Measurement of Protease Release by a Fluorogenic Casein Assay in Human Cells Exposed In Vitro to Sulfur Mustard

Sulfur mustard (HD) exposure causes severe ocular, respiratory, and blistering injuries to skin. The mechanism(s) of injury has eluded identification, although a protease(s) may be involved in the dermal-epidermal separation seen in blister formation. Recent research using in vitro systems has demonstrated that protease release occurs in these models as a function of HD exposure. A number of protease inhibitors have been proposed as candidate anti-vesicant medical countermeasures and need to be screened in a rapid and cost-effective in vitro assay to determine efficacy before being transitioned to an in vivo model for further testing. New fluorogenic casein substrates that are sensitive to a number of proteases have been developed by Molecular Probes and may be helpful in developing a rapid assay for protease analysis. This report describes the use of these substrates in assays for screening compounds to develop medical countermeasures against this vesicant agent The data suggest that these substrates are not appropriate to the development of a rapid protease assay.