Experimental Anti-Inflammatory Drug Semapimod Inhibits TLR Signaling by Targeting the TLR Chaperone Gp96

Experimental Anti-Inflammatory Drug Semapimod Inhibits TLR Signaling by Targeting the TLR Chaperone gp96

This information is current as Jin Wang, Anatoly V. Grishin and Henri R. Ford of September 29, 2021. J Immunol 2016; 196:5130-5137; Prepublished online 18 May 2016; doi: 10.4049/jimmunol.1502135 http://www.jimmunol.org/content/196/12/5130 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Experimental Anti-Inﬂammatory Drug Semapimod Inhibits TLR Signaling by Targeting the TLR Chaperone gp96

Jin Wang,* Anatoly V. Grishin,*,† and Henri R. Ford*,†

Semapimod, a tetravalent guanylhydrazone, suppresses inﬂammatory cytokine production and has potential in a variety of inﬂam- matory and autoimmune disorders. The mechanism of action of Semapimod is not well understood. In this study, we demonstrate that in rat IEC-6 intestinal epithelioid cells, Semapimod inhibits activation of p38 MAPK and NF-kB and induction of cyclooxygenase-2 by

TLR ligands, but not by IL-1b or stresses. Semapimod inhibits TLR4 signaling (IC50 0.3 mmol) and acts by desensitizing cells to LPS; it fails to block responses to LPS concentrations of ‡5 mg/ml. Inhibition of TLR signaling by Semapimod is almost instanta- neous: the drug is effective when applied simultaneously with LPS. Semapimod blocks cell-surface recruitment of the MyD88 adapter, one of the earliest events in TLR signaling. gp96, the endoplasmic reticulum–localized chaperone of the HSP90 family

critically involved in the biogenesis of TLRs, was identified as a target of Semapimod using ATP-desthiobiotin pulldown and mass Downloaded from spectroscopy. Semapimod inhibits ATP-binding and ATPase activities of gp96 in vitro (IC50 0.2–0.4 mmol). On prolonged exposure, Semapimod causes accumulation of TLR4 and TLR9 in perinuclear space, consistent with endoplasmic reticulum retention, an anticipated consequence of impaired gp96 chaperone function. Our data indicate that Semapimod desensitizes TLR signaling via its effect on the TLR chaperone gp96. Fast inhibition by Semapimod is consistent with gp96 participating in high-affinity sensing of TLR ligands in addition to its role as a TLR chaperone. The Journal of Immunology, 2016, 196: 5130–5137. http://www.jimmunol.org/ emapimod (CNI-1493, N, N’-bis [3, 5-diacetylphenyl] The mechanism of action of Semapimod is not well understood. decanediamide tetrakis [amidinohydrazone]) was initially It inhibits activating phosphorylation of MAPKs of p38, JNK, and S designed as a bulky arginine mimetic to limit arginine trans- ERK families in response to inflammatory stimuli (21–23), but it port and NO production during inflammation (1). In addition to the does not directly inhibit these kinases. Although Semapimod di- expected inhibition of inflammatory cytokine-induced arginine rectly inhibits c-Raf (21), a MAPK kinase kinase upstream of transport in macrophages, Semapimod attenuated inflammation and ERK, this does not explain blockade of activation of p38 and JNK, protected against lethal endotoxemia (1). Inhibition of arginine which are independent of c-Raf. Semapimod has been found to uptake and NO production was not the only mechanism responsible directly inhibit deoxyhypusine synthase, an enzyme that catalyzes for the anti-inflammatory effect of Semapimod: the drug inhibited posttranslational modification of the translation initiation factor by guest on September 29, 2021 LPS-induced inflammatory cytokine release by macrophages at 5A, which might explain the antiviral and antimalaria effects (9, concentrations at least 10 times lower than that required for the 24), but not the blockade of inflammatory cytokine production. inhibition of arginine uptake (2). Semapimod inhibits inflammatory Upon intracranial injection, Semapimod potently activates the responses not only in macrophages/monocytes, but also in other cholinergic anti-inflammatory pathway involving the vagus cell types, including endothelial cells (3), dendritic cells (4), and nerve (25, 26); however, this does not explain the drug’s anti- enterocytes (5). Since its discovery, Semapimod has been reported inflammatory effects in vitro. to have a beneficial effect in a broad range of experimental and The intestinal epithelium becomes largely refractory to the TLR clinical inflammatory conditions, such as acute endotoxemia (6, 7), ligands following bacterial colonization, which has been exten- bacterial infection (8), malaria (9), arthritis (10, 11), autoimmune sively demonstrated in the adult, microbiota-associated intestine. encephalomyelitis (12), Alzheimer disease (13), pancreatitis (14), However, the naive epithelium of the neonates possesses TLR re- allograft rejection (15), cancer (16, 17), postoperative ileus (18, 19), sponsiveness similar to that of the professional innate immune cells and Crohn disease (20, 21). (5, 27–31). TLR signaling in the epithelium plays critical role in the pathogenesis of necrotizing enterocolitis, a disease coincident with the onset of bacterial colonization of the gut (29, 32–34). We are *Division of Pediatric Surgery, Children’s Hospital Los Angeles, Los Angeles, CA interested in Semapimod because it improves outcomes of experi- † 90027; and Department of Surgery, University of Southern California, Los Angeles, mental necrotizing enterocolitis (35). Because Semapimod is not CA 90027 absorbed in the intestine (19), it is an attractive drug for organ- ORCID: 0000-0001-9118-9512 (H.R.F.). targeted therapy of intestinal inflammatory disorders. Received for publication October 1, 2015. Accepted for publication April 18, 2016. In this study, we demonstrate that in enterocytes, Semapimod This work was supported by National Institutes of Health Grant R01 AI014032 inhibits TLRs by targeting their common molecular chaperone gp96. (to H.R.F.). Address correspondence and reprint requests to Dr. Anatoly V. Grishin, Division of Pediatric Surgery, Children’s Hospital Los Angeles MS35, 4661 Sunset Boulevard, Materials and Methods Los Angeles, CA 90027. E-mail address: [email protected] Cell culture and reagents The online version of this article contains supplemental material. Abbreviations used in this article: COX-2, cyclooxygenase-2; ER, endoplasmic re- IEC-6, HEK293, and SW480 cell lines were grown as recommended by the ticulum; iNOS, inducible NO synthase; NECA, N-ethyl carboxamidoadenosine; supplier (American Type Culture Collection, Manassas, VA). IEC-6 cells NP-40, Nonidet P-40; Pam3CSK4, tripalmytoyl cysteine-serine-(lysine)4. were used at passages 17–28. For all experiments, cells were plated at 4 to 5 3 104/cm2 and grown overnight to 70–90% confluence. Cell viability Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 was determined by Trypan blue staining. Reagents were purchased from www.jimmunol.org/cgi/doi/10.4049/jimmunol.1502135 The Journal of Immunology 5131 the following suppliers: Semapimod, Medkoo Biosciences (Chapel Hill, activated charcoal was removed by centrifugation. Radioactivity of the clear NC); recombinant canine gp96, catalog number ADI-SPP-766, Enzo Life supernatant was measured by Cerenkov counting. In control reactions, gp96 Sciences (Farmingdale, NY); recombinant human HSP90, catalog number was substituted for equivalent amount of autoclaved porcine collagen. Fol- SPR-101A, StressMarq Biosciences (Victoria, BC, Canada); LPS from lowing background subtraction, data were expressed as percent of charcoal- Escherichia coli 0127:B8, MG132, geldanamycin, radicicol, and N-ethyl absorbed radioactivity in the absence of inhibitor. carboxamidoadenosine (NECA), Sigma-Aldrich (St. Louis, MO); tripalmytoyl Statistical methods cysteine-serine-(lysine)4 (Pam3CSK4), Tocris Bioscience (Bristol, U.K.); ultrapure flagellin from Salmonella typhimurium, Invivogen (San Diego, Quantitative data were expressed as means 6 SD. Data were compared CA); recombinant rat IL-1b, PeproTech (Rocky Hill, NJ); peroxynitrite, using unpaired t test. Cayman Chemical (Ann Arbor, MI); and the ATP-desthiobiotin kit, Thermo Scientific (Rockford, IL). Abs were from the following sources: gp96 (H-212) and TLR4 (H80), Santa Cruz Biotechnology (Santa Cruz, Results CA); TLR9 (SAB2104136) and FLAG M2, Sigma-Aldrich; phospho-p38, Semapimod specifically blocks responses to a subset of TLR p38, phospho-MAPK kinase 3/6, and IkBa, Cell Signaling Technology ligands (Danvers, MA); cyclooxygenase-2 (COX-2), Cayman Chemical; inducible NO synthase (iNOS), BD Biosciences (San Jose, CA); MyD88, Because Semapimod has been reported to inhibit a variety of in- Abcam (Cambridge, MA); and HSP90, StressMarq Biosciences. Syn- flammatory responses, it was not clear what aspects of inflammation thetic oligonucleotide 59-TCGTCGTTTCGTCCGGCGCGCCGG-39 was are affected. To define targets of Semapimod in the inflammatory used as CpG DNA. cascade, we examined effects of this drug on inflammatory responses in TLR4 plasmid and transient transfection intestinal epithelial cells elicited by a variety of stimuli. The IEC-6 cell line was chosen because these are primary untransformed

The mTLR4-Flag plasmid (catalog number 13087; constructed by Downloaded from R. Medzhitov) was obtained from Addgene (Cambridge, MA). HEK293 epithelioid cells with presumably intact innate immune machinery. human embryonic kidney cells were transiently transfected using the calcium Pretreatment with Semapimod blocked LPS-induced, but not IL-1b– phosphate precipitate method. Control cells were transfected with the empty induced, activating phosphorylation of p38 MAPK and its up- pFLAG-CMV2 vector (Sigma-Aldrich). stream activators MAPK kinases 3/6 (Fig. 1A). These protein kinases Western blots and immunofluorescence are critical mediators of transcriptional induction of COX-2, the rate- Standard procedures were used as described previously (5). For quantitative limiting enzyme in the biosynthesis of inflammatory prostanoids. http://www.jimmunol.org/ protein measurements, band densities on underexposed Western blots MAPK kinases 3/6 mediate inflammatory cytokine- and stress- were determined using GelDoc imaging system and Quantity One software induced, but not LPS-induced, expression of COX-2 in entero- (Bio-Rad, Hercules, CA). Immunofluorescence images were acquired on cytes (5). As expected, Semapimod also blocked LPS-induced but LSM 700 confocal system (Carl Zeiss Microimaging, Thornwood, NY). not IL-1b–induced expression of COX-2 (Fig. 1B). Semapimod For comparisons, sections were mounted and processed on the same slide. Identical acquisition settings and image adjustments were used. Surface failed to block IL-1b–induced expression of another key inflam- and 1-mm subsurface signal intensity was measured using the ImageJ matory factor, iNOS. iNOS was not appreciably induced by LPS software (National Institutes of Health) by scanning randomly chosen cells either in the presence or absence of Semapimod (Fig. 1B). Induction along a horizontal line across the center of the nucleus. of COX-2 by osmotic shock, oxidative stresses, or proteasome blockade was unaffected (Fig. 1C). Semapimod blocked activating Immunoprecipitation by guest on September 29, 2021 phosphorylation of p38 MAPK induced by Pam3CSK4 and CpG The standard procedure was used (Santa Cruz Biotechnology), except that DNA, the agonists of TLR2 and -9, respectively (Fig. 1D), but not cells were lysed in Nonidet P-40 (NP-40) buffer (1% NP-40, 100 mmol NaCl, 20 mmol Tris [pH 8], and 0.5 mmol PMSF). by the TLR5 agonist flagellin (Fig. 1F). Semapimod did not significantly affect cell viability (Fig. 1E), which rules out effects of ATP-desthiobiotin pulldown and identification of gp96 alarmons released from dead cells. Thus, Semapimod appears to

IEC-6 cells were lysed with NP-40 buffer containing 2.5 mmol MgCl2 specifically block responses mediated by a subset of TLRs includ- for 10 min at 4˚C, and the lysate was cleared by high-speed centrifuga- ing TLR2, -4, and -9. tion. Incubation with ATP-desthiobiotin, adsorption to streptavidin-agarose beads, washing, and elution were performed as recommended by the Semapimod blocks TLR-mediated activation of NF-kB manufacturer. Eluted proteins were separated on 20-cm 10% polyacryl- amide gel, and protein bands were visualized by silver staining. Protein To elucidate whether Semapimod blocks responses mediated by bands were identified at CHLA Proteomics Core. Briefly, bands excised proinflammatory signaling pathways other than p38, we examined from gel were digested with trypsin, and resulting peptides were identified effects of this drug on activation of the NF-kB pathway, as judged using liquid chromatography-mass spectroscopy. Protein identity was by the levels of the inhibitory subunit IkBa.IkBa is rapidly established by database search for matching peptides. degraded in response to inflammatory stimuli, which relieves in- gp96 ATP binding hibition of the NF-kB and leads to transcriptional activation of inflammatory genes. Semapimod blocked LPS-induced (Fig. 2A), but The 20-ml reactions (20 mmol HEPES [pH 7.2], 50 mmol NaCl, 2.5 mmol 35 not IL-1b–induced (Fig. 2B), degradation of IkBa. Semapimod also MgCl2, 0.1 mmol DTT, 200 mmol [100 mCi/ml] ATP-g-[ S], and 100 mg/ml gp96, with or without Semapimod) were incubated 30 min at 37˚C and blocked degradation of IkBa induced by CpG DNA and Pam3CSK4 loaded onto drained and precooled 1 ml Sephadex G25 spin columns (GE (Fig. 2D), but not by flagellin (Fig. 2E). Thus, Semapimod blocks Healthcare Life Sciences). The columns were spun for 2 min at 2000 rpm TLR ligand–induced activation of both p38 MAPK and NF-kB and 4˚C. Radioactivity of flow-through was determined by scintillation signaling, suggesting that the blockade occurs early in the signaling counting. In control reactions, gp96 was substituted for equivalent amount of autoclaved porcine collagen. Following background subtraction, data cascade, before branching into the p38 and NF-kBpathways. 35 were expressed as percent of ATP-g[ S] binding in the absence of the Semapimod desensitizes responses to TLR ligands inhibitor. According to Fig. 2A, Semapimod inhibits degradation of IkBa ATPase induced by LPS concentrations up to 1 mg/ml, but is ineffective at The 20-ml reactions (20 mmol HEPES [pH 7.2], 50 mmol NaCl, 2.5 mmol 5 mg/ml, suggesting that inhibition can be overcome by high con- 32 MgCl2, 0.1 mmol DTT, 20 mmol [100 mCi/ml] g-[ P] ATP, and 100 mg/ml centration of LPS. To examine this effect in more detail, we studied gp96 or HSP90, with or without Semapimod) were incubated 3 h at 37˚C. 0.5 ml 5% w/v suspension of activated charcoal (Norit A) equilibrated with inhibition at various concentrations of LPS and Semapimod. In the 20 mmol HEPES (pH 7.2), 50 mmol NaCl, 5 mmol EDTA, and 200 mmol absence of Semapimod, LPS caused a dose-dependent increase in ATP was mixed with samples, and after 10-min agitation at room temperature, p38 phosphorylation at concentrations between 1 and 100 ng/ml 5132 SEMAPIMOD INHIBITS gp96 Downloaded from

FIGURE 2. Semapimod blocks activation of NF-kB by a group of TLR ligands. (A and B)LevelsofIkBa in cells pretreated with or without 10 mmol http://www.jimmunol.org/ Semapimod and treated with LPS or IL-1b as indicated. (C)LevelsofIkBa after pretreatment with 10 mmol Semapimod and treatment with 100 ng/ml

LPS, 1 mg/ml CpG DNA, or 1 mg/ml Pam3CSK4,asindicated.(D) Levels of IkBa following pretreatment with or without Semapimod and treatment with 100 ng/ml LPS or indicated concentrations of ﬂagellin. Results are representative of at least three independent experiments.

FIGURE 1. Effects of Semapimod on proinflammatory responses in Therefore, Semapimod acts almost instantaneously, but is ineffective once the response has been initiated. IEC-6 cells. (A) Activating phosphorylation of p38 MAPK and MAPK by guest on September 29, 2021 kinase (MKK) 3/6 MAPKK following 15 min pretreatment with 10 mmol Semapimod blocks LPS-induced cell surface localization of Semapimod and 15 min treatment with 100 ng/ml LPS or 1 ng/ml IL-1b, MyD88 as indicated. (B) Levels of iNOS, COX-2, and b-actin proteins following 15 min pretreatment with 10 mmol Semapimod and 12 h treatment with Recruitment of the cytosolic adapter protein MyD88 to the plasma 100 ng/ml LPS or 1 ng/ml IL-1b, as indicated. (C) Levels of COX-2 membrane-localized TLRs is one of the earliest events in in- protein following 15 min pretreatment with (filled boxes) or without (open flammatory signaling (36). To test whether Semapimod affects boxes) 10 mmol Semapimod and 12 h treatment with: 1 ng/ml IL-1b; 100 this event, we examined changes in intracellular localization of ng/ml LPS; osmotic stress (Osm) with 0.5 mol glycerol; oxidative stress MyD88 following stimulation with LPS, with or without pre- with 200 mmol H2O2 or 20 mmol peroxynitrite (PN); or protein misfolding treatment with Semapimod. In untreated cells, MyD88 localized stress with 3 mmol proteasome inhibitor MG132. COX-2 levels are relative to myddosomes, granule-like structures dispersed throughout the to b-actin levels. (D) Activating phosphorylation of p38 MAPK after pretreatment with or without 10 mmol Semapimod for 15 min and treat- cell (37). LPS caused pronounced localization of MyD88 to the cell surface in the absence, but not in the presence of Semapimod ment for 15 min with 100 ng/ml LPS, 1 mg/ml Pam3CSK4,or1mg/ml CpG DNA, as indicated. (E) Levels of cell death following 24-h incubation of (Fig. 4). Therefore, Semapimod blocks LPS-induced cell surface IEC-6 cells with or without 10 mmol Semapimod as indicated. (F) Acti- localization of MyD88, consistent with blockade of MyD88 re- vating phosphorylation of p38 MAPK after pretreatment with 10 mmol cruitment to the TLR4 complexes on the cell surface. Semapimod and treatment with LPS or indicated concentrations of flagellin. All data are representative of at least three independent experi- Semapimod targets glycoprotein-96 ments. Ctrl, control, untreated cells. Because TLRs associate with multiple ATP-binding proteins including protein kinases TNFR-associated factor 6, TAK1, RIP2 (38, 39), and heat shock proteins (40, 41), we performed a pulldown (Fig. 3A). Semapimod concentrations from 0.02 to 10 mmol pro- assay for ATP-binding proteins in the presence or absence of gressively shifted the response curve to the right (Fig. 3A). At any Semapimod. Cell lysates prepared from IEC-6 cells treated with or concentrationupto10mmol, Semapimod failed to significantly without LPS in the presence or absence of Semapimod were in- inhibit response to $5 mg/ml LPS. The IC50 of Semapimod for cubated with ATP-desthiobiotin, a reagent that covalently binds to response to 20–1000 ng/ml LPS is ∼0.3 mmol (Fig. 3B). Similar ATP-binding sites in proteins. ATP-desthiobiotin–modified proteins results were obtained for inhibition of responses to CpG DNA, the were then collected on streptavidin-agarose beads, and bound pro- agonist of TLR9 (Supplemental Fig. 1). Thus, Semapimod appears teins were analyzed by gel electrophoresis and silver staining. LPS to desensitize responses to TLR ligands in a dose-dependent fashion. treatment did not cause any detectable change in the spectra of We next examined the time course of inhibition. Semapimod bound proteins. However, Semapimod treatment resulted in disap- inhibited LPS-induced IkBa degradation when applied before or pearance of a prominent band of ∼100 kDa, regardless of LPS simultaneously with, but not after application of LPS (Fig. 3C). treatment (Fig. 5A). This band was excised, and the corresponding The Journal of Immunology 5133

FIGURE 4. Semapimod blocks LPS-induced recruitment of MyD88 to cell surface. Top panel, Anti-MyD88 immunostaining following 10-min treatment with 100 ng/ml LPS, with or without 15-min pretreatment with 2 mmol Semapimod as indicated. Arrowheads indicate localization of MyD88 to the cell surface. Scale bar, 5 mm. Similar results were obtained in four independent experiments. Bottom panel: Ratios of surface to sub- Downloaded from surface MyD88 signal in cells treated with or without LPS and Semapimod, as indicated. n = 40 in each treatment group. *Signiﬁcant difference from other groups, p , 0.0001. NS, normal rabbit serum.

Semapimod inhibits ATP-binding and ATPase activities of

gp96 http://www.jimmunol.org/ Protection from modification by ATP-desthiobiotin by Semapimod indirectly indicates that the latter inhibits ATP-binding and/or ATPase activity of gp96. To test such inhibition directly, we examined effects of Semapimod on ATP binding and ATPase activities of purified gp96 in vitro. ATP binding was measured by incubating the gp96 protein with 35S-labeled ATP-gS, a non- hydrolysable analog of ATP, followed by spin column size- exclusion chromatography on Sephadex G25 (GE Healthcare Life Sciences), and determining radioactivity of the high m.w. by guest on September 29, 2021 A FIGURE 3. Semapimod desensitizes responses to LPS. ( ) Phospho-p38/ (flow-through) fraction. Semapimod inhibited ATP binding in a p38 ratio as function of LPS concentration in the presence of 0, 0.2, 0.5, and B dose-dependent fashion (IC50 0.4 mmol, Fig. 6A). ATPase ac- 10 mmol Semapimod. ( ) Phospho-p38/p38 ratio following treatment with 32 indicated concentrations of LPS in mg/ml as function of Semapimod tivity was measured by incubating gp96 with g-[ P] ATP, fol- concentration. Values are percentages of full activation (100 ng/ml LPS lowed by adsorption of released inorganic phosphate on activated for 15 min) in the absence of Semapimod. SD of the individual points charcoal and determining the radioactivity of unhydrolysed ATP. are #20%. (C) Time course of inhibition by 2 mmol Semapimod of Semapimod inhibited gp96 ATPase in a dose-dependent fashion IkBa degradation induced by 15-min treatment with 100 ng/ml LPS. (IC50 0.3 mmol, Fig. 6B), which is close to the IC50 for ATP Time of addition of Semapimod is relative to the beginning of LPS binding and innate immune response inhibition in IEC-6 cells treatment. (Fig. 3, Supplemental Fig. 1). At the concentration that completely inhibits gp96 ATPase, Semapimod did not appreciably inhibit the ATPase activity of HSP90, the cytosolic paralog of gp96 (Fig. 6B, protein was identified as gp96 using trypsin digestion, liquid inset). Therefore, Semapimod directly and specifically inhibits chromatography, and database-linked mass spectroscopy. The ATP-binding and ATPase activities of gp96. identity of the ∼100-kDa species as gp96 was further con- firmed by Western blot. gp96-immunoreactive species was not Comparisons of Semapimod to other gp96 inhibitors recovered by ATP-desthiobiotin pulldown if cells were treated To gain an additional insight into the mechanism of action of with Semapimod (Fig. 5B). Interestingly, Semapimod did not Semapimod, we sought to elucidate whether known gp96 inhibitors interfere with the ATP-desthiobiotin pulldown of HSP90, the cy- block LPS signaling like Semapimod and whether Semapimod tosolic paralog of gp96 (Fig. 5B). These results demonstrate that inhibits trafficking of TLR signaling complexes similar to other Semapimod specifically abrogates modification of gp96 with ATP- gp96 inhibitors. To answer the first question, we examined LPS- desthiobiotin. induced IkBa degradation and p38 MAPK phosphorylation in gp96 is a glycoprotein chaperone of the HSP90 family that IEC-6 cells pretreated with geldanamycin, radicicol, or NECA. facilitates folding, assembly, and trafficking of a limited number of These drugs have been shown to inhibit gp96 by associating with client proteins, most notably TLR signaling complexes (42–46). its nucleotide-binding pocket (51–55). Geldanamycin and radicicol gp96 resides in the endoplasmic reticulum (ER) and possesses indeed inhibited responses to LPS, however, only upon prolonged intrinsic ATPase activity, which is required for its chaperone exposure of $3 h (Fig. 7A). By contrast, Semapimod blocked LPS function (47–50). gp96 deficiency obliterates TLR signaling due signaling almost instantaneously (Fig. 3C). NECA failed to block to failure of trafficking of functional TLR complexes to the cell LPS signaling at any concentration tested up to 20 mmol (Fig. 7A). surface (44). It was therefore plausible that Semapimod inhibits Although geldanamycin and radicicol inhibit LPS signaling, their multiple TLRs via its effect on gp96. effect is slow, which implies a different mechanism of action and is 5134 SEMAPIMOD INHIBITS gp96

FIGURE 5. Semapimod abrogates modification of gp96 with ATP- desthiobiotin. (A) Silver-stained gel of IEC-6 proteins modified by ATP- desthiobiotin and collected on streptavidin-agarose. Cells were pretreated with 10 mmol Semapimod for 15 min and then treated with LPS for 15 min as indicated. The prominent protein band that is present or absent depending on Semapimod treatment is indicated by arrowhead. (B) Im- munoblot analysis of IEC-6 proteins modified by ATP-desthiobiotin. Cells were treated with or without Semapimod, and ATP-desthiobiotin-modified proteins were analyzed by Western blotting with anti-gp96, anti-HSP90, and anti–b-actin Abs. b-Actin is pulled down by ATP-desthiobiotin be- Downloaded from cause it is an ATP-binding protein. Data are representative of at least three independent experiments. M, marker lanes; positions of protein size markers are indicated on the left.

consistent with the blockade of TLR trafficking to the cell surface. http://www.jimmunol.org/ To answer the second question, we examined effects of Semapimod on subcellular localization of TLR4 and TLR9 in SW480 enterocytes. This cell line of human origin allowed the use of proven anti- FIGURE 6. Semapimod inhibits ATP-binding and ATPase activities of human TLR4 and TLR9 Abs. Responses of SW480 cells to LPS gp96. (A) Effect of Semapimod on ATP-binding activity of gp96. (B)Ef- and CpG DNA are similar to those of IEC-6 cells. The bulk of TLRs fect of Semapimod on ATPase activity of gp96. Inset:effectofSemapimod localized to granules dispersed throughout the cell body, as previ- on ATPase activity of HSP90. Values are percentages of activities in the ously reported (56, 57). Exposure to Semapimod caused dramatic absence of Semapimod. Error bars show SD. The data are from the four accumulation of both TLRs in the perinuclear space (Fig. 7B), independent assays. which is consistent with retention in the ER. Thus, Semapimod by guest on September 29, 2021 affects subcellular localization of TLRs in a manner expected of an purified gp96 in a dose-dependent fashion in vitro. Like the known inhibitor of gp96 chaperone function. In contrast, Semapimod is gp96 inhibitors geldanamycin and radicicol, Semapimod impedes different from geldanamycin and radicicol in its rapid effect on trafficking of TLR4. However, Semapimod inhibits TLR4 signaling TLR4 signaling, which is consistent with inhibition of the pre- much faster than geldanamycin or radicicol, which is consistent existing TLR4 signaling complexes. Semapimod is similar to the with direct inhibition of TLR signaling, but not with indirect effect other two gp96 inhibitors in its ability to block intracellular traf- via impaired receptor trafficking. ficking of the TLRs (Fig. 8). The fact that Semapimod does not alter the dose-response curves Geldanamycin has been reported to disrupt gp96-TLR com- for TLR ligands, but rather shifts them to higher ligand concen- plexes, as judged by coimmunoprecipitation (42). To test whether trations, indicates that this drug acts by reducing the affinity of Semapimod acts by dissociating gp96-TLR complexes, we tran- TLRs to their ligands rather than by inhibiting receptor signaling. siently transfected HEK293 cells with FLAG-tagged TLR4 and Even at highest Semapimod concentrations tested, the responses, examined effect of Semapimod on coimmunoprecipitation of gp96 including p38 phosphorylation or IkBa degradation, were not and TLR4. Semapimod failed to block coimmunoprecipitation of inhibited as long as high concentrations of TLR ligands were used. these two proteins (Fig. 7C), indicating that its effects on TLR4 This mode of action is consistent with a model by which the high, does not involve physical dissociation of gp96-TLR4 complexes. but not the low affinity of TLR receptor complexes to their cog- nate ligands depends on gp96 (Fig. 8). Discussion Several lines of evidence argue that Semapimod blocks innate In this report, we demonstrate that in primary enterocyte cell immune responses at the level of TLRs. First, because this drug culture, Semapimod inhibits signaling by agonists of TLR2, -4, and inhibits neither IL-1b–induced activation of NF-kB or expression -9, but not by TLR5 agonist, the inflammatory cytokine IL-1b,or of iNOS, nor IL-1b– or stress-induced activation of p38 MAPK cellular stresses. Effects of Semapimod on responses to LPS are or expression of COX-2, it does not appear to directly target dose-dependent with regard to concentrations of both Semapimod the intracellular NF-kB or p38 MAPK signaling cascades. and LPS. In cells treated with Semapimod, higher concentrations The blockade of TLR ligand–induced IkBa degradation and p38 of LPS are required to elicit the same response than in untreated MAPK phosphorylation indicates that Semapimod targets a cells, and Semapimod fails to block responses to LPS applied at common upstream activator shared by NF-kB and p38 MAPK concentrations of $5 mg/ml. gp96, an ER-associated chaperone of pathways, which is consistent with blockade at the receptor level. the HSP90 family, which is critically involved in assembly and Second, Semapimod apparently decreases the affinity of the TLR4 trafficking of the TLR signaling complexes, was identified as a signaling complex to its ligand, LPS. At concentrations up to 10 target of Semapimod using a pulldown assay for the ATP-binding mmol, Semapimod has no effect on responses to LPS applied at proteins. Semapimod inhibits ATP binding and ATPase activity of high concentrations. Such behavior is most easily explained by The Journal of Immunology 5135

FIGURE 8. Model of Semapimod effects on TLR receptor complexes. Downloaded from Semapimod rapidly shifts cell-surface TLR complexes into the low-afﬁnity state by inhibiting cell-surface gp96. In addition, by inhibiting intracellular gp96 (dashed arrow), Semapimod blocks TLR trafﬁcking from the ER to the cell surface (vertical arrows).

via its effect on gp96. Close IC50 values for inhibition of TLR sig- http://www.jimmunol.org/ naling and for inhibition of gp96 ATP binding and ATPase activities FIGURE 7. Comparisons between Semapimod and other gp96 inhibi- provide further support for this idea. Insensitivity of IL-1b responses tors. (A)IkBa and phospho-p38 levels after pretreatment with 8 mmol to Semapimod is in agreement with known independence of IL-1R geldanamycin, 8 mmol radicicol, or 20 mmol NECA and treatment with biogenesis from gp96 (45). Although our data strongly implicate 100 ng/ml LPS as indicated. (B) TLR4 and TLR9 immunofluorescence in gp96, they cannot formally rule out other targets of Semapimod in SW480 cells before (left panel) and after (right panel) 3-h treatment with 2 TLR signaling. However, auxiliary proteins shared by IL-1R and mmol Semapimod. Scale bar, 5 mmol. (C) Coimmunoprecipitation of TLRs, as well as receptor-specific auxiliaryproteinssuchasMD-2or FLAG-tagged TLR4 and gp96 from lysates of HEK293 cells transiently CD14, can be excluded as targets. If main receptor subunits (TLR2, expressing FLAG-TLR4, with or without 10 mmol Semapimod. Cells were by guest on September 29, 2021 -4, and -9 proteins) are targets, one would expect different IC50 for treated as indicated with Semapimod for 2 h prior to lysis, and the drug each receptor, which was not the case. was also added to cell lysates. All data are representative of at least three Semapimod is similar to geldanamycin and radicicol, the two independent experiments. NS, normal rabbit serum. known gp96 inhibitors, in its ability to block intracellular trafficking of the TLRs and thus to act as one would expect of a true targeting the receptor complex, but it is inconsistent with blockade gp96 inhibitor. However, unlike the other two inhibitors for which of a downstream signaling mediator. Third, Semapimod blocks the effect on TLR4 signaling is slow, Semapimod inhibits TLR4 recruitment of the MyD88 adapter, the earliest detectable event in signaling fast. Unlike geldanamycin or radicicol, Semapimod fails TLR signaling. Because TLRs and IL-1R share the key elements to inhibit the ATPase activity of the closely related HSP90 and is of their downstream signaling cascades (58), the failure of Semapimod thus not a generic HSP90 family inhibitor. A plausible explanation to inhibit signaling from the IL-1R also argues against a downstream for Semapimod’s fast effect on responses to LPS is inhibition by signaling mediator as target. this drug of gp96 associated with cell-surface high-affinity TLR Using a pulldown assay for ATP-binding proteins, we have signaling complexes. This explanation is consistent with increased identified gp96, the ER paralog of the HSP90 chaperone, as a sensitivity to LPS upon surface expression of gp96 protein (43) or direct target of Semapimod. Judging by abrogation of gp96 treatment with low concentrations of extrinsic gp96 (59), as well pulldown by Semapimod in the ATP-desthiobiotin-streptavidin as competition between gp96 and LPS for cell-surface binding assay, this drug interferes with modification of gp96 by ATP- (60). desthiobiotin, an ATP derivative that covalently attaches to the Our results provide mechanistic explanation as to why Sem- ATP-binding pockets in proteins. Blockade of ATP-desthiobiotin apimod is effective in experimental NEC. The neonates are gen- modification indirectly indicated that Semapimod inhibits the erally supersensitive to the TLR ligands (31). The naive intestinal ATP-binding activity of gp96. To corroborate inhibition of ATP epithelium of the neonates responds to TLR ligands (27, 28, 30), binding, we examined effects of Semapimod on ATP-binding and and these responses may play critical role in the pathogenesis ATPase activities of purified gp96 and found that both were inhibited of NEC (32–34, 61). Thus, Semapimod may prevent NEC by in a dose-dependent fashion and with similar IC50. It remains un- blocking TLR ligand signaling in the neonatal intestine. known whether Semapimod inhibits ATP binding/ATPase activities Our study provides an insight into the roles of gp96 in TLR of gp96 by interaction with the ATP-binding pocket or an allosteric signaling complexes. It is generally believed that gp96 participates site. X-ray crystallography is needed provide an answer to this in TLR signaling by facilitating correct folding, plasma membrane question. insertion, and trafficking of receptor signaling complexes to the The facts that Semapimod inhibits TLR signaling at the receptor cell surface (40). If this is true, one could expect gp96 inhibitors to level and that it directly targets gp96, the essential chaperone for block TLR signaling slowly, as pre-existing plasma membrane TLRs (45, 46), strongly argue that Semapimod inhibits TLR signaling TLR complexes should not be affected. Slow (hours) inhibition of 5136 SEMAPIMOD INHIBITS gp96 responses to LPS is what we indeed observed using the classic 10. Larsson, E., H. E. Harris, K. Palmblad, B. Ma˚nsson, T. Saxne, and L. Klareskog. 2005. CNI-1493, an inhibitor of proinflammatory cytokines, retards cartilage de- gp96 inhibitors geldanamycin and radicicol. However, unlike struction in rats with collagen induced arthritis. Ann. Rheum. 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