Journal: bioRxiv
Article Title: Nonspecific membrane bilayer perturbations by ivermectin underlie SARS-CoV-2 in vitro activity
doi: 10.1101/2023.10.23.563088
Figure Lengend Snippet: (A) A gramicidin-based fluorescence assay (GFA) demonstrating how the influx of the heavy ion quencher Tl + into fluorophore-loaded LUVs increases with IVM ( 1a ) or remdesivir. Left: stopped-flow fluorescence quench traces of normalized fluorescence decay over 1 s with light dots representing individual replicates (n = 9 technical replicates) and dark dots representing replicate mean. Right: representative time courses of the initial 100 ms with dots denoting individual time points and solid lines denoting exponential fits (2–100 ms). (B) GFA concentration-response curves demonstrate IVM significantly perturbs membrane bilayers at low micromolar concentrations. 5% EtOH, positive control. Data are mean ± SD for three biological replicates (independent LUV batches). (C) IVM induces changes in gramicidin A (gA) function. Left: representative single-channel traces recorded in a planar bilayer doped with the right-handed AgA(15) and the left-handed gA − (13) analogs, which form channels of different lengths and helix sense. Note that 3 µM IVM produces increases in channel lifetimes and a decrease in channel appearance rate. Right: representative single-channel survivor plots for gA − (13) channels (top) and AgA(15) channels (bottom) with single-exponential fits to the survivor distributions. IVM produced concentration-dependent increases in the average single-channel lifetimes (1). Data are from one of seven independent experiments each with at least three separate measurements. See also for experimental summary. (D) IVM causes voltage-dependent inhibition of the peak Na + current. Time-course of drug wash-in showing inhibition of voltage-dependent sodium channels in neuroblastoma ND7/23 cells using an alternating two-pulse protocol. Left: representative Na + current traces from an alternating two-pulse protocol. A test pulse to 0 mV to elicit peak Na + current ( I Na ) was preceded by a 300 ms prepulse to holding potentials of either V 0 (−130 mV, solid lines) or V ½ (dashed lines, see insert; V ½ = −69 ± 3 mV). Black lines, DMSO control; red lines, 10 µM IVM. IVM significantly reduced peak I Na to 0.91 ± 0.01 mV (prepulse to V 0 , p < 0.0001) and peak I Na to 0.78 ± 0.02 mV (prepulse to V ½ , p < 0.0001). Data are mean ± SD from four to six biological replicates. (E) IVM promotes inactivation of voltage-dependent sodium channels. Left: representative Na + current traces from a double-pulse protocol in which a test pulse to 0 mV was preceded by a 300 ms conditioning prepulse to potentials ranging from –130 mV to –30 mV. The data from each experiment were fitted with a standard two-state Boltzmann equation to calculate the individual V ½ values. Right: fitted data showing IVM causes a hyperpolarizing shift of the voltage-dependance of half-maximal inactivation (ΛV ½ = –7.9 mV, p < 0.0001). Data are mean ± SD from four to six biological replicates. Source data provided as a Source Data file.
Article Snippet: We also tested a blinded (to the experimenter) library of IVM analogs and remdesivir at 10 µM concentration ( Supplementary Table 3 ); the results are unmarkable (flat SAR), except for the avermectin B 1a aglycone ( 12 ), which has a two-fold lower bilayer-modifying potency than avermectin B 1b 10 (but the ivermectin B 1a aglycone 3 is equipotent to IVM and the ivermectin B 1a monosaccharide 4 ).
Techniques: Fluorescence, Concentration Assay, Membrane, Positive Control, Produced, Inhibition
