Nazartinib (EGF816) (EGF816, NVS-816) is a covalent, irreversible, mutant-selective EGFR inhibitor that has nanomolar inhibitory potency against activating mt (L858R, ex19del) and T790M mt, with up to 60-fold selectivity over wild type (wt) EGFR in vitro.

EGFR (mutant):0.031 μM(Ki)

Nazartinib是一种新型共价的、突变选择性EGFR抑制剂，对致癌基因(L858R和ex19del)和T790M耐药性突变具有几乎等效的活性，并对WT EGFR具有良好的选择性。Nazartinib在体外对最常见的EGFR突变L858R、Ex19del和T790M表现出强大的抑制作用。通过使用携带L858R、Ex19del和L858R/T790M突变的三种特征明确的细胞系H3255、HCC827和H1975，评估了Nazartinib对EGFR突变体的细胞活性。与细胞孵育3小时后，Nazartinib在H3255、HCC827和H1975细胞中显示出对pEGFR水平的强效抑制，其EC50值分别为5、1和3nmol/L。基于细胞的测定显示Nazartinib对突变型EGFR比对WT EGFR具有选择性。

Nazartinib在小鼠中具有良好的耐受性、有利的理化性质以及良好的口服生物利用度。在啮齿动物中, Nazartinib的分布量适中，清除率从低到中等（分别为大鼠和小鼠肝脏血流的30%和35%）。在狗中，Nazartinib表现出高清除率和高分布量。此外，Nazartinib在exon 20插入突变模型中显示出抗肿瘤活性。在高于有效剂量的水平上，Nazartinib治疗对WT EGFR的抑制作用最小且有良好的耐受性。在单剂量研究中，Nazartinib能持续抑制EGFR的磷酸化，这与其不可逆结合的能力相一致。Nazartinib在人体中的半衰期比在小鼠中更长，目前正在进行I/II期临床试验，评估其在携带EGFR突变（包括T790M）的患者中的效果。

Recombinant kinase domain of EGFR L858R and T790M-L858R mutants are incubated with EGF816 to confirm covalent modification of EGFR and site of adduction. Recombinant enzyme is incubated at room temperature with a 20-fold molar excess of compound in 40 mM Tris, pH 8, 500 mM NaCl, 1% glycerol, 5 mM TCEP for 1 h. The reaction is quenched by addition of dithiothreitol (DTT, 80-fold excess to compound) and transfer to ice. A third of the reaction (10 μL) is processed for intact MS by adding an equal volume of 6 M Guan HCl, 100 mM Tris, pH 8, 20 mM DTT, 10 mM TCEP and incubating at room temperature for 15 min. Intact MS analysis is performed on an Agilent 6520 QToF mass spectrometer equipped with a dual spray ion source (IS of 4500 V, fragmentor of 250 V, fas temp of 350°C, and skimmer of 75 V). The samples are injected onto a PLRP-S column (2.1 mm × 50 mm), heated to 60°C, and desalted for 2 min at 500 μL/min and 3% B prior to elution with a fast gradient of 3-50% B in 3 min (B, 0.1% formic acid). The data are analyzed in MassHunter for automatic peak selection, integration, and spectral deconvolution with a mass range of 15?000-75?000 Da.

H1975, H3255, HCC827, A431, and HaCaT cells are maintained in RPMI media supplemented with antibiotics and 10% FBS, maintained in a 37°C, 5% CO2 humidified incubator. After an overnight incubation in 384-well plates, serial diluted compounds are transferred to cells and incubated for 3 hours. HaCaT cells are stimulated with 10 ng/mL EGF (50 ng/mL EGF for A431) for 5 minutes. Cells are lysed in 1% Triton X-100 buffer containing protease and phosphatase inhibitors. Lysates are analyzed by sandwich ELISA utilizing goat anti-EGFR capture antibody, anti-phospho-EGFR(Y1173), and anti-rabbit HRP. Signal is measured by chemiluminescent detection.(Only for Reference)