Tranilast Blocks the Interaction between the Protein S100A11 and Receptor for Advanced Glycation End Products (RAGE) V Domain and Inhibits Cell Proliferation*

The human S100 calcium-binding protein A11 (S100A11) is a member of the S100 protein family. Once S100A11 proteins bind to calcium ions at EF-hand motifs, S100A11 changes its conformation, promoting interaction with target proteins. The receptor for advanced glycation end products (RAGE) consists of three extracellular domains, including the V domain, C1 domain, and C2 domain. In this case, the V domain is the target for mutant S100A11 (mS100A11) binding. RAGE binds to the ligands, resulting in cell proliferation, cell growth, and several signal transduction cascades. We used NMR and fluorescence spectroscopy to demonstrate the interactions between mS100A11and RAGE V domain. The tranilast molecule is a drug used for treating allergic disorders. We discovered that the RAGE V domain and tranilast would interact with mS100A11 by using 1H-15N HSQC NMR titrations. According to the results, we obtained two binary complex models from the HADDOCK program, S100A11-RAGE V domain and S100A11-tranilast, respectively. We overlapped two binary complex models with the same orientation of S100A11 homodimer and demonstrated that tranilast would block the binding site between S100A11 and the RAGE V domain. We further utilized a water-soluble tetrazolium-1 assay to confirm this result. We think that the results will be potentially useful in the development of new anti-cancer drugs.

Human S100 proteins are a family of low molecular weight, homodimeric, and acidic proteins that contain two EF-hand motifs that can bind to calcium ions (1,2). Human S100A11 (S100C) protein is a member of the S100 family. S100A11 was first discovered in chicken gizzards and is also called calgizzarin (3). S100A11 protein can be expressed in the heart, kidney, liver, lung, and other tissues. It is most abundant in smooth muscle tissues (4). In previous studies, it has been shown that S100A11 protein can interact with various other proteins, such as annexin A1 and A2, ATPase Rad54B, and RAGE. 2 The inter-actions induce activities or conformational changes of these target proteins and further promote the specific physiological functions. Calcium-bound S100A11 homodimer acts as a bridge to link two N termini of annexin proteins, inducing plasma membrane vesiculation in cells (5,6). Interaction between S100A11 and DNA repair and recombination protein Rad54B participates in the repair of double-stranded DNA and regulation of the cell cycle (7). Furthermore, S100A11-RAGE interaction is correlated with the inflammation of chondrocytes (8). It has been reported that overexpression of S100A11 protein is associated with a number of cancers, including breast (9), colon (10), pancreatic (11), and papillary thyroid carcinoma (12). However, overexpression of S100A11 protein results in the contrary effect in ovarian (13) and bladder cancer (14). Reduced expression of S100A11 protein is correlated with progression of ovarian and bladder cancer, which has been investigated in previous studies.
RAGE, the receptor of the immunoglobulin for signal transductions at the cell surface, is a 35-kDa protein (15,16). RAGE consists of one cytoplasmic domain at the C terminus, one transmembrane domain, two distinct constant domains (C1 and C2), and one variable domain at the N terminus (V) (17). The variable domain is predominantly related to ligand binding, such as that seen in most S100 family proteins (18), advanced glycation end products (19,20), amyloid-␤ protein (21), and amphoterin (HMGB1 (high mobility group protein 1)) (22,23). Some ligands can interact with both the variable domain and constant domain simultaneously, such as S100B (24) and S100A12 protein (25,26). Furthermore, S100A6 protein can even interact with V, C1, and C2 domains (27,28). However, the mechanisms by which some S100 family proteins bind to RAGE are still shrouded in mystery. The cytoplasmic domain is a tail domain with various charged residues that are correlated to autophosphorylation and activation of intracellular signal transduction pathways (29). Once ligands bind to the RAGE, they would trigger homodimerization of two RAGEs, followed by mutual phosphorylation of two cytoplasmic domains and signal transduction cascades (30 -32). These signal transductions induce inflammation, cell proliferation and migration, and even tumor growth. However, which pathway will be activated depends on the type and concentration of binding ligands in different cells (33). RAGE has been reported in many diseases, such as Alzheimer disease (34), diabetic complication (35), chronic vascular inflammation (36), and even some cancers (37)(38)(39). Therefore, insight into the interactions between RAGE and its ligands is important for development of treatments for RAGE-dependent diseases.
The tranilast molecule (N-(3, 4-dimethoxycinnamoyl) anthranilic acid) is a drug used for anti-allergy treatment (40). Tranilast was initially developed to treat bronchial asthma in children (41). Subsequently, it also has been used for the treatment of other allergic disorders, such as allergic rhinitis (42) and allergic dermatitis (43). Recently, tranilast has been developed to be an anti-proliferative drug for neurofibroma cells in vitro (44).
Different S100 proteins were reported to bind to different domains of RAGE. However, the V domain of RAGE was reported to bind different S100 proteins. For example, S100B was reported to bind the V domain (24,45). With a surface plasmon resonance experiment, Heizmann and co-workers (18) showed that S100A2, S100A5, and S100A12 bound to the V domain of RAGE. Although the interaction between S100A11 and RAGE has been mentioned in previous studies, they were not characterized at the molecular level (46). In this study, we seek to find out insight into the interaction of S100A11 with the RAGE V domain and construct a model for demonstrating a S100A11-RAGE V domain heterotetrameric complex at the molecular level. Binding interfaces of mutant S100A11 (mS100A11) and the RAGE V domain are identified using NMR spectroscopy (47). We determined the binding constant (K d ) of the mS100A11-RAGE V domain complex by fluorescence spectroscopy. The HADDOCK (high ambiguity-driven protein-protein docking) program was used to approach the residues at the interfaces of two proteins that we obtained from NMR 1 H-15 N HSQC titrations (48). Furthermore, tranilast was identified to be an inhibitor that could interact with mS100A11 and block the interface of S100A11 with the RAGE V domain in this study. These findings were sustained by the results of NMR 1 H-15 N HSQC titrations, fluorescence experiments, and HAD-DOCK modeling. We further proved the inhibition properties of tranilast by a water-soluble tetrazolium-1 (WST-1) assay (49). The putative complex models and result of the WST-1 assay provide prospective insight into the development of new drug treatments for RAGE-dependent diseases.

Experimental Procedures
mS100A11 Expression and Purification-There are two free cysteines in the S100A11 molecule. In this case, DTT must be used in the buffer for NMR experiments to ensure that S100A11 contains cysteines in its free form. However, when the structure determination of the S100A11-RAGE V domain complex is performed by NMR, DTT should be avoided because the RAGE V domain contains one disulfide bond. This disulfide bond would be broken by DTT. To solve this problem, we constructed mS100A11. Residues Cys-13 and Cys-91 were mutated to serine, so that the NMR experiments could be executed without DTT. Because the 1 H-15 N HSQC spectra of wild type and the mutant form of S100A11 are similar, we believe that the three-dimensional structure of mutant and wild-type are alike. The cDNA of the mutant S100A11 (C13S and C91S) was constructed and purchased from Mission Biotech Co. We used pET20b (ϩ) as a vector that was transformed and overexpressed in Escherichia coli BL21 host cells (Novagen). We pre-pared 15 N-labeled mS100A11 protein and cultured the E. coli cells in M9 medium, containing [ 15 N]ammonium chloride as the sole nitrogen source. The cells were cultured at 310 K until the A 600 nm reached ϳ0.7, and then 1 mM isopropyl ␤-D-thiogalactopyranoside was added for the induction of protein expression with 200-rpm shaking at 298 K. After 21 h of overnight induction, the cells were then centrifuged at 6,000 rpm for 20 min, followed by resuspension in buffer A (25 mM Tris-HCl, 100 mM NaCl, 5 mM CaCl 2 , pH 7.5). The cell lysis was performed three times by using a French press at 1,000 psi. We removed the lysate by centrifugation at 13,000 rpm and 4°C for 45 min. The supernatant was then purified by a Hi-Prep Phenyl FF 16/10 column on an AKTA FPLC system (GE Healthcare). The unwanted proteins were washed out by buffer A, and mS100A11 would bind to the column because of hydrophobic interactions. After that, buffer B (elution buffer containing 25 mM Tris-HCl, 100 mM NaCl, 5 mM EDTA, pH 7.5) was used to detach the target protein mS100A11 from the column. Finally, we used a Hi-load 16/60 Superdex 75 column (GE Healthcare) that was equilibrated with buffer A as the second column for buffer exchange and final purification.
RAGE V Domain Expression and Purification-Recombination of RAGE V domain was performed using pET-32b (ϩ) vector and transformed into Rosetta host cells (Novagen). 15 N-Labeled RAGE V domain proteins were also prepared in M9 medium. The cells were cultured at 310 K until the A 600 nm reached 0.7; then we added 1 mM isopropyl ␤-D-thiogalactopyranoside for induction with 200-rpm shaking at 298 K for 3 h. After induction, the cells were centrifuged at 6,000 rpm for 20 min, followed by resuspension in buffer C (20 mM Tris-HCl, 300 mM NaCl, pH 8.0). The cell lysis was performed by using the sonicator (Qsonica) for 45 min in an ice bath. The lysate was removed by centrifugation at 13,000 rpm and 4°C for 45 min. The supernatant was purified using an Ni Sepharose 6 fast flow column (GE Healthcare), which was designed for scaling up purification of histidine-tagged proteins. At first, unwanted impurities that had no binding affinity with the nickel column were washed out with 50 ml of buffer C. Then we used 50 ml of buffer D (20 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, pH 8.0) to wash out the unwanted proteins that had weak binding with the nickel column, followed by elution of the target protein, which shows strong binding with the nickel column using 50 ml of buffer E (elution buffer; 20 mM Tris-HCl, 300 mM NaCl, 500 mM imidazole, pH 8.0). We concentrated the collection of elution to 2 ml, and then the His tag, which contained six continuous histidine units at the N terminus, was removed by using thrombin cleavage (1 unit/mg protein) in a water bath at 25°C for 3 h. After that, the RAGE V domain-containing fraction was purified using Hitachi L-7000 system HPLC with an Atlantis dc18 Prep Column (Waters) as the final purification. The molecular weights of both the mS100A11 and RAGE V domain proteins were confirmed by electrospray ionization-mass spectrometry.
Fluorescence Experiments-All of the fluorescence experiments were performed under the same buffer conditions, using buffer A. The RAGE V domain has three tryptophan residues that could be excited and emit fluorescence. The fluorescence experiments were performed using the F-2500 fluorescence spectrophotometer (Hitachi). We used a wavelength of 295 nm as the excitation light source, and the RAGE V domain has a maximum emission band at a wavelength of 351 nm. We detected the wavelength of emission in the range of 310 -440 nm. Tranilast also has fluorescence emission. We found that tranilast has an absorption band at 310 nm. Therefore, we used a wavelength of 310 nm to be the excitation light source, and tranilast has a maximum emission band at a wavelength of 335 nm. We added increasing concentrations of mS100A11 (0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, and 11.5 M) to the tranilast solution at a concentration of 5 M.
The data that we acquired from both fluorescence experiments were plotted as 1/[mS100A11] versus 1/(I Ϫ I 0 ). We utilized the following equation to a fit linear curve in the Origin program, which was used to calculate the dissociation constant of two samples (50), where I 0 , I, and I 1 are representations of emission intensities in the absence of mS100A11, in the middle concentration of mS100A11, and in the maximal concentration of mS100A11 that we used, respectively. ⌬F is the difference of fluorescence intensity between I and I 0 . ⌬F max is the maximal fluorescence change, and K d is the dissociation constant.

Results
Tryptophan Residue Fluorescence Measurements-The intrinsic fluorescence of tryptophan residues is very sensitive to structural changes of proteins and the environmental polarity around the tryptophan when substrates are binding (51). Tryptophan residues exhibit maximum emission ranging from 340 to 360 nm with excitation at 295 nm in highly polar environments. However, maximum emission of tryptophan residues is 330 -350 nm in hydrophobic environments (52). There are three tryptophan residues in the RAGE V domain, Trp-51, Trp-61, and Trp-72, respectively. Excited by a wavelength of 295 nm, the maximum emission of the RAGE V domain is at 351 nm (53). Residue Trp-61 of the RAGE V domain is at the binding site; therefore, we monitored the change of fluorescence intensity at 351 nm upon excitation at 295 nm. Increasing mS100A11 resulted in a decreasing RAGE V domain fluorescence intensity in titration experiments (Fig. 1A). According to the curve that we fitted with the fluorescence intensity changes versus the mS100A11 concentrations as one binding site model, we obtained a K d of ϳ0.5 M (Fig. 1B). Overall, the fluorescence results showed the binding between the RAGE V domain and mS100A11 that is on the lower micromolar level, provided that the conformation of the protein complex is very stable under normal conditions of physiology.
Tranilast Fluorescence Measurements-Tranilast has two benzyl groups, so that it also has fluorescence emission. We monitored the change of tranilast fluorescence intensity at 335 nm upon excitation at 310 nm. The increasing fluorescence intensities of tranilast were detected by increasing the concentration of mS100A11 (Fig. 1C). The dissociation constant that we obtained was 11.75 M (Fig. 1D). This dissociation constant is also present at the micromolar level, indicating the formation of a stable complex.
Molecular Docking-We used the HADDOCK program to dock S100A11 and RAGE V domain (or tranilast) and obtain a binary S100A11-RAGE V domain (or S100A11-tranilast) complex model (54). In this case, we utilized the structure of wildtype S100A11 (Protein Data Bank entry 2LUC) taken from the Protein Data Bank as the structure of mS100A11 (55). The three-dimensional structure of RAGE V domain was also taken from the Protein Data Bank (entry 2E5E) (56). The three-dimensional structure of tranilast was taken from DrugBank (accession number DB07615), and its structure was given for reference (Fig. 2). The signals of residues that exhibited chemical shift perturbations or intensities decreasing on HSQC titration experiments were performed. It indicated that these residues were located in the interface region of two proteins. We used HADDOCK to identify these residues as ambiguous interaction restraints. The active and passive residues of the proteins were predicted by the NACCESS program (57). NACCESS defines that the area of active residues for accessible surface is Ͼ30%, including the side chain and backbone. In contrast, the residues whose area is Ͻ30% are defined as passive. For docking S100A11 with the RAGE V domain, the 2,000 total structures were set for rigid body docking by the standard HADDOCK protocol with optimized potential for liquid simulation parameters (58). Then 800 structures were set for semiflexible refinement, and the 200 lowest energy structures were analyzed. On the other hand, the 8,000 total structures for rigid body docking, 1,000 structures for semiflexible refinement, and 800 lowest energy structures were finally analyzed for docking S100A11 with tranilast. Results of the calculation were classified into clusters that contained similar structures with small root mean square deviation. We demonstrated the results of structures with the PyMOL program (59). mS100A11 and RAGE V Domain Binding Interface-Using the two-dimensional 1 H-15 N HSQC NMR spectrum is very useful for identifying the binding interface of the protein-protein complex (60). In the interfacial residues of the Ca 2ϩ -mS100A11 and RAGE V domain complex, it was observed that the chemical shift perturbations and intensities of peaks were changed compared with free mS100A11 without the RAGE V domain on 1 H-15 N HSQC. We overlapped HSQC of 15 N-labeled free mS100A11 and of the 15 N-labeled mS100A11 complex with the unlabeled RAGE V domain (Fig. 3A). Decreasing intensities and chemical shift perturbations of NMR signals were observed at a ratio of 1:1 for the [ 15 N]mS100A11 complex with unlabeled RAGE V domain. The decreasing intensities and shift perturbations of signals result from complex formation, which makes the environment of the residues change at the binding interface. This formation caused an obvious decrease in the intensities and perturbations of amide cross-peaks on 1 H-15 N HSQC spectra. To find out which residues interacted with the RAGE V domain, the peak intensities of 15 N-labeled mS100A11 in complex with unlabeled RAGE V domain (I) on 1 H-15 N HSQC compared with free mS100A11 (I 0 ) and chemical shift perturbations were plotted in bar diagrams (Fig. 3, B and C, respectively). The equation for the perturbation calculation was derived by the NMR spectroscopy research group of Utrecht University (61). Bar diagram analyses indicated that most of the residues that exhibited decreasing intensities or perturbations were located at the hydrophobic region of mS100A11, including Ala-48, Ala-49, Thr-51, Lys-52, Asn-53, Gln-54, Asp-68, Glu-79, Phe-80, Gly-85, Leu-87, Ala-88, and Ala-90. These residues are colored in red (Fig. 3D). Most of these residues are at helix 2 and 4 of S100A11. In reverse titration, we observed signal decreasing on 1 H-15 N HSQC only using the 15 N-labeled RAGE V domain with unlabeled mS100A11 at a ratio of 1:1 (Fig. 4A). We also used a bar diagram to find the affected residues, such as Asn-54, Thr-55, Trp-61, Lys-62, Val-63, Leu-64, Ser-65, Asp-73, Gly-95, and Arg-98 (Fig. 4B). These residues are labeled in red on the structure of the RAGE V domain (Fig. 4C). The role of these residues, which are accessible for solvent as predicted by NACCESS, was further substantiated by the analysis of the S100A11 complex with the RAGE V domain. Overall, labeled residues on each protein exhibited the binding interface of the protein-protein complex.
The S100A11-RAGE V Domain Complex Model-By HSQC titration experiments, we identified the binding regions of pro-  Block Interaction between S100A11 and RAGE V Domain JULY 1, 2016 • VOLUME 291 • NUMBER 27 teins and looked for the structural model of the S100A11 in complex with the RAGE V domain. Most of these residues of mS100A11 are buried in the hydrophobic site, forming a binding interface upon calcium binding. Most affected residues also form a continuous binding interface in the structure of the RAGE V domain. We set these two binding interfaces of proteins to form a binary complex model using HADDOCK 2.2. The 2,000 complex structures were produced by using rigid body minimization, followed by further water refinement to determine the best 200 structures with the lowest total energy. We chose 20 backbone structures with the lowest energy, which were overlapped to demonstrate the similarity of the calculated structures (Fig. 5A). The mS100A11 residues Asn-53, Gln-54, and Ala-90 (shown in red) interact with residues Trp-61, Val-63, Ser-65, and Asp-73 of the RAGE V domain (shown in yellow), as shown in Fig. 5B. We used the PROCHECK analysis of the European Bioinformatics Institute on their Web site to check the structure stereochemistry of the complex (62). The Ramachandran plot and the results show that area of the disallowed regions is only 0.6%, and that of the most favored regions is about 81.1%. The overall score average of G-factors is 0.13; it is thus in the usual range (data not shown).
Binding Interface between mS100A11 and Tranilast-For the residues at the interface of Ca 2ϩ -mS100A11 in complex with tranilast, it was observed that only chemical shifts were changed compared with free mS100A11 without tranilast in 1 H-15 N HSQC spectra. We overlapped HSQC of 15 N-labeled free mS100A11 and 15 N-labeled mS100A11 complex with tranilast (Fig. 6A). In this case, no decreasing signals were observed, and thus we can choose the perturbed residues directly. We found that most residues were located at helix 2, helix 3, and helix 4 of S100A11. These residues are colored in red: Leu-47, Ala-48, Thr-51, Asn-53, Leu-60, Arg-62, Leu-87, and Ala-90 (Fig. 6B).
Structural Model of the mS100A11 in Complex with Tranilast Molecule-Most affected residues of mS100A11 are also at the hydrophobic site and form a continuous interface to interact with tranilast. We set S100A11 and the tranilast molecule to form a binary complex model using the HADDOCK with restraints obtained from chemical shift perturbations in HSQC spectra. The 8,000 complex structures were produced from rigid body minimization, followed by further water refinement to the best 800 structures with the lowest total energy. HAD-DOCK analyzed these 800 lowest energy structures and grouped them into a number of clusters. We chose the complex with the best symmetry between two tranilast molecules in the first cluster and demonstrated S100A11 in complex with tranilast in PyMOL. Labeling affected residues on the S100A11 homodimer reveals that two tranilast molecules bind to the hydrophobic site of each S100A11 monomer symmetrically (Fig. 7A). The mS100A11 residues Asn-53, Leu-87, and Ala-90 The selected residues are shown in red bars. C, bar diagram analysis of the perturbations of the cross-peaks of the mS100A11 complex with RAGE V domain compared with free mS100A11. The dark red line represents the criterion of selected residues that exhibited greater perturbations (Ͼ0.06). The selected residues are shown in red bars. Perturbations were calculated by using the equation, combined shift difference ϭ ((proton shifts) 2 ϩ (nitrogen shifts/6.51) 2 ) 0.5 (60). D, selected residues were labeled in red on the three-dimensional structure of homodimeric S100A11 (green) using the PyMOL program.
interact with tranilast (Fig. 7B). We also used the PROCHECK analysis to check the structure stereochemistry of S100A11 in complex with tranilast. We produced a Ramachandran plot, and the results show that the area of the disallowed regions is 0.0%, and that of the most favored regions is about 81.9%. The overall score average of G-factors is Ϫ0.04; it is also in the usual range (data not shown).
WST-1 Cell Proliferation Assay-For the cell proliferation assay, WST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) was used as a reagent for the experiments. The SW480 cell is a cell line with high expression of RAGE and low expression of S100A11 protein. Therefore, we can clearly observe cell activity change and prevent the influence of endogenous S100A11 in this cell. The increasing cell activity was observed with increasing concentration of exogenous mS100A11 (Fig. 8, lanes 2-4). However, the cell activity was reduced upon adding exogenous RAGE V domain (Fig. 8, lane 5). The reduction resulted from the competition of exogenous RAGE V domain with endogenous RAGE V domain. This observation can prove that the signal transduction pathways indeed arise from the interaction between S100A11 and the RAGE V domain. On the other hand, we added 1 M tranilast to 100 nM mS100A11 and compared this with free 100 nM mS100A11. We observed decreasing cell activity in mS100A11 with tranilast compared with free mS100A11 (Fig. 8, lane 6). This indicated that tranilast can successfully act The dark red line represents the criterion of selected residues that exhibited greater decreasing signals (Ͻ0.5). The selected residues are shown in red bars. C, selected residues were labeled in yellow on the three-dimensional structure of RAGE V domain (cyan) using the PyMOL program. as an inhibitor of cell proliferation for mS100A11-endogenous RAGE V domain interaction.

Discussion
The calcium-binding S100A11 contains two EF-hand motifs, which are located at the helix 1-loop-helix 2 and helix 3-loophelix 4 regions. These regions mediate many signal transduction cascades that are calcium-dependent. Thus, S100A11 is associated with a large number of functions, including intracellular and extracellular functions, in various cell types (63). The binding of calcium ion to S100A11 homodimeric protein results in conformational changes that promote the interaction between the hydrophobic site of S100A11 and target proteins and further mediate the activity of proteins (64). The S100 protein family is one of the ligands of RAGE. The binding of one site of S100A11 dimer to RAGE V domain induces homodimerization of two adjacent RAGEs and autophosphorylation of their cytoplasmic domains. This autophosphorylation would trigger a series of signal transduction cascades, lead-ing to cell proliferation and survival (Fig. 9). The interactions between mS100A11 and RAGE V domain were characterized by using techniques such as fluorescence spectroscopy and NMR spectroscopy. The NMR titration experiments and plotting bar diagrams identified the residues of binding sites of mS100A11 homodimer and RAGE V domain. Mapping these affected residues on three-dimensional structures elucidates the binding sites between mS100A11 and RAGE V domain. According to the experimental results, we utilized the HAD-DOCK program to calculate the S100A11-RAGE V domain heterotetrameric complex model, which demonstrated the binding sites between mS100A11 and the RAGE V domain. The results show that the binding site of S100A11 is at helix 2 and helix 4. This region is similar to the regions that have been reported as the binding interfaces of other S100 family proteins complexed with the RAGE V domain. For instance, S100P homodimer was shown to interact with the RAGE V domain by helix 1 of one monomer and helix 4Ј of another monomer in a previous study (65). It has been reported that the binding site of  . The binary complex model of S100A11-tranilast determined from the HADDOCK program. A, the results of the HADDOCK calculations show the binding interfaces of S100A11 with tranilast by overlapping 13 stick structures of tranilast molecules at both sides of the S100A11 homodimer. The S100A11 homodimer is colored in green, and binding sites with tranilast are labeled in red. B, the expansion region is shown as the binding interface between S100A11 and tranilast with side chains of binding residues. The stick structures of resides Asn-53, Leu-87, and Ala-90 of one of the S100A11 monomers (green) are shown in red. S100A6 to the RAGE V domain is located at loops 1 and 3 and helix 4 (27). For S100B, the binding region on loop 1, loop 3, helix 3, and helix 4 has been investigated to determine its interaction with the RAGE V domain (24). These S100 proteins generally have similar regions in binding interfaces as a result of their high structural similarity in three dimensions. Some parts of the minor differences in binding sites between each S100 protein and the RAGE V domain may result from distinct identification of RAGE V domain with S100 proteins due to the differences in the hydrophobic region, polarity, net charge, or properties of S100 proteins. Overall, our results reveal that the S100A11 homodimer interacts with two RAGE V domain molecules symmetrically and forms a heterotetrameric complex model. This model of S100A11-RAGE V domain complex would be useful for designing drugs to block the interaction between mS100A11 and the RAGE V domain. Furthermore, we discovered that the tranilast molecule could be a potential drug for blocking the interaction between mS100A11 and the RAGE V domain based on our results of experiments, including fluorescence, NMR, HADDOCK modeling, and WST-1 cell prolif-  . Schematic diagram for mechanism of interaction between S100A11 and RAGE. The proposed mechanism elucidates that the binding of extracellular S100A11 dimer to RAGE would trigger the homodimerization of two RAGEs and autophosphorylation of their cytoplasmic domains in RAGE. These result in activation of signal transduction pathways and promotion of the downstream pathways, followed by cell proliferation. eration assays. Fig. 10 shows the overlap of the following two complexes that were generated by our HADDOCK results: 1) S100A11-RAGE V domain complex, shown in green, and 2) S100A11-tranilast complex (S100A11 is shown in green, and tranilast molecules are shown in multicolor). This clearly shows that tranilast blocked the binding sites between mS100A11 and the RAGE V domain. Our findings give prominence to the inhibition of cell proliferation resulting from the blocking interaction of S100A11 with RAGE using the tranilast molecule. This will also be helpful for improving therapeutic strategies to treat RAGE-dependent diseases or even cancers.