Synthesis of bio-MTX (3).

<p>The reaction gave bio-MTX <b>3a</b> and <b>3b</b> as a 1∶1 mixture.</p

SPR analysis of the interaction between MTX and HMGB1.

<p>(A) Full length map of HMGB1 and truncated recombinant versions of the protein engineered in <i>E. coli</i>. The displayed sequence of the MTX-binding T7 phage particle (K86-V175) includes the Box B domain (K89-Y161), TLR4-binding domain (F88-E107) and part of the RAGE-binding domain (K149-V175). (B) SDS-PAGE of AlBj, Al and Bj proteins after purification by affinity chromatography. The bands were stained with CBB. (C, D) Representative SPR sensorgram with a global fitting curve between bio-MTX and Al (C) or Bj (D). Solutions containing various concentrations of Al (0.31–5 µM) or Bj protein (0.16–2.5 µM) were injected over the immobilized MTX-biotin on a SA sensor chip for 120 s and then dissociation was monitored for a further 120 s at a flow rate of 30 µl/min. Response curves were generated by subtraction of the background signals generated simultaneously on the control flow cell (bio-MTX-non-immobilized cell) and the injection of vehicle (0 µM analyte). (E) Concentration-response curve between bio-MTX and AlBj obtained from SPR analysis. R_max = 66. (F) Scatchard-plot analysis of AlBj binding to MTX. (G) Hill-plot analysis of AlBj binding. Hill coefficients n = 1.1. RU: resonance unit. 1 RU = 1 pg/mm².</p

Identification and Characterization of the Direct Interaction between Methotrexate (MTX) and High-Mobility Group Box 1 (HMGB1) Protein

<div><p>Background</p><p>Methotrexate (MTX) is an agent used in chemotherapy of tumors and autoimmune disease including rheumatoid arthritis (RA). In addition, MTX has some anti-inflammatory activity. Although dihydrofolate reductase (DHFR) is a well-known target for the anti-tumor effect of MTX, the mode of action for the anti-inflammatory activity of MTX is not fully understood.</p><p>Methodology/Result</p><p>Here, we performed a screening of MTX-binding proteins using T7 phage display with a synthetic biotinylated MTX derivative. We then characterized the interactions using surface plasmon resonance (SPR) analysis and electrophoretic mobility shift assay (EMSA). Using a T7 phage display screen, we identified T7 phages that displayed part of high-mobility group box 1 (HMGB1) protein (K86-V175). Binding affinities as well as likely binding sites were characterized using genetically engineered truncated versions of HMGB1 protein (Al G1-K87, Bj: F88-K181), indicating that MTX binds to HMGB1 <i>via</i> two independent sites with a dissociation constants (K_D) of 0.50±0.03 µM for Al and 0.24±0.01 µM for Bj. Although MTX did not inhibit the binding of HMGB1 to DNA <i>via</i> these domains, HMGB1/RAGE association was impeded in the presence of MTX. These data suggested that binding of MTX to part of the RAGE-binding region (K149-V175) in HMGB1 might be significant for the anti-inflammatory effect of MTX. Indeed, in murine macrophage-like cells (RAW 264.7), TNF-α release and mitogenic activity elicited by specific RAGE stimulation with a truncated monomeric HMGB1 were inhibited in the presence of MTX.</p><p>Conclusions/Significance</p><p>These data demonstrate that HMGB1 is a direct binding protein of MTX. Moreover, binding of MTX to RAGE-binding region in HMGB1 inhibited the HMGB1/RAGE interaction at the molecular and cellular levels. These data might explain the molecular basis underlying the mechanism of action for the anti-inflammatory effect of MTX.</p></div

Effect of MTX on the truncated HMGB1 (Bj)-elicited TNF-α release and mitogenic activity in RAW 264.7 cells.

<p>(A) Bj protein-dependent TNF-α release. RAW 264.7 cells, in a 24-well culture dish format, were stimulated with the indicated concentrations of Bj protein for 6 h. The amount of TNF-α released into the conditioned medium was then determined by ELISA. N ≥2. (B) Influence of MTX alone for TNF-α release. RAW 264.7 cells were stimulated with 0–10 µM of MTX for 6 h. N ≥3. (C) Inhibition of Bj protein-elicited TNF-α release, in a 24-well culture dish format, stimulated with 0.5 µM of Bj protein for 6 h in the presence of 0–10 µM MTX. N ≥3. (D) Bj protein-elicited mitogenic activity for 10 h. Results are given in terms of relative cell growth. N ≥3. (E) Cell growth in the presence or absence of Bj protein (0.5 µM), or MTX (100 µM). N ≥3. (F) Time course of MTX cytotoxicity. RAW 264.7 cell growth was elucidated using the WST-8 cell proliferation assay and is shown as relative cell growth (%). Data represented as mean ± SE. *P<0.05, ****P<0.001.</p

Effect of MTX for the HMGB1 binding to DNA or to RAGE.

<p>(A) Electrophoretic mobility shift assay (EMSA). The linearized form III pGEM DNA vector (0.5 ng) and AlBj protein was complexed for 1 h at various molar ratios of AlBj/DNA (0–800) in the absence (−) or presence (+) of MTX (1 mM). (B) The relative retarding distance of the DNA band was plotted against the molar ratio (0–800) of AlBj protein to DNA. Relative retardation = Distance from applied well to DNA band/Distance from applied well to control DNA band. N = 3; data represented as mean ± SE. (C–E) Representative SPR sensorgram with global fitting curve between Bj protein and RAGE (purity >90%) immobilized on a CM5 sensor chip. Six or seven different concentrations of Bj protein were injected over the immobilized RAGE for 120 s and then dissociation was monitored for a further 120 s at 30 µl/min. (C, D) Interaction between Bj protein (0.31–10 µM) with immobilized RAGE in the absence (C) or presence (D) of MTX (1 mM). (E) Interaction between MTX (1–63 µM) with immobilized RAGE. (F) Concentration-response curve and Lineweaver-Burk plot between Bj protein and RAGE in the absence (open circle) or presence (filled circle) of MTX (1 mM). Linear equations in Lineweaver-Burk plot are as follows; [MTX (−)]: y = 0.0083x +0.0061 (r² = 1), K_D = 1.35 µM, R_max = 163, [MTX (+)]: y = 0.0686x +0.0493 (r² = 0.90), K_D = 1.39 µM, R_max = 33, K_I = 142 µM. (G, H) Computer-aided binding model between MTX and HMGB1. (G) Molecular modeling of the MTX binding site in HMGB1 (K81–K164) potentially involved in the interference of HMGB1/RAGE interaction was derived using the NMR structure of fragments associated with previously published data (PDB accession code 2gzk). The predicted binding structure was solved using DS 1.7 with the CDOCKER application program by calculating the binding energy in an aqueous environment. (H) Expansion diagram of the MTX-binding site within part of RAGE-binding region (K149-V175). N92, D157, Y161 and R162 form hydrogen bonds with MTX. The binding energy is −29.31 kcal/mol.</p

Kinetic parameters of each ligand-analyte interaction.

<p>Analytical conditions: PBS, 25°C. <i>k</i>_a: association rate constant, <i>k</i>_d: dissociation rate constant, K_D: dissociation constant, R_max: maximum binding amount. RU: resonance unit. 1 RU = 1 pg/mm².</p