Lipid tail length dictates membrane disruption by carmofur analogs and reveals noncanonical modes of action.

Carmofur, a lipidated urethane derivative and prodrug of 5-fluorouracil, has an established clinical application as an antineoplastic agent for the treatment of colorectal cancer, primarily through inhibition of acid ceramidase and subsequent intracellular drug release. Beyond this oncological application, recent drug repurposing efforts have identified covalent inhibition of the SARS-CoV-2 main protease as an additional antiviral mechanism. The pronounced amphiphilic character of carmofur, arising from a polar fluoropyrimidine headgroup appended to a hydrophobic aliphatic tail, raises the possibility of an additional membrane-directed mechanism orthogonal to canonical target engagement. Driven by observations that certain lipidated carmofur analogs induce potent disruption in giant unilamellar vesicles (GUVs), which serve as model-membrane systems, we evaluated nine analogs with differing alkyl tail lengths and saturation to characterize the capacity of such lipidated compounds to directly engage with biological membranes. To efficiently quantify the membrane-disrupting capabilities of these analogs, we benchmarked the detection accuracy of a transformer-based model and a CNN-based vision model, where YOLOv11 matched RT-DETRv3's detection accuracy while running at a fraction of its computational cost. Through both cell-free and