Fig. 1. First generation quenched activity based probes development. (a) General scheme for the mechanism of action of a fluorescent quenched activity based probe (qABP). (b) Inhibition studies of recombinant caspase-3 vs. inhibition of legumain in RAW cell lysates with the different inhibitors described in Table 1. (c) Chemical structure, fluorescence intensity and quenching efficiency of probes 17, 18 and 19. (d) Direct labeling of recombinant caspase-3 (upper panel) and legumain and cathepsin B in RAW cell lysate (lower panel) by indicated qABPs. Recombinant caspase-3 was incubated with increasing probe concentrations for one hour, the reaction was stopped and separated on a SDS PAGE and scanned for Cy5 fluorescence. Samples marked with ⁺ were pretreated with a caspase inhibitor (AB46 peptide) 30 min prior to the probe treatment. Legumain and cathepsin B from RAW cell lysates were labeled by the indicated qABPs similarly to caspase labeling. Samples marked with ^{a, b} or ^c were pretreated for 30 min with the inhibitors AB46 peptide, GB111-NH₂ 25 or 5 to selectively block caspase-3; cathepsin B or legumain, respectively. (e) Direct labeling of active caspase-3 in intact MM1s cells undergoing apoptosis. The indicated qABP showed covalent binding to active caspase-3, seen at 17 kDa. Samples marked with ⁺ represent the pretreatment with a caspase-3/legumain inhibitor (AB46 peptide) or cathepsin B inhibitor (GB111-NH₂) which was added 1 h prior to the probe.