Evaluation of a fluorescence–based screening assay for the detection of silyl hydrolase activity

This study reports the development of fluorometric assays for the detection and quantification of silyl hydrolase activity, using silicatein as a model enzyme. These assays employed a series of organosilane substrates containing either the mycophenolate or umbelliferone moieties, which become fluorescent upon hydrolysis of a scissile Si–O bond. Amongst these substrates, the mycophenolate-derived molecule MycoF, emerged as the most promising candidate due to its relative stability in aqueous media, which resulted in good differentiation between the enzyme-catalysed and uncatalysed background hydrolysis. The utility of MycoF was also demonstrated in the detection of enzyme activity in cell lysates and was found to be capable of rapid identification of a positive ‘hit’ with assay times as low as 15 min. These fluorogenic substrates were also suitable for use in quantitative kinetic assays, as demonstrated by the acquisition of their Michaelis-Menten parameters.