Solid white

The selected chalcones (1c, 2a, 3e, 4e, and 4t), those with a cytotoxic IC₅₀ of less than 10 μM, plus erlotinib were screened for their ability to inhibit the tyrosine kinase activity of the enriched rEGFR using the ADP-Glo™ Kinase Assay. The first 8 µL of buffer (40 mM Tris-HCI pH 7.5, 20 mM MgCl₂, 0.1 mg/mL bovine serum albumen) was added to the wells of a 384-well plate (Promega, solid white). Then, 5 µL of 2 ng/µL of enzymes and 1 µM of the respective inhibitor was added to triplicate wells, followed by 10 µL of a mixture of 5 µM ATP and 2.5 μM poly(glu·tyr), and incubated for 1 h at room temperature. Next, 5 μL of the ADP-Glo reagent was added, incubated for 40 min and then 10 µL kinase detection reagent was added and incubated at room temperature for 30 min to convert the ADP to ATP. The ATP was then detected by measuring the luminescence using a microplate spectrophotometer system (Synergy HTX Multi-Mode reader, BioTek, Winooski, VT, USA). After screening, the compounds that inhibited EGFR-TK more than 50% were selected for determining their IC₅₀ value. All assays were performed in triplicate. The relative inhibition (%) of erlotinib and the chalcone derivatives was then calculated compared to that of the no-inhibitor control.

The IC₅₀ values of the potent PD derivatives obtained from the SIE method were measured using the ADP-Glo^TM kinase assay kit as previously reported [19 (link)]. Firstly, 8 µL of buffer (40 mM Tris-HCl pH 7.5, 20 mM MgCl₂, and 0.1 mg/mL bovine serum albumin) were added to a 384-well plate (Promega, solid white). Secondly, 5 µL of 1.25 ng/µL EGFRs enzyme (wild-type, L858R/T790M, and L858R/T790M/C797S EGFR) and 2 µL of 1 μM of compounds/known drugs were added separately to individual cell cultures, followed by 10 µL of a mixture of 5 µM ATP and 2.5 µM poly(glu-tyr), and were incubated for 1 h at room temperature. Thirdly, 5 µL of the ADP-Glo reagent was added and incubated for 40 min. Finally, 10 µL of kinase detection reagent was added and incubated at room temperature for 30 min and was detected by measuring the luminescence using a microplate reader (Infinite M200 microplate reader, Tecan, Männedorf, Switzerland). A triplicate assay was performed on all potent PD derivatives and known drugs. The percentage relative inhibition (%) of potent compounds was measured compared to the control with no inhibitor as shown in Equation (1): $$\%\mathrm{Relative} \mathrm{inhibition}=\frac{\left(\mathrm{positive}-\mathrm{negative}\right)-\left(\mathrm{sample}-\mathrm{negative}\right)}{\left(\mathrm{positive}-\mathrm{negative}\right)}\times 100.$$

The inhibitory activities of salvicine and chalcone 3d were determined by measuring the ATPase activity of rhTopoIIα-ATPase using the ADP-Glo™ Kinase Assay. Briefly, 8 µL of buffer (40 mM Tris–HCI pH 7.5, 20 mM MgCl₂, 0.1 mg/mL BSA) was added to each well of a 384-well plate (Promega, solid white) with 5 µL of enzymes (10 ng/µL) and 2 µL of the test compound at different concentrations. The reaction was initiated by the addition of 5 µL of 12.5 µM ATP, allowed to proceed for 1 h at room temperature and then stopped by the addition of 5 µL of ADP-Glo™ Reagent and incubating at room temperature for 40 min. Next, 10 μL of Detection Reagent was added and incubated for 30 min prior to the addition of luciferase and luciferin to detect the ATP by measuring the luminescence of each well with a microplate spectrophotometer system (Synergy HTX Multi-Mode reader, BioTek, Winooski, VT). All assays were performed in triplicate. The percentage relative inhibition of salvicine and 3d was calculated as shown in Equation (1);
$\text{\% relative inhibition }=\frac{[\left(\mathit{\text{positive}}-\mathit{\text{negative}}\right)\hspace{0.17em}-\hspace{0.17em}(\mathit{\text{sample}}\hspace{0.17em}-\hspace{0.17em}\mathit{\text{negative}}\mathrm{ }\left)\right]\times \mathrm{ }100}{\mathrm{ }(\text{positive}\hspace{0.17em}-\hspace{0.17em}\text{negative})},$
where negative and positive are the luminescence without and with the enzyme activity, respectively, and sample is luminescence with the sample. Finally, the IC₅₀ curve was determined by GraphPad Prism version 6 (GraphPad Sofware, La Jolla, CA).