T-Cell Activation-Inhibitory Assay to Screen Caloric Restriction Mimetics

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550 Biol. Pharm. Bull. 44, 550–556 (2021) Vol. 44, No. 4 Regular Article

T-Cell Activation-Inhibitory Assay to Screen Caloric Restriction Mimetics Drugs for Drug Repositioning Shouma Ishikawa, Atsushi Sawamoto, Satoshi Okuyama, and Mitsunari Nakajima* Department of Pharmaceutical Pharmacology, College of Pharmaceutical Sciences, Matsuyama University; 4–2 Bunkyo-cho, Matsuyama, Ehime 790–8578, Japan. Received November 4, 2020; accepted February 3, 2021

We previously reported a screening method for caloric restriction mimetics (CRM), a group of plant- derived compounds capable of inducing good health and longevity. In the present study, we explored the possibility of using this method to screen CRM drugs for drug repositioning. The method, T-cell activation- inhibitory assay, is based on inductive logic. Most of CRM such as resveratrol have been reported to suppress T-cell activation and have anti-inﬂammatory functions. Here, we assessed the activity of 12 antiallergic drugs

through T-cell activation-inhibitory assay and selected four that showed the lowest IC50 values—ibudilast (IC50 0.97 µM), azelastine (IC50 7.2 µM), epinastine (IC50 16 µM), and amlexanox (IC50 33 µM)—for further in- vestigation. Because azelastine showed high cytotoxicity, we selected only the remaining three drugs to study their biological functions. We found that all the three drugs suppressed the expression of interleukin (IL)-6, an inflammatory cytokine, in lipopolysaccharide-treated macrophage cells, with ibudilast being the strongest suppressor. Ibudilast also suppressed the secretion of another inflammatory cytokine, tumor necrosis factor (TNF)-α, and the expression of an inflammatory enzyme, cyclooxygenase-2, in the cells. These results suggest that T-cell activation-inhibitory assay can be used to screen potential CRM drugs having anti-inflammatory functions for the purpose of drug repositioning. Key words caloric restriction mimetics; drug repositioning; interleukin-6; inflammation

INTRODUCTION suggesting the possibility of repositioning this drug to treat coronavirus pneumonia disease (COVID-19). In this report, Caloric restriction (CR) is known to contribute to good we propose another approach of drug repositioning using an health and longevity.1) We are exploring practical approaches experimental assay system. to identify caloric restriction mimetics (CRM) that can mimic In a previous study, we developed a bioassay for screening the CR benefits without actually restricting caloric intake, plant-derived CRM.8) The screening system, denominated as considering that restricting calories could be difficult for hu- T-cell activation-inhibitory assay, is based on inductive logic. mans in the long term. According to Lane et al.2) and Ingram Most of the CRM known to induce good health and longevity, et al.,3) a CRM should 1) mimic the metabolic, hormonal, and such as resveratrol, butein, and fisetin, have been reported to physiological effects of CR; 2) significantly reduce long-term suppress T-cell activation, promote the signalling of the lon- food intake; 3) activate CR-like stress response pathways gevity gene SIRT1, and perform other anti-inflammatory func- and provide protection against a variety of stressors; and 4) tions.9–12) We tried to apply the screening method for plant- produce CR-like effects on longevity, reduction of age-related derived substances to medicinal drugs, because some groups diseases, and function maintenance. reported, using microarray-based gene expression analysis, that Ashburn and Thor4) recently defined drug repositioning several drugs that modulate glucose and lipid metabolism, or as the process of finding new uses outside the scope of the inflammation, could be potential CRM.13,14) For this purpose, original medical indication for existing drugs. There are ap- we examined 12 antiallergic drugs in the T-cell activation- proximately 16000 items listed in the Drug Price Standard inhibitory assay and found that all the three drugs showing in Japan. However, the compound targets and mechanisms T-cell activation-inhibitory activity among the 12 drugs have of action of 70–80% of these drugs are not fully known.5) In anti-inflammatory properties like plant-derived CRM. addition, most drugs are characterized according to their pri- mary therapeutic effects and side effects. Thus, there is scope MATERIALS AND METHODS for finding new targets and therapeutic benefits for these drugs other than those listed initially. Several approaches for drug Animals Female BALB/c mice were purchased from SLC repositioning have been described previously. Kaneko and (Shizuoka, Japan) at 6–7-weeks of age, maintained under con- Nagashima6) proposed the idea of drug repositioning based on trolled temperature and light (23 °C and 12-h light–dark, re- clinical evidence from a voluntary reporting database, such spectively) and used at 7–8-weeks of age, while receiving food as the U.S. Food and Drug Administration (FDA) Adverse and water ad libitum. All experimental procedures followed Event Reporting System (FAERS). Yamamoto et al.7) reported the Guidelines for Animal Experimentation of the Animal that nafamostat mesylate, a drug used for disseminated in- Care and Use Committee of Matsuyama University (Matsu- travascular coagulation (DIC), inhibits severe acute respira- yama, Japan). The numbers of certifications that verified the tory syndrome coronavirus 2 (SARS-CoV-2) infection in vitro, approval of the study were 20-009 on 2020.3.24, 18-007 on

2019.3.22, 17-008 on 2018.4.1, and 16-014 on 2017.4.1. signal-regulated kinase (ERK) (Cell Signalling #9101), rabbit Spleen Cell Culture To examine T-cell activation, single- anti-total ERK (Merck #ABS44, Darmstadt, Germany), rab- cell suspensions were prepared by mincing the mouse spleen bit anti-p-inhibitor of kappa B (IκB) (Cell Signalling #2859), tissues and passing them through a 40-µm nylon mesh (Fal- rabbit anti-total IκB (Cell Signalling #4812) antibodies; all pri- con, Corning, NY, U.S.A.). The cell suspensions were then mary antibodies were detected at 1 : 2000 dilution with horse treated with ACK lysis buffer (0.15 M NH4Cl, 1 mM KHCO3, radish peroxidase (HRP)-conjugated anti-rabbit immunoglobu- and 0.1 mM Na2 ethylenediaminetetraacetic acid (EDTA)) for lin G (IgG) antibody (Cell Signalling #7074) and re-probed 5 min, and red blood cells were removed from the spleen cell with HRP-conjugated anti-α-tubulin (11H10) (Cell Signalling suspensions. After washing with Hank’s balanced salt solution #9099). Immunoreactive bands were visualized using ECL- (Thermo Fisher Scientific, Waltham, MA, U.S.A.), the result- prime (GE Healthcare #RPN2236, Little Chalfont, U.K.) and ing pellets were used as spleen leukocytes, including a small the band intensities were quantified using a ChemiDoc™ percentage of T cells. For T-cell activation-inhibitory assay, the touch imaging system (Bio-Rad #1708370, Hercules, CA, spleen leukocytes were stimulated with 1 µg/mL each of coated U.S.A.). Data are presented as the fold-change of control anti-CD3 (#100314, BioLegend, San Diego, CA, U.S.A.) and mean ± standard error of the mean (S.E.M.). soluble anti-CD28 (#102112, Bio-Legend) antibodies for 48 h in Enzyme-Linked Immunosorbent Assay (ELISA) the presence of assay samples. The assay was performed using RAW264.7 cells were treated with/without samples at the culture medium containing indicated concentrations (0–100 µM) indicated concentrations. Three hours later, LPS (0111E. coli of samples and/or 0.1% dimethyl sulfoxide (DMSO), and incuba- B4, L2630; Sigma-Aldrich, St. Louis, MO, U.S.A.) was added tions were performed at 37 °C in a humidified atmosphere con- to the culture at a concentration of 10 ng/mL and the culture taining 5% CO2. The culture medium contained 45 mL of RPMI media were harvested 24 h later for ELISA. Tumor necrosis 1640 (Thermo Fisher Scientific), 50 µL of 2-mercaptoethanol factor (TNF)-α and interleukin (IL)-6 in the culture media (Thermo Fisher Scientific) (final concentration: 55 µM), 0.5 mL were measured with Mouse TNF-α ELISA MAX Deluxe Set of penicillin/streptomycin/glutamine (Thermo Fisher Scien- (Bio Legend #430904) and Mouse IL-6 ELISA MAX Deluxe tific) (final concentrations: 120 Units/mL, 100 µg/mL, 2 mM, Set (Bio Legend #431304), respectively. respectively), and 5 mL of heat-inactivated foetal calf serum Assay Samples Sodium cromoglycate, tranilast, pemi- (FCS) (Thermo Fisher Scientific) (final concentration: 10%). rolast, amlexanox, ibudilast, ketotifen fumarate, azelastine To assess T-cell activation, we used a 3-(4,5-dimethylthiazol- hydrochloride, epinastine hydrochloride, fexofenadine hy- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation drochloride, ozagrel hydrochloride, and seratrodast were kit (Roche, Basel, Switzerland), following the manufacturer’s commercially obtained from Tokyo Chemicals Industry instructions. MTT is reduced to form purple formazan crystals (Tokyo, Japan). Ramatroban was obtained from CAYMAN in metabolically active cells. Thus, the appearance of formazan CHEMICAL (Ann Arbor, MI, U.S.A.), U0126 was obtained crystals suggests the existence of anti-CD3/CD28 antibody- from Nacalai Tesque, and SB203580 was obtained from Ther- activated T-cells in spleen leukocyte cultures. The formazan mo Fisher Scientific. crystals were solubilised in 78% DMSO-containing medium and Statistical Analyses Data are expressed as mean ± S.E.M. the optical density (OD) value (570/655) was measured. We de- The data were analysed using one-factor analysis of variance

fined IC50 as the concentration of compounds needed to inhibit followed by Tukey’s multiple comparison test. p < 0.05 was anti-CD3/CD28 antibody-induced T-cell activation by 50%, as considered significant. measured by the OD value. Mouse Monocyte Macrophage RAW264.7 Cell Culture Mouse monocyte macrophage RAW264.7 cells were purchased from DS Pharma Biomedical (Osaka, Japan) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Nacalai Tesque, Kyoto, Japan) supplemented with 10% FCS and 0.1% penicillin/streptomycin/glutamine (Thermo Fisher Scientific, San Diego, CA, U.S.A.). Cytotoxicity Assay RAW264.7 cells were treated with/ without samples at the indicated concentrations. Three hours later, lipopolysaccharide (LPS; 0111E. coli B4, L2630; Sigma- Aldrich, St. Louis, MO, U.S.A.) was added to the culture at a concentration of 10 ng/mL, and the cells were cultured for 24 h. To assess the cytotoxicity of drugs, we used the MTT cell proliferation kit (Roche, Basel, Switzerland), following the manufacturer’s instructions. Western Blot Analysis RAW264.7 cells were treated with/without samples at the indicated concentrations. Three hours later, LPS was added to the culture at a concentration of 10 ng/mL and the cells were harvested 3 or 24 h later for West- Fig. 1. Summary of T-Cell Activation-Inhibitory Assay ern blotting. Blots were probed at 1 : 1000 dilution with rabbit Mouse spleen leukocytes were stimulated with anti-CD3/CD28 antibodies for anti-cyclooxygenase (COX)-2 (D5H5) (Cell Signalling #12282, 48 h in the presence of samples, including the CRMs and the antiallergic drugs, and Tokyo, Japan), rabbit anti-inducible nitric oxide synthase the MTT assay was performed. IC50 values of the 12 antiallergic drugs are plotted. Note the low IC50 values of ibudilast, azelastine, epinastine, and amlexanox, com- (iNOS) (Cell Signalling #13120), rabbit anti-p-extracellular parable to those of typical CRM such as resveratrol (IC50 12 µM). 552 Biol. Pharm. Bull. Vol. 44, No. 4 (2021)

RESULTS inhibitory assay.8) We assessed 12 antiallergic drugs using this assay, including mediator isolation suppressing agents (cro- In a previous study, we developed a bioassay for screen- moglycate, tranilast, pemirolast, amlexanox, and ibudilast), ing plant-derived CRM. The screening system, called the H1 blockers (ketotifen, azelastine, epinastine, fexofenadine), T-cell activation-inhibitory assay, is based on inductive logic. and thromboxane signalling inhibitors (ozagrel, seratrodast,

Most typical CRM, such as resveratrol, butein, and ﬁsetin, ramatroban). Of the 12 drugs, four had IC50 values of less than show IC50 values of less than 20 µM in the T-cell activation- 40 µM in the assay, including ibudilast (IC50 0.97 µM), azelas-

Fig. 2. Microscopic Observation of the Cultures of T-Cell Activation-Inhibitory Assay Mouse spleen leukocytes were stimulated with anti-CD3/CD28 antibodies for 48 h in the presence of the test samples. MTT was added to the cells, which changed to purple formazan crystals in activated T-cells. (A) No antibody stimulation and no sample. (B) Antibody stimulation without sample. Formazan crystals are visible in the activated T cells or around the cells. (C) Ibudilast (20 µM) with antibody stimulation. (D) Azelastine (20 µM) with antibody stimulation. (E) Epinastine (100 µM) with antibody stimulation. (F) Amlexanox (100 µM) with antibody stimulation.

Fig. 3. Cytotoxicity of Ibudilast, Azelastine, Epinastine, and Amlexanox Towards RAW264.7 Cells RAW264.7 cells were cultured with ibudilast, azelastine, epinastine, or amlexanox in the presence of LPS (10 ng/mL) for 24 h and the cytotoxicity was accessed with the MTT assay. Note that azelastine showed a strong cytotoxicity towards RAW264.7 cells in the range of 25–100 µM. n = 4. * indicates the significant difference (p < 0.05) between the control cultures treated with LPS only and the test cultures treated with LPS and drug. Vol. 44, No. 4 (2021) Biol. Pharm. Bull. 553 tine (IC50 7.2 µM), epinastine (IC50 16 µM), and amlexanox while amlexanox did not show significant change in viability (IC50 33 µM) (Fig. 1). T-cells among spleen leukocytes were at up to 100 µM (Fig. 3D). Azelastine caused serious decrease activated with anti-CD3/CD28 antibodies (Figs. 2A, B). The in viability at not less than 25 µM (Fig. 3B). We then assessed T-cell activation was blocked by the four drugs (Figs. 2C–F). the effect of ibudilast, epinastine, and amlexanox on the secre- Because the T-cell activation-inhibitory assay also detects tion of inflammatory cytokines (TNF-α and IL-6) from LPS- cytotoxic agents, we examined the cytotoxicity of the four induced RAW264.7 cells (Fig. 4). We ceased the assessment drugs in a macrophage cell line, RAW264.7, which is also of azelastine on the cytokine secretion, because the serious used in the following experiments to assess biological func- cytotoxicity complicates our understanding concerning the tions of the drugs. Ibudilast caused slight increase (Fig. 3A) effect of this drug on the cytokine secretion. The LPS-induced and epinastine caused slight decrease (Fig. 3C) in viability, secretion of TNF-α and IL-6 was largely attenuated by ibu-

Fig. 4. Effect of Ibudilast, Epinastine, and Amlexanox on the Secretion of Inflammatory Cytokines from RAW264.7 Cells RAW264.7 cells were cultured with ibudilast, epinastine, or amlexanox in the presence of LPS (10 ng/mL) for 24 h and TNF-α and IL-6 secreted from RAW264.7 cells into culture media were measured with ELISA. Note that all the three drugs attenuated the secretion of IL-6. n = 3. * indicates the significant difference (p < 0.05) between the control cultures treated with LPS only and the test cultures treated with LPS and drug.

Fig. 5. Ibudilast Inhibited COX-2 Expression but Not iNOS Expression RAW264.7 cells were cultured with ibudilast in the presence of LPS (10 ng/mL) for 24 h and analysed with Western blotting. COX-2 levels were significantly reduced by ibudilast (C, D), while iNOS levels were slightly increased. n = 4. * indicates the significant difference (p < 0.05) between the control cultures treated with LPS only and the test cultures treated with LPS and drug. 554 Biol. Pharm. Bull. Vol. 44, No. 4 (2021) dilast (Figs. 4A, B). Amlexanox also reduced the expression ment in the LPS-induced Raw264.7 cells (Fig. 6). We then of these cytokines, although to a lesser extent (Figs. 4E, F), examined the possible involvement of ERK signalling in the and epinastine reduced the secretion of IL-6 only slightly anti-inflammatory effects of ibudilast. We found that the phos- (Figs. 4C, D). As ibudilast was prominent on the reduction of phorylation levels of p-ERK were increased by LPS treatment, inflammatory cytokine secretion, we decided to explore addi- and the increase was significantly countered by ibudilast treat- tional anti-inflammatory functions of this drug. ment, although the levels of total ERK were not affected by Next, we examined the effect of ibudilast on the expres- LPS and ibudilast (Fig. 7). To confirm the role of ERK signal- sion of inflammation-related enzymes, iNOS and COX-2, ling in the anti-inflammatory effects of ibudilast, we treated and found that ibudilast largely attenuated the expression of the LPS-induced RAW264.7 cells with two mitogen-activated COX-2 and increased the expression of iNOS in the LPS- protein kinase signalling inhibitors, U0126 (ERK signalling induced RAW264.7 cells (Fig. 5). inhibitor) or SB203580 (p38 signalling inhibitor). We found Nuclear factor-kappa B (NF-κB) signalling and ERK sig- that the LPS-induced COX-2 expression was reduced by the nalling have been reported to be involved in the secretion of treatment of U0126 or SB203580 at the concentration of 10 µM, inflammatory cytokines induced by LPS. To probe the mecha- which concentration of the inhibitors was used for the experi- nism underlying the anti-inflammatory effects of ibudilast, we ments in monocyte-lineage cells.15,16) However, U0126 was more studied the effect of the drug on IκB expression which modulate effective on the reduction of COX-2 expression than SB203580 NF-κB signaling. We found that the levels of phosphorylated (Fig. 8), suggesting that LPS-induced inflammation conditions IκB and total IκB were not largely altered by ibudilast treat- in RAW264.7 cells are mainly mediated by ERK signalling.

Fig. 6. Ibudilast Did Not Signiﬁcantly Aﬀect NF-κB Signalling RAW264.7 cells were treated with ibudilast in the presence of LPS (10 ng/mL) for 3 h and analysed with Western blotting. Neither the degree of phosphorylation of IκB (A, B) nor the total IκB levels (C, D) were largely altered by ibudilast. n = 4.

Fig. 7. Ibudilast Inhibited ERK Signalling RAW264.7 cells were treated with ibudilast in the presence of LPS (10 ng/mL) for 3 h and were analysed with Western blotting. The levels of ERK phosphorylation (A, B) were significantly reduced, while total ERK levels in RAW264.7 cells were not changed (C, D). n = 3. * indicates the significant difference (p < 0.05) between the control cultures treated with LPS only and the test cultures treated with LPS and drug. Vol. 44, No. 4 (2021) Biol. Pharm. Bull. 555

inhibitory assay in screening potential CRM drugs having anti-inﬂammatory functions for drug repositioning. In the present study, we demonstrated that three drugs

showing lower IC50 values in T-cell activation-inhibitory assay reduced the secretion of inﬂammatory cytokine IL-6 from cultured macrophage cells. However, we have found that there exist diﬀerences in the sensitivity of the drugs between the two experiments of T-cell activation-inhibitory assay and IL-6

secretion assay. IC50 of ibudilast, epinastine and amlexanox in the T-cell activation-inhibitory assay were 0.97, 16, and 33 µM, respectively (Fig. 1), while those in the IL-6 secretion assay were approximately 10, 100, and 100 µM, respectively (Fig. 4). The T-cell activation-inhibitory assay using mouse spleen T-cells might highly sensitively respond to the drugs and the IL-6 secretion assay using a mouse macrophage cell line insensitively. We examined four proinflammatory indicators, the secretion of IL-6 and TNF-α and the protein expression of COX-2 Fig. 8. COX-2 Expression Was Completely Blocked by ERK Signalling and iNOS using LPS-treated macrophage cell line, for evalu- Inhibitor, but Not by p38 Signalling Inhibitor ating the anti-inflammatory effect of potential CRM drugs RAW264.7 cells were cultured with MAP kinase signalling inhibitors (U0126: ERK signalling inhibitor; SB203580: p38 signalling inhibitor) in the presence of screened with T-cell activation-inhibitory assay. LPS is known LPS (10 ng/mL) for 24 h and analysed with Western blotting. COX-2 expression to activate toll-like receptor 4 (TLR4), a transmembrane pro- induced by LPS was completely eliminated by U0126, but incompletely eliminated by SB203580. n = 3. * indicates the significant difference (p < 0.05) between the tein, which belongs to the pattern recognition receptor family. control cultures treated with LPS only and the test cultures treated with LPS and The activation of TLR4 by LPS stimulates NF-κB signaling drug. # indicates the significant difference (p < 0.05) between the cultures treated with LPS and U0126 and with LPS and SB203580. pathway and three mitogen-activated protein kinase (MAPK) pathways, leading to proinflammatory responses.19) The most interesting point in the present study is that the three drugs DISCUSSION of ibudilast, epinastine, and amlexanox each showed different anti-inflammatory responses. The only consistent feature The T-cell activation-inhibitory assay was originally de- of the three drugs is that all drugs showed the reduction of veloped to screen plant-derived potential CRM.8) In the pres- IL-6 secretion induced by LPS. Also, it is very interesting that ent study we examined the possibility of using the T-cell ibudilast reduced the expression of COX-2, but not iNOS, in- activation-inhibitory assay to screen CRM drugs for drug duced by LPS, while many anti-inflammatory compounds, but repositioning.4,5) Among the 12 antiallergic drugs that we not all, are reported to reduce the expression of both COX-2 20,21) tested through the assay, four showed IC50 values comparable and iNOS. Because NF-κB signaling pathway is known to the plant-derived candidate CRM.8) Three of these drugs to activate a promotor inducing the expression of both COX-2 that showed the lowest IC50 values, without showing serious and iNOS, it is presumed that the function of ibudilast is not cytotoxic effects, led to a reduction in the expression of the mediated by NF-κB signaling. In fact, the possibility of the inflammatory cytokine IL-6 in cultured mouse macrophage involvement of NF-κB signaling in the action mechanism of RAW264.7 cells. Ibudilast, which showed the highest inhibi- ibudilast was negated by the experiments of Fig. 6. tion of T-cell activation in the assay, was found to have the Although various studies have reported the anti-inflammatory most prominent anti-inflammatory properties. These results properties of ibudilast, its anti-inflammatory effects on suggest that the T-cell activation-inhibitory assay can be used RAW264.7 cells have never been reported before. We found for drug repositioning to screen potential CRM drugs, based that ibudilast reduced the secretion of inflammatory cyto- on an inductive logic, because most of CRM are known to kines TNF-α and IL-6 from LPS-induced RAW264.7 cells, be inflammation modulators.9–12) It could be possible that the and the expression of an inflammatory enzyme COX-2 in the potential CRM drugs screened with the T-cell activation- cells. Furthermore, in the experiments of COX-2 expression inhibitory assay could target various inflammatory diseases with an ERK inhibitor (Fig. 8), we demonstrated that the anti- such as sepsis, COVID-19, a chronic inflammatory bowel dis- inflammatory effects of ibudilast might be mainly mediated by ease, multiple sclerosis, neuropathic pain et al. the attenuation of ERK signalling. To confirm the hypothesis of Ibudilast was originally developed for the treatment of the action mechanism of ibudilast, we must perform additional asthma and dizziness in Japan; however, the drug’s applica- experiments concerning TNF-α and IL-6 secretions with the tion has been considered for various inflammatory diseases, ERK inhibitor in a future study. Our study shows that ibudilast such as multiple sclerosis17) and neuropathic pain.18) Recently, may be useful in the treatment of additional diseases related to an investigational new drug (IND) application for the use of inflammation other than asthma; however, further studies must ibudilast against COVID-19 was submitted to the US-FDA be performed before clinical applications. (https://www.biospace.com/article/releases/medicinova- The anti-inflammatory properties of amlexanox have also announces-opening-of-investigational-new-drug-applica- been reported previously, in which the anti-inflammatory tion-for-mn-166-ibudilast-for-prevention-of-acute-respira- effects of amlexanox in LPS-induced RAW264.7 cells were tory-distress-syndrome-in-patients-with-covid-19/). These mediated by phosphodiesterase 4B.22) Phosphodiesterases are developments reinforce the usefulness of T-cell activation- also known targets of ibudilast.23) This may suggest that drug 556 Biol. Pharm. Bull. Vol. 44, No. 4 (2021) screening through the T-cell activation-inhibitory assay may metabolic disorders by enhancing fat oxidation. 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