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. Author manuscript; available in PMC: 2025 Apr 18.
Published in final edited form as: Cell Chem Biol. 2023 Nov 28;31(4):712–728.e9. doi: 10.1016/j.chembiol.2023.10.026

Figure 5. Semapimod disrupts the bacterial outer membrane by binding LPS.

Figure 5.

A) Growth inhibition checkerboard of Ec BW with semapimod and rifampicin. Darker shading indicates higher bacterial growth. Shown is the mean of two biological replicates. B) Uptake of a 1-N-phenylnaphthylamine (NPN) fluorescent probe during semapimod, colistin, and ampicillin treatment in Ec BW. Data are representative of two biological replicates; error bars show s.e.m. C) Semapimod hemolysis was tested against rat Sprague-Dawley red blood cells. Data are representative of two biological replicates; error bars denote s.e.m. D) Time-lapse microscopy profiling of semapimod-treated cells. Stationary-phase Ec BW cells were treated with vehicle (DMSO) and semapimod (4X MIC) in the presence of SYTOX Blue, a membrane damage-sensitive dye. Brightfield and fluorescence images of a population of cells 8 hours after treatment indicate extensive SYTOX Blue fluorescence in semapimod-treated cells. Results shown are representative of at least 12 different fields of view from three biological replicates, and the total number of cells analyzed was >2,500 per condition. Scale bar denotes 10 μm. See also Movies S1–S4. E) Quantification of the fraction of SYTOX-Blue positive cells across all fields of view in the experiments shown in Figure 5C. Thick curves represent average values across all fields of view, and error bars denote standard deviation. F) Mutants raised against semapimod have a single-point mutation in the pmrB gene, a known colistin-resistance mechanism. The chemical structure of colistin is shown in the inset. G) Semapimod killing activity against stationary-phase E. coli carrying colistin-resistance determinant mcr-1 in biological duplicate; error bars denote s.e.m. H) Schematic of LPS binding assay, where displacement of BODIPY-cadaverine from LPS isolated from E. coli is monitored by an increase in fluorescence. I) BODIPY-cadaverine displacement assay of semapimod binding E. coli-derived LPS. Data are representative of two biological replicates; error bars indicate s.e.m.