Inhibitors testing by prevention of hemoglobin release in erythrocytes. Recombinant PLY was expressed with a 10-fold N-terminal His-tag for initial testing. Native cytolytic activity of the protein product was analyzed prior to the examination of virtual hits and was found to lyze cells efficiently at a concentration of 15 nM. For the primary screening of PLY inhibitors, the hemolysis of defibrinated sheep erythrocytes was investigated. The membranes of human and sheep erythrocytes are well-suited as targets of PLY as they contain an equally high amount of cholesterol (ca. 23%) as human cells. The inhibitory potential of virtual hits (VH) was investigated at three different concentrations (250 µM, 500 µM and 1 mM) against PLY. One molecule, a substituted 5-benzylidene-pyrimidine-2,4,6-trione, was actively hindering hemoglobin release at all three concentrations without precipitation and thus was named poreblock-1 (PB-1) (Supplementary Figure ). The IC50 of PB-1 toward PLY was determined in the same assay and was 38 µM (Figure , Table ). Molecular dynamics (MD) simulations supported the proposed binding mode of PB-1 and revealed the most important interactions by applying dynamic pharmacophores 34 (Figure ). Thus, PB-1 was selected for structural variation and functional optimization. Several structural analogs of PB-1 were designed and prepared in order to scrutinize the activity profile of PB-1 (Table , Supplementary Figure ). PB-1 was synthesized through a Knoevenagel condensation of 3-(2,4-difluorophenoxymethyl)-4-methoxy-benzaldehyde with pyrimidine-2,4,6-trione (barbituric acid). Modification of the benzylidene substituents to 3-(2,4-dichlorobenzyloxy) and 4-ethoxy in PB-2.5 improved the IC50 value toward PLY about 3-fold to 13 µM (Supplementary Figure ). Additional Nsubstitution of the pyrimidine-trione residue with 3,5-dimethyl phenyl in PB-2 increased the potency about 8-fold relative to PB-1 (Figure ). N-substitution of the pyrimidine ring desymmetrized the double bond and accordingly PB-2 was obtained as a 1:1 equilibrium of the E/Z isomers which were interconverted rapidly and thus could not be separated by chromatographic methods. The observed isomerism of PB-2 can be rationalized by a plausible hydration/dehydration mechanism (Supplementary Figure ). All precursor fragments of PB-2, the aromatic aldehyde PB-2.2, the N-substituted barbituric acid PB-2.3 and the truncated precursor PB-2.4 were inactive toward PLY in the hemolysis assay with 10 min incubation.