Fig 1: ALDH1A1 is differentially expressed in cancer cell lines and its activity can be indirectly visualized by BAA fluorescence.(A) Protein extracts from pancreatic cancer cell lines MIA PaCa-2 (lane 1) and PANC-1 (lane 2), colon cancer cell lines HT-29 (lane 3) and SW480 (lane 4) and glioma cell lines LN-18 (lane 5) and LN-229 (lane 6) were analyzed by immunoblot for ALDH1A1, ALDH1A2, ALDH1A3, ALDH2 and ALDH3A1 expression. ß-Actin was used as loading control. The corresponding recombinant protein was loaded for reference (lane 7). (B) Representative fluorescent images of MIA PaCa-2 and HT-29 cells incubated with 500 nM of BAAA substrate and treated with 22 µM DEAB or DMSO for 30 minutes. Green fluorescent images indicate intracellular BAA and blue fluorescent images indicate nuclei staining.
Fig 2: (a) Binding mode of N-benzylaniline-based activator C6 with ALDH2. The ALDH2 protein structure template is derived from the crystal structure (PDB ID: 3INJ). The protein is shown as a gray ribbon and a solvent-accessible surface, and the residues important for binding are shown as gray stick models. The activator is shown as a purple stick model, and the hydrogen bonds are shown as blue dashed lines. (b) The design of novel N-benzylaniline-based ALDH2 activators.
Fig 3: Binding mode of N-benzylaniline-based activator D10 with ALDH2 shown as solvent-accessible surface models (a) or gray ribbon models (b). The ALDH2 protein structure template is derived from the crystal structure (PDB ID: 3INJ). The residues important for binding are shown as gray stick models. The activators are shown as purple stick models, and the hydrogen bonds are shown as blue dashed lines.
Fig 4: (a) Structures of representative activators; and (b) topological structure of ALDH2. The protein structure in the figure is shown using a ribbon model, with the two subunits represented in pink and red, respectively. Three regions of each subunit are labeled. The mutated amino acids in the ALDH2 variant are shown using a yellow CPK model, and the small-molecule activators are demonstrated using a purple stick model.
Fig 5: Four synthetic routes I–IV of novel N-benzylaniline-based ALDH2 activators. Reagents and conditions: (a) DMSO, K2CO3, 80 °C, reflux, 1–4.5 h, 79–99%; (b) DMAP, EDCI, DCM, r. t. 1.5–6 h, 67–95%; (c) EtOH, NH4Cl, Fe, H2O, 80 °C, reflux, 1–3 h, 71–97%; (d) DCE, NaBH(OAc)3, 80 °C reflux, r. t., 2–12 h, 59–95%; (e) DCM, TFA, r. t., 0.5–2.5 h, 73–90%; (f) NaOH, MeOH, THF, H2O, 1–2 h, 91%; (g) conc-HCl, EA, 1.5 h, r. t., 69%; (h) HATU, DIPEA, DMF, 80 °C, 12 h, 42.8%; (i) PdXPhosG2, Pd/C, K3PO4, Dioxane/H2O (4:1), 80 °C, 4 h; then MeOH, NH4COOH, r. t. to 60 °C, 16 h, 75%; and (j) Pd(dppf)Cl2, K2CO3, Dioxane/H2O (3:1), 100 °C, 12 h, ~70%.
Supplier Page from Abcam for Recombinant human ALDH2 protein (Active) (Tag Free)