Fig 2: ALKBH5 and the secondary m6A modification are involved in regulating NPC autophagy by BMSC coculture.a The m6A/A ratio and m6A/AUCG ratio of total mRNA were determined by LC–MS/MS. Data are shown as mean ± SD. Statistical analysis was conducted by unpaired Student’s t test (n = 3, *P < 0.05, **P < 0.01; Co-NPCs + CPR vs. NPCs + CPR). b RNA dot blot was performed to analyze the m6A level of mRNA in NPCs with or without BMSC coculture. Methylene blue staining served as a loading control. c The relative m6A level in NPCs with or without BMSC coculture, as determined by colorimetric assay. Data are shown as mean ± SD. Statistical analysis was conducted by unpaired Student t test (n = 3, **P < 0.01, Co-NPCs + CPR vs. NPCs + CPR). d Relative mRNA levels of methyltransferases and demethylases were determined by RT-qPCR. The expression levels were normalized to the control group; the results are presented as mean ± SD. Statistical analysis was conducted by unpaired Student t test (n = 3, *P < 0.05, **P < 0.01, #no significance, NPCs + BMSCs + CPR vs. NPCs + CPR). e Protein levels of the methyltransferases METTL3, METTL14, and WTAP, and the demethylases FTO and ALKBH5, were measured by Western blot. Protein levels were normalized to GAPDH; the results are presented as mean ± SD. Statistical analysis was conducted by unpaired Student’s t test (n = 3, *P < 0.05, **P < 0.01, NPCs + BMSCs + CPR vs. NPCs + CPR). f The ALKBH5 knockdown efficiency by siALKBH5 was confirmed by Western blot. Expression levels were normalized to GAPDH; the results are presented as mean ± SD. Statistical analysis was conducted by unpaired Student t test (n = 3, **P < 0.01, siALKBH5 vs. siControl). g Protein levels of apoptosis- and autophagy-associated proteins after silencing of ALKBH5 with or without BMSC coculture, as determined by Western blot. h m6A/A ratio and m6A/AUCG ratio of the total mRNA in three groups, as determined by LC–MS/MS. Data are shown as mean ± SD. Statistical analysis was conducted by unpaired Student’s t test (n = 3, *P < 0.05, **P < 0.01, Co-NPCs + CPR vs. NPCs + CPR; **P < 0.01, Co-NPCs + CPR + siFIP200 vs. Co-NPCs + CPR). i The m6A level of mRNA in NPCs with or without BMSC coculture upon ALKBH5 knockdown was analyzed by RNA dot-blot analysis. Methylene blue staining served as a loading control. j Colorimetric assay to determine the relative m6A level in NPCs with or without BMSC coculture upon ALKBH5 knockdown. Data are shown as mean ± SD. Statistical analysis was conducted by unpaired Student t test (n = 3, *P < 0.05, Co-NPCs + CPR vs. NPCs + CPR; **P < 0.01, Co-NPCs + CPR + siFIP200 vs. Co-NPCs + CPR).

Fig 3: Rational design of oxetanyl FTO inhibitors. (A) Optimization strategy identifying lead FTO-43 N. (B) Docking pose of hit FTO-04. FTO-04 forms a hydrogen bond with Arg 96, and a water-mediated hydrogen bond is observed with Lys 216. Optimization of the benzothiazole ring was directed toward catalytic residues His 231 and Glu 234. (C) Docking pose of FTO-04 with the benzothiazole ring replaced by 5-fluoroindole. New interactions are observed with catalytic His 231, Glu 234, and Arg 322, while interactions are maintained with Arg 96. (D) Docking pose of a pyrrolidine amide replacement of the pyrimidine ring. The amide forms a weak hydrogen bond to the backbone of Ser 229. (E) Comparison of the pyrrolidine amide (green) and oxetane (gray) scaffolds. The oxetane substitution is observed to strengthen the hydrogen bond with Ser 229 without significantly altering the interactions with residues Arg 96, His 231, Glu 234, or Arg 322.

Fig 4: Synthesis of Oxetanyl FTO Inhibitors

Fig 5: Oxetanyls are potent inhibitors of recombinant FTO in vitro. (A) LLE plot for select FTO inhibitors. pIC50 is determined from in vitro inhibition assay against recombinant FTO. Oxetanyl libraries (red squares) show a significant improvement in pIC50 and LLE from pyrimidine libraries (blue spheres). Hit FTO-04 is shown as a yellow triangle. (B) Dose–response curves for FTO-09 N against recombinant FTO and ALKBH5. (C) Dose–response curves for FTO-43 N against recombinant FTO and ALKBH5. (D) Double-reciprocal plot for FTO-11 N. All data sets converge upon a common y-intercept, consistent with competitive inhibition. (E) Double-reciprocal plot for FTO-35 N. All data sets converge upon a common y-intercept, consistent with competitive inhibition. (F) Double-reciprocal plot for FTO-38 N. All data sets converge upon a common y-intercept, consistent with competitive inhibition. (G) Double-reciprocal plot for FTO-43 N. All data sets converge upon a common y-intercept, consistent with competitive inhibition. (H) Double-reciprocal plot for FTO-49 N. All data sets converge upon a common y-intercept, consistent with competitive inhibition.