Figure 5. Bithionol inhibits anthrax toxin lethality.

(a) Bithionol was tested for its ability to inhibit anthrax toxin-mediated cytotoxicity. RAW264.7 cells were incubated with the indicated doses of Bithionol for 1 hour, followed by 6 hours intoxication with anthrax toxin PA-LF. Cell viability was determined by MTT assay and is shown as the percentage of survivors relative to cells not treated with drugs. (b) Bithionol inhibits LF-PA–induced activity of cellular caspase-1. RAW264.7 cells were treated with LF-PA for 1 hour, and then treated either with 33 μM Bithionol or DMSO for 1 hour prior to lysis and determination of caspase-1 activity. The activity of caspase-1 was measured by FRET assay. (c) MAPKK2 immunoblotting showing that Bithionol does not block proteolysis of cellular MAPKKs by anthrax LF toxin. While MAPKK2 was cleaved in LF-PA treated RAW264.7 cells, treatment with Bithionol did not affect this process. RAW264.7 cells were incubated with Bithionol or DMSO for 1 hour before addition of vehicle control or 1 μg/ml PA + LF for up to 60 minutes. Cells were lysed and analyzed via immunoblotting with a MAPKK2–specific antibody. Tubulin was used as a loading control. (d,e) Bithionol reduces cell death induced by the hybrid toxin FP59, which has been widely used as an anthrax LF surrogate and contains the PA binding site of LF, as well as a toxin domain derived from PE. Bithionol-treated cells were found to be less sensitive to treatment with PA + FP59. PA was either in the native 83 kDa form (d), or used as 63 kDa–lacking 20 kDa Furin cleavage domain (e). RAW264.7 cells were preincubated with a titration of Bithionol for 1 hour, followed by a 6 hours intoxication with 0.5 μg/ml 83 kDa PA + FP59 or Furin processed 63 kDa PA + FP59. Cell viability was measured via MTT. (f) Bithionol doesn’t inhibit cathepsin B protease activity in RAW264.7 cells. RAW264.7 cells were treated with 33 μM Bithionol of DMSO for 1 hour prior to lysis, and determination of cathepsin B activity was assessed by FRET assay.