Biophysical validation of binding of PB-3 to PLY. Bio-layer interferometry (BLI) was applied to validate binding of PB-2 (Supplementary Figure ) and the best inhibitor PB-3 to PLY protein and to quantify the binding kinetics. BLI is a surface-based protein-binding assay, which uses the recorded shifts in the interference pattern of white light in order to measure the adsorption and desorption of ligand to a sensor surface loaded with protein. Here, His-tagged wild-type PLY and a non-binding PLY-C428A mutant as protein reference (see below) were loaded onto a Ni-NTA-coated sensor surface and the protein-loaded biosensors were dipped in buffer solutions with three different concentrations (0.6, 1 and 6 µM) of PB-3 prepared from a PB-3 stock solution in DMSO. Buffer including equivalent amounts of DMSO (up to 5%) were employed as reference samples without inhibitor. Association of PB-3 to wild-type PLY and dissociation from PLY were recorded over time when the sensor was dipped in the inhibitor solution and buffer solution without inhibitor. Protein and inhibitor reference data were subtracted and the binding isotherm was plotted with time on x-axis and response (nm) on yaxis (Figure ). The 1:1 binding model was applied and kinetics were calculated by using ForteBio data analysis 9 software, revealing that PB-3 bound with an affinity or KD value of 2.56×10 -7 M, or 256 nM, to wild-type PLY (Figure ). The high binding affinity was the result of a high on-rate (1800 M -1 s -1 ) and small off-rate (0.00046 s -1 ).