Photoaffinity Labeling Reveals a Role for the Unusual Triply Acylated Phospholipid N-Acylphosphatidylethanolamine in Lactate Homeostasis

From Bioorganic Class 2025: The authors identify proteins that recognize and whose function is modulated by N-acylphosphatidylethanolamine (NAPE) through incorporation of bioorthogonal moieties. They employ a reasonable strategy with modifications to accommodate metabolism of their lipid, allowing them to propose a novel model for the physiological function of NAPE during ischemia. Major comments: Figure 1B and C show probe-protein binding, but more experiments are needed to confirm that only proteins binding NAPE are detected, not partially hydrolyzed components of their probe. We suggest modifying the probe to include two different clickable moieties. Detection of target complexes could then be demonstrated through colocalization of fluorescent signals. Another suggestion is to replace the ester on the alkyne handle with a more inert functional group to minimize NAPE degradation. Figure 3 is insufficient to support the conclusion “substantial increases in intracellular NAPE”. Control showing NAPE levels in the cell line without doxycycline is needed. Authors conclude NAE “formation via alternate NAPE degradation pathways” in Figure S2. This statement needs further experimental evaluation to elucidate specific mechanisms, especially since it might undermine the validity of their probe as a selective reporter. Minor comments: Explanation of benefits of using clickable moieties over directly linking affinity label onto fatty acid is needed. Figure 2 shows photoaffinity labeling targets of NAPE, however authors lack rationale for choosing CD147, CD44 and BZW2 for further analysis. More explanation of their protein selection parameters is needed. The 1H-NMR spectrum of Compound 7 has impurity peak merged with product peak at around 3.4 ppm. Analytical HPLC spectrum for final compound is suggested. Figure 4 needs clearer labeling to indicate replicates. Figure 5 is misnumbered.