Small-Molecule Modulators of Lipid Raft Stability and Protein-Raft Partitioning

The functions of phase-separated ordered membrane domains, colloquially known as lipid rafts, have yet to be well-defined, in part because of a lack of tools to manipulate raft formation and protein affinity for rafts. Human peripheral protein 22 (PMP22) exhibits a high affinity for lipid rafts in giant plasma membrane vesicles (GPMVs). We screened 24,000+ small molecules in search of compounds that alter the raft affinity of PMP22. Hits were counter-screened against another raft partitioning protein, MAL, and also were tested for their impact on raft formation. We describe the discovery of two classes of compounds. Class I molecules altered the raft partitioning of both PMP22 and MAL and also reduced raft formation in a protein-dependent manner. Class II molecules either increased or decreased raft formation in a manner independent of the presence of PMP22 and MAL. Both classes of compounds altered membrane fluidity in live cells, with no observed toxicity. We further demonstrated that these compounds modulate TRPM8 ion channel function but not phosphorylation of the receptor tyrosine kinase, EGFR. These compounds deepen our understanding of raft biophysics and present a new set of tools for probing lipid raft function.