Prolonged incubation of aromatic aldehyde PB-2.2 with PLY, however, resulted in inhibition of hemolysis, although with slower inhibition kinetics and lower efficacy requiring an incubation time of 1.5 h for an IC50 of 18 µM in the hemolysis assay (Supplementary Figure ). PB-2.1, comprising a PB-2 derivative with a reduced olefinic double bond, was inactive. In contrast, the condensation product PB-2.12, obtained from aldehyde PB-2.2 and malonic acid diamide, displayed a residual activity with an IC50 value of 51 µM (Supplementary Figure ). These results suggested that the reactivity of the olefinic double bond in both PB-1 and PB-2 was essential for PLY inhibition. At the same time, a gradual reduction of potency of inhibitors PB-1 and 2 was observed in phosphate buffer saline (PBS) and in cell culture medium, presumably via hydration followed by a retro-aldol reaction, while the molecules were entirely stable in DMSO. Systematic variation of the substituents of the reactive double bond led the discovery of the more potent and stable molecule PB-3. Replacement of the benzylidene residue by 5-(2,4-dichlorophenyl)-furfuryl-2-methylene in PB-3 increased the chemical stability of the PLY inhibitor and improved potency to an IC50 of 3.1 µM (Figure ). The precursor fragments of PB-3, aldehyde PB-3.1 and barbiturate PB-2.3, were both inactive toward PLY, even with prolonged incubation times. In the presence of PLY protein, however, these fragments underwent protein-templated Knoevenagel condensation, a reaction reported recently for the formation of protease inhibitors, resulting in the formation of PB-3 and inhibition of hemolysis (Supplementary Figure ).