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Shine, N., Eaton, L., Crawford, K.

Lethal factor (LF) is the enzymatic component of anthrax lethal toxin which specifically cleaves the MAPK-kinase proteins. Molecular mechanisms that link this proteolysis with pathogenesis of the toxin remain unclear, however, LF presents as an ideal target for therapeutic inhibitors. Identification of potential inhibitory drug leads is dependent on a sensitive and rapid method for screening large numbers of compounds. One of the most efficient methods for this type of assay is based on the use of internally quenched fluorescent substrates. These substrates contain a fluorescent group paired with a suitable chromogenic group so that before cleavage the fluorescence is quenched by fluorescence resonance energy transfer (FRET). Fluorescence is restored as cleavage occurs and enzymatic activity can be monitored continuously. A FRET substrate for LF, MAPKKide, has been designed at LIST laboratories. The Km value is 4.7 M. This substrate is ideally suited for evaluating the IC(50)s and inhibitor modality for large screens of potential inhibitors. Several metalloprotease inhibitors, including the antibiotic, antinonin, have been examined using MAPKKide. The IC(50) measured for actinonin with LF is 18 M. A Dixon plot indicated noncompetitive inhibition. Inhibitor specificity for LF was confirmed using an unrelated enzyme that also cleaved MAPKKide. One disadvantage associated with FRET substrates is that the fluorophores absorb in the range of aromatic compounds that are potential inhibitors, thus complicating the interpretation of the data. Another FRET substrate for LF, using a fluorophore excited at longer wavelengths, has been designed at LIST laboratories. This FRET substrate can also be detected using an argon ion laser.