| Description |
Lorlatinib is a potent, dual ALK/ROS1 inhibitor, with Kis of 0.02 nM, 0.07 nM, and 0.7 nM for ROS1, wild type ALK, and ALK-L1196M, respectively.
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| Related Catalog |
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| Target |
Ki: < 0.02 nM (ROS1), < 0.07 nM (ALK WT), 0.7 nM (ALK L1196M)
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| In Vitro |
Lorlatinib demonstrates significant cell activity against ALK and a large set of ALK clinical mutations with IC50 ranging from 0.2 nM-77 nM[1]. Lorlatinib significantly inhibits cell proliferation and induces cell apoptosis in the HCC78 human NSCLC cells harboring SLC34A2-ROS1 fusions and the BaF3-CD74-ROS1 cells expressing human CD74-ROS1[2]. Lorlatinib also shows potent growth inhibitory activity and induces apoptosis in the NSCLC cells harboring either non-mutant ALK or mutant ALK fusions[3].
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| In Vivo |
In rats, Lorlatinib displays low plasma clearance, a moderate volume of distribution, a reasonable half-life, low propensity for p-glycoprotein 1-mediated efflux and a bioavailability of 100%[1]. In vivo, Lorlatinib shows cytoreductive antitumor efficacy in the NIH3T3 xenograft models expressing human CD74-ROS1 and Fig-ROS1 via inhibition in ROS1 phosphorylation and the downstream signaling molecules, as well as inhibition of the cell cycle protein Cyclin D1 in tumors[2]. In vivo, Lorlatinib also demonstrates marked antitumor activity in mice bearing tumor xenografts expressing EML4-ALK, EML4-ALK-L1196M, EML4-ALK-G1269A, EML4-ALK-G1202R or NPM-ALK[3].
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| Kinase Assay |
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.
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| Cell Assay |
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 Lorlatinib 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.
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| Animal Admin |
De novoGBM tumorigenesis is initiated in LSL-FIG-ROS1;Cdkn2a−/−;LSL-Luc mice through intracranial stereotactic injections of Adeno-Cre as described previously. Tumor development is monitored using BLI as described below. Once tumors reach a given size (107 p-1·s-1·cm-2·sr-1), animals are randomLy enrolled into vehicle control or 3-, 7-, or 14-d treatment with the indicated doses of Lorlatinib. Drug is administered through s.c. implanted Alzet osmotic pumps. After treatment, mice are killed, GBM tumors are microdissected, and tissues are flash-frozen in liquid N2. The remaining brains are processed for histology.
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| References |
[1]. Johnson TW, et al. Discovery of (10R)-7-amino-12-fluoro-2,10,16-trimethyl-15-oxo-10,15,16,17-tetrahydro-2H-8,4-(metheno)pyrazolo[4,3-h][2,5,11]-benzoxadiazacyclotetradecine-3-carbonitrile (PF-06463922), a macrocyclic inhibitor of anaplastic lymphoma kinas [2]. Zou HY, et al. PF-06463922 is a potent and selective next-generation ROS1/ALK inhibitor capable of blocking crizotinib-resistant ROS1 mutations. Proc Natl Acad Sci U S A. 2015 Mar 17;112(11):3493-8 [3]. Zou HY, et al. PF-06463922 is a potent and selective next-generation ROS1/ALK inhibitor capable of blocking crizotinib-resistant ROS1 mutations. Proc Natl Acad Sci U S A. 2015 Mar 17;112(11):3493-8.
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