Screening for bilayer-active and likely cytotoxic molecules reveals bilayer-mediated regulation of cell function

Abstract

A perennial problem encountered when using small molecules (drugs) to manipulate cell or protein function is to assess whether observed changes in function result from specific interactions with a desired target or from less specific off-target mechanisms. This is important in laboratory research as well as in drug development, where the goal is to identify molecules that are unlikely to be successful therapeutics early in the process, thereby avoiding costly mistakes. We pursued this challenge from the perspective that many bioactive molecules (drugs) are amphiphiles that alter lipid bilayer elastic properties, which may cause indiscriminate changes in membrane protein (and cell) function and, in turn, cytotoxicity. Such drug-induced changes in bilayer properties can be quantified as changes in the monomer↔dimer equilibrium for bilayer-spanning gramicidin channels. Using this approach, we tested whether molecules in the Pathogen Box (a library of 400 drugs and drug-like molecules with confirmed activity against tropical diseases released by Medicines for Malaria Venture to encourage the development of therapies for neglected tropical diseases) are bilayer modifiers. 32% of the molecules in the Pathogen Box were bilayer modifiers, defined as molecules that at 10 µM shifted the monomer↔dimer equilibrium toward the conducting dimers by at least 50%. Correlation analysis of the molecules’ reported HepG2 cell cytotoxicity to bilayer-modifying potency, quantified as the shift in the gramicidin monomer↔dimer equilibrium, revealed that molecules producing <25% change in the equilibrium had significantly lower probability of being cytotoxic than molecules producing >50% change. Neither cytotoxicity nor bilayer-modifying potency (quantified as the shift in the gramicidin monomer↔dimer equilibrium) was well predicted by conventional physico-chemical descriptors (hydrophobicity, polar surface area, etc.). We conclude that drug-induced changes in lipid bilayer properties are robust predictors of the likelihood of membrane-mediated off-target effects, including cytotoxicity.

Modulation of Acid-Sensing Ion Channels by Tannic Acid and Green Tea via a Membrane-Mediated Mechanism

“…In fact, a recent study showed that amphiphilic drugs, which change membrane properties, cause indiscriminate changes in membrane protein function and, in turn, even cytotoxicity. 57 The present work has some limitations. First, while our findings strongly suggest a membrane-mediated mechanism for the modulation of ASICs by TA, we cannot prove actual interaction with the membrane nor can we exclude direct interaction with ASICs.…”

“…Our data showing that TA, PG, and GT lack selectivity for membrane proteins, particularly ion channels, suggest that they have limited potential for clinical applications. In fact, a recent study showed that amphiphilic drugs, which change membrane properties, cause indiscriminate changes in membrane protein function and, in turn, even cytotoxicity …”

Nonspecific membrane bilayer perturbations by ivermectin underlie SARS-CoV-2 in vitro activity

“…Drug-induced changes in the ANTS quench rate thus reflect the bilayer-modifying potency of a compound, 49, 50 and changes in rate / rate control > 1.5 are associated with increased risk that the compound may be cytotoxic. 21 In initial testing, IVM significantly increased the ANTS quench rate at 10 µM ( Figure 5A ). In concentration-response testing, IVM increased the ANTS quench rate in a concentration-dependent manner, with 1 μM IVM increasing the rate relative to DMSO control 12-fold indicating that IVM is a potent bilayer modifier at this concentration ( Figure 5B ).…”

“…This is consistent with previous observations showing a correlation between membrane bilayer-modifying properties and cytotoxicity. 21…”

“…This is consistent with previous observations showing a correlation between membrane bilayer-modifying properties and cytotoxicity. 21 The effect of IVM on cellular health was also examined using publicly available datasets. IVM decreased cell counts after 72 h compound treatment in the Broad PRISM viability screen, a multiplexed cell proliferation assay using 480 different cancer cell lines (Figure 2C).…”