LAUSR

It remains a challenge to predict whether a new drug candidate will have undesirable side effects. Many biologically active molecules, including drugs and drug-leads, are amphiphiles that partition into lipid bilayers, which will tend to alter bilayer physical properties, thereby modulating membrane protein function. Such bilayer-modifying molecules may be promiscuous modifiers of membrane protein function, raising the possibility that they have off-target effects. Consequently, it may be possible to predict whether a compound will have off-target effects based on quantitative studies on the compound's bilayer-modifying potential. To address this question, we developed an assay to quantify the bilayer-modifying potential of large numbers of compounds using a gramicidin-based fluorescence assay (GBFA), which reports how a compound alters the gramicidin monomer ↔ dimer equilibrium. Using this assay, we have shown that many drug and drug-leads alter lipid bilayer properties at the concentrations where these compounds become indiscriminate modifiers of membrane protein function. Such indiscriminate modifiers of membrane protein function are likely to have off target effects; we pursued this question in a study on libraries of compounds that had been tested for toxicity in “high-content” screening assays that quantified cellular ATP levels, nuclear morphology, nuclear membrane integrity, and apoptosis in immortalized liver, lung, and neuronal cell lines. We tested 524 total compounds comprising four libraries (one non-toxic library and three mixed libraries of non-toxic and toxic compounds; the libraries were “blinded” until the results of the GBFA were known) and found that the GBFA predicts cellular toxicity, with minimal assignment of false positives. These results suggest that in vitro measurement could be used as a warning sign for off-target biological effects in drug discovery efforts and, further, provides a mechanism by which amphiphiles exert their toxicity, namely by altering lipid bilayer physical properties.