Lorlatinib (PF-6463922) is an orally available, ATP-competitive inhibitor of the receptor tyrosine kinases, anaplastic lymphoma kinase (ALK) and C-ros oncogene 1 (Ros1), with potential antineoplastic activity.

ROS1:<0.02 nM(Ki), ALK:<0.07 nM(Ki), TYK1/LTK:2.7 nM, ALK (L1196M):0.07 nM(Ki), FER:3.3 nM

Recombinant human wild-type and mutant ALK kinase domain proteins (amino acids 1093–1411) are produced in-house using baculoviral expression, preactivated via autophosphorylation with MgATP, and assayed for kinase activity using a microfluidic mobility shift assay. The reactions contained 1.3 nM wild-type ALK or 0.5 nM mutant ALK (appropriate to produce 15-20% phosphorylation of peptide substrate after 1 h of reaction), 3 μM 5-FAM-KKSRGDYMTMQIG-CONH2), 5 mM MgCl2, and the Km level of ATP in 25 mM Hepes, pH 7.1. The inhibitors are shown to be ATP-competitive from kinetic and crystallographic studies. The Ki values are calculated by fitting the conversion (%) to a competitive inhibition equation. ROS1 enzyme is assayed as described above for ALK, except using 0.25 nM recombinant human ROS1 catalytic domain (amino acids 1883-2347). Kinase inhibitor selectivity is evaluated using a 206-kinase panel.

Cells are seeded in 96-well plates in growth medium containing 10% FBS and are cultured overnight at 37°C. The following day, serial dilutions of PF-06463922 or appropriate controls are added to the designated wells, and cells are incubated at 37°C for 72 h. A CellTiter-Glo assay is performed to determine the relative cell numbers. IC50 values are calculated by concentration-response curve fitting using a four-parameter analytical method.

劳拉替尼, PF-6463922, PF-06463922, Loratinib

Ethanol: 40.6 mg/mL (99.9 mM), Sonication is recommended.

DMSO: 13 mg/mL (31.99 mM), Sonication is recommended.

10% DMSO+40% PEG300+5% Tween 80+45% Saline: 2 mg/mL (4.92 mM), Sonication is recommended.