Figure 4. Bithionol reduces pathogenicity of toxins by inhibiting host caspases.

(a–c) Bithionol inhibits caspases. FRET data showing fluorescence emission from two reactions, where caspase-containing cellular lysate cleaves fluorescently labeled substrate peptide without drugs, or in the presence of 33 μM Bithionol. FRET substrates were specific for cleavage by caspase-3 (a), caspase-3/7 (b), and caspase-6 (c). (d–f) Bithionol was tested for its ability to inhibit cytotoxicities mediated by toxins of cholera, diphtheria, and Pseudomonas. RAW264.7 cells were incubated with indicated doses of Bithionol for 1 hour, followed by 12 hours intoxication with Pseudomonas and cholera toxins. Diphtheria toxin was added to C32 cells for 24 hours. Cell viability was determined by MTT assay and is shown as the percentage of survivors relative to cells not treated with drugs. (g) Different concentrations of Bithionol are tested for their ability to inhibit caspase activity in cellular lysate of cells. Cells were pre-treated with Pseudomonas aeruginosa exotoxin A to induce caspases. FRET was done using substrates cleaved by caspases-3, -6, and -3/7. (i) Bithionol inhibits cytotoxicity mediated by P. aeruginosa exotoxin A in sensitive human B-lymphocytes. B-cells were seeded at 1 × 10⁴ cells/well on 96-well plates and were incubated with indicated doses of Bithionol for 1 hour, and then challenged with the toxin for 6 hours. Cell viability was determined by Alamar Blue assay and is shown as the percentage of survivors relative to cells not treated with drugs. (i) Bithionol inhibits caspases-1, -3, -6, -7, and -9. Bithionol was tested at 33 μM for its ability to inhibit FRET reactions of purified human caspases-1 through 10. Percent inhibition values are shown, and compared to activity of caspases untreated with Bithionol. Phenogram of ten human caspases, assembled by Multalin using Dayhoff alignment parameters, is used to demonstrate relative homology of caspases.