exposed from mixed glial cells and maintained in DMEM supplemented with 10% FBS to LPS for 1 h and then fixed with 4% paraformaldehyde for 30 min at room and penicillin-streptomycin. Primary cortical neuron cultures were prepared temperature and with cold methanol for 10 min at −20 °C. After permeabiliza- from E15 C57BL/6 mice. Briefly, mouse embryos were decapitated, and the tion with 0.3% Triton X-100 and PBS for 10 min, the fixed cells were blocked brains were removed rapidly and placed in a culture dish with cold PBS. The with 1% normal horse serum for 1 h and incubated with mouse anti-p65 anti- cortices were isolated and then transferred to a culture dish containing 0.25% body (1:100 dilution, sc-8008, Santa Cruz) at 4 °C overnight. After washing trypsin-EDTA in PBS for 30 min at 37 °C. After two washes in serum-free with PBS containing 0.05% Tween-20 (PBST), Alexa Fluor-488–labeled goat neurobasal medium (Gibco-BRL), dissociated cortical cells were seeded onto anti-mouse IgG antibody was added to the sample, incubated for 1 h at room poly D-lysine–coated plates using neurobasal medium containing 10% FBS, temperature and washed with PBST. Nuclei were visualized by 4′,6-diamidino- 0.5 mM glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, N2 supple- 2-phenylindole (DAPI) staining (Vector Laboratories). Samples were analyzed ment and B27 supplement. Cells were maintained by changing the medium by fluorescence microscopy. every 2–3 d and grown at 37 °C in a 5% CO2 humidified atmosphere. For E15 striatum cell culture, striatal tissue was isolated from E15 C57BL/6 mice and Microglia and neuron coculture. For the coculture of microglial cells and neuro placed in a culture dish with cold Hank’s balanced salt solution (HBSS) buffer. blastoma cells, HAPI microglial cells were exposed to ICM and LPS (100 ng/ml) The tissue was transferred to a culture dish containing 0.025% trypsin-EDTA for 8 h. The medium was replaced with fresh medium containing B35-eGFP

Nature America, Inc. All rights reserved. America, Inc. © 201 4 Nature in HBSS for 15 min at 37 °C and then washed with FBS-containing medium. neuroblastoma cells, and the cocultures of microglia and neuroblastoma were Striatal tissues were mechanically dissociated with a Pasteur pipette and sus- incubated for 24 h. At the end of the cocultures, the B35-eGFP neuroblastoma pended in 10 ml of neurobasal medium containing 1% FBS, 100 U/ml penicil- cells were counted to assess cell viability. The coculture of primary microglial lin, 100 mg/ml streptomycin, 2 mM glutamine, 10 ng/ml nerve growth factor cells and cortical neurons was done in a similar manner. Cortical neurons were npg (NGF) and B27 supplement (Gibco-BRL). Cells were counted with a hemo- labeled with CMFDA (C2925, Molecular Probe) before initiating the coculture. cytometer and seeded onto poly D-lysine–coated 96-well plates. After 1 d, the The viability of cortical neurons was determined after the coculture for 48 h. cultures were treated with 2 μM Ara-C (C1768, Sigma) to remove glial cells for some experiments. Assessment of neurogenesis in SVZ. C57BL/6 mice (10 weeks) were given an intraperitoneal (i.p.) injection of 200 mg BrdU per kg body weight (B5002, Animals. C57BL/6 mice (25–30 g) were supplied by Samtaco. The animals were Sigma) every 12 h for 2.5 days (total of five injections) and perfused 12 h after maintained in temperature- and humidity-controlled conditions with a 12-h the final injection. Mice received a single intracerebroventricular (posterior, light, 12-h dark cycle. All animal experiments were approved by the Institutional 0.5 mm; lateral, 1 mm; vertical, 1.75 mm) injection of vehicle or ICM (10 or Review Board of Kyungpook National University School of Medicine and were 50 nM) using a stereotaxic apparatus before the first injection of BrdU. Mice carried out in accordance with the guidelines in the US National Institutes of were anesthetized with diethyl ether, transcardially perfused with cold saline Health Guide for the Care and Use of Laboratory Animals. and perfused with 4% paraformaldehyde (PFA) diluted in 0.1 M PBS. The brains were fixed using 4% PFA for 3 d and then cryoprotected with 30% Griess assay. The Griess assay was used for quantification of the secretion of sucrose solution for 3 d. Fixed brains were embedded in optimal cutting tem- nitrite. Cells were treated with compounds in the absence or presence of LPS perature (OCT) compound for frozen sectioning and then sectioned sagit- (100 ng/ml). After 24 h of incubation, the cell culture media were reacted with tally at 20 μm. To detect cell proliferation in vehicle- or ICM-injected brains, Griess reagent (0.1% naphthylethylenediamine dihydrochloride and 1% sulfa- sections were washed with PBS, and BrdU antigen retrieval was performed nilamide in 2% phosphoric acid). Absorbance was measured at 550 nm using a using 2 N HCl treatment for 30 min at 37 °C followed by three washes in microplate reader, and the level of nitrite was estimated by comparison with a 0.1 M borate buffer (pH 8.0) and three washes in PBS. All sections were sodium nitrite standard curve. blocked for 1 h at room temperature with 5% donkey serum, 0.3% BSA and 0.3% Triton X-100 in PBS solution and then stained with rat anti-BrdU anti- Cell viability assay. Cell viability was measured with MTT (3-(4,5- body (1:500 dilution, MCA2060, Serotec). dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; M-6494, Life Techno logies) or WST (water-soluble tetrazolium salt; W201-12, Dojindo) assay kits, Synthesis. The synthetic approach for ICM and its derivatives, including and the experimental procedure was based on the manufacturer’s manual. ICM-BP, is described in detail in the Supplementary Note.

doi:10.1038/nchembio.1669 nature CHEMICAL BIOLOGY In-gel analysis for identification of the protein target. BV-2 cells were treated sucrose, 20 mM HEPES, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT with the compounds for 30 min and then with LPS for 2 h. Then the cells and protease inhibitor cocktail (Roche)), passed through a 25-G needle sev- were irradiated with 365-nm UV light for 30 min on ice. The cells were washed eral times and incubated on ice for 20 min. After centrifugation at 3,000 r.p.m. with PBS and stored at −80 °C. Cells were lysed in RIPA buffer containing for 5 min, the pellet and supernatant were separated. The nuclear pellet was a protease inhibitor cocktail, and the protein concentration was adjusted to dispersed in buffer, centrifuged to remove the washing buffer and then resus- 1 mg/ml. The proteome was labeled with Cy5-azide (40 μM), Tris[(1-benzyl- pended in standard lysis buffer with 10% glycerol and 0.1% SDS. The cytosolic

1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) (100 μM), CuSO4 (1 mM), Tris(2- fraction was obtained by centrifugation of the separated supernatant at 8,000 carboxyethyl)phosphine (TCEP) (1 mM) and t-BuOH (5%) for 1 h. Acetone r.p.m. for 15 min. The conditioned medium was collected for the analysis of the was added to the mixture to precipitate the proteins, and the mixture was kept extracellular level of HMGBs and concentrated with Amicon Ultra 10K filters at −20 °C for 20 min. After centrifugation at 4 °C, 14,000 r.p.m. for 10 min, (Millipore). The samples were analyzed by SDS-PAGE and western blotting. the pellet was washed twice with cold acetone. For 1D gel analysis, the pellet was dissolved in Laemmli sample buffer and analyzed by SDS-PAGE. For Immunoprecipitation analysis. For the analysis of the PTM levels of phospho- 2D gel analysis, the pellet was dissolved in rehydration buffer. The proteome rylation and acetylation on HMGB, immunoprecipitation and western blot- labeled with ICM-BP (labeled with Cy5-azide) and the control (labeled with ting were performed. After a 4-h incubation with a compound in the absence Cy3-azide) were mixed (1:1 ratio). The mixed proteomes were analyzed by 2D or presence of LPS, cells were harvested and lysed in IP buffer containing a gel electrophoresis. In-gel fluorescence was scanned with a Typhoon Trio. protease inhibitor cocktail and the appropriate inhibitor, i.e., PMSF, NaF and

Na3VO4 for phosphorylation or nicotinamide and trichostatin A for acetyla- Mass analysis. The residual peptides in desired bands and spots were extracted tion. The concentration of protein was measured by bicinchoninic acid assays, by trypsin in-gel digestion and then subjected to LC/MS/MS analysis. In detail, and the lysates were incubated with anti-phosphoserine antibody (1:1,000 selected protein spots and bands were excised and dehydrated in acetonitrile dilution, ab9332, Abcam) or anti–acetylated lysine antibody (1:1,000 dilution, for 10 min. The acetonitrile was removed and dried under vacuum. For mass 9441, Cell Signaling) at 4 °C overnight and subsequently precipitated using a analysis, the resulting gel pieces were re-swelled at 4 °C for 45 min in buffer protein G immunoprecipitation kit (Sigma) following the manufacturer’s pro- containing trypsin and 50 mM (NH4)2CO3 and incubated overnight at 37 °C tocol. Samples were analyzed with the identical procedure used for the western for trypsin digestion. The samples were centrifuged, and the supernatants were blot assays with anti-HMGB2 as the primary antibody. collected for mass analysis. The residual peptides in gel pieces were further

extracted with 50% acetonitrile containing 20 mM (NH4)2CO3 and 5% formic LPS neuroinflammation model. All experiments were carried out on acid three times at room temperature. The combined peptide samples were 11-week-old male C57BL/6 mice (25–30 g). The animals were divided into condensed down in a SpeedVac until the desired sample concentration was four experimental groups in each experiment: group 1, treated with vehicle; reached. LC/MS/MS experiments were performed at National Instrumentation group 2, treated with ICM; group 3, treated with LPS and vehicle; and group Center for Environmental Management (NICEM) at Seoul National University. 4, treated with LPS and ICM. The ICM (2 or 10 mg per kg body weight) or A hybrid quadrupole-TOF LC/MS/MS spectrometer (Q-Star Elite) had a nano- vehicle (distilled water containing 5% DMSO and 40% polyethylene glycol) electrospray ionization source and was fitted with a fused silica emitter tip. For was administered i.p. daily for 4 d. LPS (from Escherichia coli 055:B5; Sigma) each LC/MS/MS run, 1–2 μg of the fractionated peptides was injected into was administered i.p. at a dose of 5 mg per kg body weight on day 2 for a the LC/MS/MS system, and the peptides were trapped and concentrated on an single challenge. Agilent Zorbax 300SB-C18 column. The peptide mixture was separated on an Agilent Zorbax 300SB nanoflow C18 column at a flow rate of 300 nl per min, EAE induction. C57BL/6 mice (7–8 weeks old, female) were immunized

and eluted peptides were electrosprayed through a coated silica tip (ion spray subcutaneously with 200 μg oMOG35–55 (MEVGWYRSPFSRVVHLYRNGK) voltage at 2,300 eV). ProteinPilot Software 2.0.1 (Software Revision Number: (GLBiochem) in 100 μl of a solution containing 50% complete Freund’s 67476) was used to identify peptides and proteins and quantify differentially adjuvant with 10 mg/ml of the heat-killed H37Ra strain of Mycobacterium expressed proteins. tuberculosis (Difco) in areas draining into the axillary and inguinal lymph nodes. Pertussis toxin (200 ng per mouse; List Biological Laboratories) in Nature America, Inc. All rights reserved. America, Inc. © 201 4 Nature Pulldown assay. Cell lysates were prepared according to the same protocol PBS was administered i.p. on the day of immunization and again 48 h later. used for the in-gel analysis before click chemistry. The proteome was reacted Animals were weighed and examined for disease symptoms daily. Clinical μ with biotin-azide (50 M) instead of Cy5-azide. The mixture was precipitated signs of disease were scored using a 0–5 scale, as follows: 0, no clinical signs; 1, according to the same protocol used for the in-gel analysis. The pellets were

npg limp tail; 2, weakness and incomplete paralysis of one or two hindlimbs; 3, dissolved in PBS containing 1.2% SDS using sonication and then diluted with complete hindlimb paralysis; 4, forelimb weakness or paralysis; and 5, mori- PBS containing 0.2% SDS. The samples were incubated with avidin beads for bund state or death. Vehicle or ICM was injected i.p. daily for 15 d after 2 h at room temperature and washed with PBS several times. Samples were MOG immunization. denatured in Laemmli sample buffer with heating and analyzed by SDS-PAGE and immunoblotting for HMGB2. Histological analysis. Mice were anesthetized with diethyl ether, transcardially perfused with cold saline and then perfused with 4% PFA diluted in Western blot analysis. The proteomes were analyzed by SDS-PAGE and trans- 0.1 M PBS. Brains or lumbar spinal cords were fixed using 4% PFA for 3 d ferred to PVDF membranes. The membranes were blocked with 2% BSA and then cryoprotected with a 30% sucrose solution for 3 d. Three animals in TBST for 1 h. The membranes were incubated overnight at 4 °C with the were used per experimental group. Fixed brains and spinal cords were embed- specific primary antibody (MAPK signaling: ERK, JNK, p38 (1:1,000 dilu- ded in OCT compound (Tissue-Tek, Sakura Finetek) for frozen sectioning tion, 4695, 9252, 9212, Cell Signaling) and their phosphorylated forms (9101, and then sectioned coronally at 20 μm. To detect microglial activation in 9251, 9211, Cell Signaling); HMGB1 and HMGB2 (1:2,000 dilution, ab67282, LPS-injected brains or microglial activation, neurites and demyelination in ab18256, Abcam)) to detect the desired proteins and then washed with TBST. EAE spinal cords, sections were incubated with rabbit anti–Iba-1 antibody The resulting membranes were exposed to HRP-conjugated secondary anti- (1:500 dilution, MB100-1028, Novus), FluoroMyelin (1:300 dilution, F34651, body (1:5,000 dilution, 7074, Cell Signaling) for 1 h at room temperature. After Invitrogen), anti–MAP-2 (1:200 dilution, M9942, Sigma) or anti-MBP anti- washing, the membranes were developed using an enhanced chemilumines- bodies (1:200 dilution, ab24567, Abcam). Sections were visualized directly or cence (ECL) detection kit, and the chemiluminescent signal was detected by incubated with Cy3-conjugated anti-rabbit IgG antibody (711-165-152, Jackson an imaging system. Laboratory). Sections were stained with hematoxylin and eosin to assess siRNA-mediated knockdown assay. The siRNA transfection of BV-2 micro- inflammatory lesions. glial cells was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. At 48 h after transfection, the cells were used Spinal cord microglia isolation. Spinal cords were homogenized in HBSS for experiments. with collagenase and DNase. The resulting homogenates were passed through a nylon cell strainer and centrifuged at 500g for 6 min. Supernatants were Subcellular fractionation and secretion analysis. After treatment with a com- removed, and cell pellets were resuspended in 37% isotonic Percoll (Amersham pound or LPS for 18 h, cells were lysed in subcellular fraction buffer (250 mM Biosciences) at room temperature. A discontinuous Percoll density gradient

nature chemical biology doi:10.1038/nchembio.1669 was set up as follows: 70%, 37%, 30% and 0% isotonic Percoll. The gradient SPSS software. Data represent the mean and s.d or s.e.m., as indicated in the was centrifuged for 20 min at 2,000g, and microglia cells were collected from individual figure legends. The P value summary related to DMSO, mock trans- the interphase between the 37% and 30% Percoll layers. Microglial cells were fection or vehicle is indicated in the individual figure legends. washed and then resuspended in sterile HBSS. Databases. MS results in the target identification were searched against a Statistics. Analyses of in vitro experiments and LPS-induced in vivo experi- National Center for Biotechnology Information (NCBI) or International Protein ments were performed with Student’s t test using GraphPad Prism. Analysis Index (IPI) database using the Paragon and Pro Group algorithms. The X-ray of the EAE experiment was performed with the Mann-Whitney U test using crystal used in the docking simulation is from Protein Data Bank (PDB). Nature America, Inc. All rights reserved. America, Inc. © 201 4 Nature npg

doi:10.1038/nchembio.1669 nature CHEMICAL BIOLOGY