Pf 6463922

The half maximal inhibitory concentrations (IC₅₀) of lorlatinib (SelleckChem PF-6463922 Cat. No. S7536) and crizotinib (SelleckChem PF-02341066 Cat. No. S1068) for ALK-TKD variants were determined using the assay conditions above with varying concentrations of inhibitor (0-10000 nM), with fixed 1 mM ATP, 10 μM peptide substrate and 5 nM kinase. Inhibitor dilutions were made in 100% DMSO, further diluted 1:10 with 1× kinase buffer [50 mM HEPES, pH 7.5, 0.01% Brij-35, 10 mM MgCl₂] and 2 μl was added per reaction so that final DMSO concentration was constant at 1%. Initial rates were determined as described under the kinase assay method. Curves were normalized against the maximal velocity (“no inhibitor” condition). IC₅₀ values were determined by fitting a 3-parameter fit [Inhibitor] vs normalized response using GraphPad Prism 9.2. IC₅₀ values are reported as mean ± SD for three biological replicates.

For laboratory assessment, the compounds crizotinib (PF-02341066, Cat. No. S1068, Selleck Chemicals LLC, Houston, TX, USA), ceritinib (LDK378, Cat. No. S7083, Selleck Chemicals LLC, Houston, TX, USA) and lorlatinib (PF-6463922, Cat. No. S7536, Selleck Chemicals LLC, Houston, TX, USA) were prepared as stock solutions of 5 mM in DMSO (J.T. Baker, Avantor Performance Materials Poland S.A., Gliwice, Poland).