The NanoLuc luciferase activity assay was performed on previous selected compounds (469 compounds) from ECBL screening library in 8 concentrations (concentration range: 0.0125 – 50 µM) in duplicates (duplicate plates). All compounds were in 10 mM stocks on LDV 384-well plate (384 LDV Labcyte # LP-0200). A 100-fold diluted compound plates were prepared by transfer 80 nL using Echo 650T (Labcyte) acoustic liquid handler to 384-well plates (384 LDV Labcyte # LP-0200) and diluted by dispense 8 µL DMSO (VWR # 83673.290) using a non-contact dispenser Multidrop Combi (ThermoFisher). Source plates and prepared plates with 100 times diluted stock solutions were thawed and centrifuged prior to transferring of 100; 32.5; 10 nL and 2.5 from 10 mM stocks and same amount from 0.1 mM stocks to the assay plates (LUMITRAC 384 white, Greiner #781075) using Echo 650T (Labcyte) acoustic liquid handler. DMSO was also transferred by ECHO to a volume of 100 nL. DMSO (VWR # 83673.290) and Isradipine (Tocris #2004) were added as negative and positive controls to the columns 23 and 24 of each assay plate to yield 0.5% and 10 µM final concentration respectively. Multidrop Combi (ThermoFisher) non-contact dispenser was used to add 10 µL solution of 20 pM NanoLuc enzyme (Promega # E499A) in PBS (ThermoFisherScientific # 10010-015) with 0,1% BSA (MP Biomedicals # 160069) per well and compounds were pre-incubated with the enzyme for 10 min at RT. Then, the enzymatic reaction was initiated by adding of 10 µL furimazine (Aobious # AOB36539) solution in PBS with 0,1% BSA to final concentration of 10 µM (final Nano-Luc concentration 10 pM) with Multidrop Combi. After 10 min luminescent signal was measured with the integration time of 0,2 sec using ClarioStar (BMG Labtech) plate reader. KNIME pipeline was generated and applied for analysis of the results. The raw data files (one txt file per pate) were read and matched with plate template followed by calculation of averages and standard deviation of controls and compound wells, and Z’ factor values for each plate. The plates were proceeded in the loop, generating one data set for all tested plates. Automatic outlier removal was used for detecting control outliers for plates with Z’ factor values below 0,5. The plates for which removal of the outliers did not lead to improvement of Z’ factor (higher then 0,5) or the algorithm was detecting more than 2 outliers, were retested. The raw data values were normalised to positive and negative control for each plate, to calculate percent inhibition following the formula: % INHIBITION = (1-(x- μp)/ (μn – μp))* 100, where X represents readout VALUE of the well, μp the mean of the positive controls and μn represents the mean of the negative controls for each tested assay plate. The heat map of % INHIBITION was then generated for each plate in order to identify plate effects or signal distribution patterns. Plates with visible patterns or abnormal distribution were retested. From the data table, the compound's concentration, % inhibition, and compound ID were selected and using a pipeline in KNIME and a node that utilizes R programming, dose-response curves were generated for each compound. All curves were generated using LL.4 regression model. The IC50 values were determined from the inflection points of the generated curves. In relation to the conducted assay aimed to identify compounds which may cause interferences with NanoLuc luciferase (commonly use in biological and biochemical assays), the following criterion was adopted. All compounds were marked as ACTIVE when they exhibited activity above 30% in two concentrations and two replicates. Other additional information was placed in the comments in results file.