4.4. IC50 Determination by the Bocillin FL Biochemical Assay

Fluorescence polarisation (FP) experiments were carried out as described before [32,35,36,47]. To determine the binding of Bocillin FL to PBP, 200 nM of ^PaPBP3 was incubated for 30 min with 100 nM of Bocillin FL (Thermo Fisher Scientific, Waltham, MA, USA). The fluorescence polarisation signal of free Bocillin FL was monitored in a Pherastar microplate reader (BMG Labtech, Ortenberg, Germany) in the polarisation mode with Ex/Em: 485/520 nm excitation and emission filters. Millipolarisation (mP) units were calculated using the MARS data analysis software v30.40.R2 (BMG Labtech, Ortenberg, Germany). All the experiments were done in Sorensen buffer at 37 °C using flat-bottom, black 96-well plates. To determine the IC₅₀ values of the potential Bocillin FL competitors, 5 µL of serial compound dilutions (100 µM to 0.8 µM compound) in 96% DMSO were incubated with PBP3 of P. aeruginosa for 30 min to promote binding of the compounds. Bocillin FL was diluted in Sorensen buffer and added to the reaction and directly measured for 30 min at 37 °C. The FP was continuously measured in the presence and absence of compound and enzyme. The differences between the mP values at 0 min and 17 min were used as the parameter to estimate the IC₅₀ values. The estimated IC₅₀ values were established by sigmoidal nonlinear regression, using Prims v.8 software.