ERLOTINIB HYDROCHLORIDE US9730934, Erlotinib US9409845, Table 1, Compound 22: erlotinib OSI-774 US10189853, erlotinib WO2022090481, Example erlotinib BDBM5446 Erotinib CHEMBL553 US11524945, Compound Erlotinib N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine US10507209, Compound Erlotinib Erlotinib Tarceva N-(3-Ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine Monohydrochloride cid_176870
ERLOTINIB HYDROCHLORIDE Erlotinib OSI-774 CP-358774-01 BDBM50311470 CHEMBL1079742
US11524945, Compound Erlotinib-4-methylphenyl analog (E4ME) BDBM582529
- Chen, KF; Pao, KC; Su, JC; Chou, YC; Liu, CY; Chen, HJ; Huang, JW; Kim, I; Shiau, CW Development of erlotinib derivatives as CIP2A-ablating agents independent of EGFR activity. Bioorg Med Chem 20: 6144-53 (2012)
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- Biochemical Assay Thus, while these compounds were extensively used in studying ILK-mediated cellular and disease processes, their reported inhibitory effects are probably due to unknown artifacts or indirect binding events. Next, we turned our attention to previously reported studies on kinase profiling and quantitative chemical proteomics. These studies suggested that a widely known lung cancer drug erlotinib, which targets EGFR, might also bind to ILK as an off target. By performing a robust fluorescence-based binding assay, we found that the FDA approved drug Erlotinib (TARCEVA) indeed binds potently to purified recombinant ILK at KD 0.43M, which is very close to the affinity of Erlotinib to EGFR measured at the same experimental conditions (KD 0.31 μM). Another erlotinib-like EGFR inhibitor Gefitinib exhibited 10-fold weaker binding affinity to ILK (KD 4.51 μM) yet 3-fold stronger affinity to EGFR (KD 0.11 μM) than erlotinib.
- ChEMBL_1538415 (CHEMBL3737962) Inhibition of EGFR T790M/del (746 to 750) deletion mutant phosphorylation in erlotinib-resistant human PC9 cells preincubated for 1 hr followed by stimulation with EGF for 8 mins by electrochemiluminescent immunoassay
- Kinase Assay JAK2 and JAK2 [V617F] Kinase Assay: In 5× Kinase Buffer A, JAK2 or JAK2 [V617F] kinase was mixed with pre-diluted compounds at different concentrations in duplicate for 10 minutes. The corresponding substrate and ATP were added and reacted at room temperature for 20 minutes (in which negative and positive controls were set: the negative control was a blank control and the positive control was erlotinib). After the reaction was completed, a detection reagent (the reagent in the HTRF Kinase TK kit) was added. After incubation at room temperature for 30 minutes, the enzyme activities in the presence of the compounds disclosed herein at each concentration were measured by an Evnvision microplate reader, and inhibitory activities of compounds at different concentrations on the enzyme activity were calculated. The inhibitory activities of compounds at different concentrations on enzyme activity were then fitted according to the four-parameter equation using Graphpad 5.0 software, and the IC50 values were calculated.
- Kinase Inhibition Assay Reagents and Materials:WT EGFR (Carna, Cat. No. 08-115), EGFR [L858R] (Carna, Cat. No. 08-502), EGFR [L858R/T790M] (Carna, Cat. No. 08-510), ATP (Sigma, Cat. No. A7699-1G), DMSO (Sigma, Cat. No. D2650), 96-well plate (Corning, Cat. No. 3365), 384-well plate (Greiner, Cat. No. 784076), HTRF Kinase TK Kit (Cisbio, Cat. No. 62TK0PEJ), Erlotinib (Selleckchem, Cat. No. S7787), EGFR [d746-750] (Life Technologies, Cat. No. PV6178), 5× Kinase Buffer A (Life Technologies, Cat. No. PV3186), Kinase Tracer 199 (Life Technologies, Cat. No. PV5830), LanthaScreen Eu-anti-GST antibody (Life Technologies, Cat. No. PV5594).Specific Experimental Protocol:Compound preparation: the test compound was dissolved in DMSO to make a 20 mM stock solution. Then, it was diluted in DMSO with a 3-fold series gradient dilution for 10 times. The dilutions were diluted 10 fold with buffer when dosing.WT EGFR and EGFR [L858R/T790M] kinase assay: WT EGFR or EGFR [L858R/T790M] kinase was mixed with different concentrations of pre-diluted compounds for 10 minutes in 5× Kinase Buffer A in duplicate. The corresponding substrate and ATP were added and reacted at room temperature for 20 minutes (in which a negative and a positive control were set: the negative control is blank and the positive control is erlotinib). After the reaction, the detection reagent (the reagent in the HTRF Kinase TK kit) was added, and after incubation at room temperature for 30 minutes, the enzyme activity in the presence of the compounds of the present disclosure at each concentration was measured by an Evnvision microplate reader, and the inhibition of the enzyme by the compound at each concentrations were calculated. The inhibitions of the enzyme activity by the compounds at different concentrations were then fitted using Graphpad 5.0 software according to the four-parameter equation, and the IC50 values were calculated.
- In Vitro Enzyme Inhibitory Activity Samples:Controls: Gefitinib, erlotinib hydrochloride, purchased from Anqing worldchem Co., LTD.; lapatinib ditosylate, purchased from Taizhou Xingcheng Chempharm Co., Ltd.; CI-1033 hydrochloride, purchased from Shanghai hanxiangchem, Co., Ltd.; andThe present compounds: lab-made, their chemical names and structural formulae are shown in the preparation examples.Assay Procedures:The abbreviations used in the following assay have the following meanings:HEPES: hydroxyethyl piperazine ethanesulfonic acid;Brij-35: polyoxyethylene lauryl ether;DTT: dithiothreitol;Coating Reagent #3: #3 coating agent;EDTA: ethylene diamine tetraacetic acid, purchased from Sigma Co. Ltd.;FAM labeled peptide: fluorescein labeled peptide 22 (GL Biochem);ATP: adenosine triphosphate (Sigma);DMSO: dimethyl sulfoxide;EGFR: human epidermal growth factor receptor (Carna);HER2: human epidermal growth factor receptor 2 (Carna);HER4: human epidermal growth factor receptor 4 (Carna).1. Formulating the agents to be used in the assay(1) 1.25-fold MnCl2-free kinase buffer (62.5 mM HEPES, PH 7.5, 0.001875% Brij-35, 12.5 mM MgCl2, 2.5 mM DTT);(2) 1.25-fold MnCl2-containing kinase buffer (62.5 mM HEPES, pH 7.5, 0.001875% Brij-35, 12.5 mM MgCl2, 12.5 mM MnCl2, 2.5 mM DTT);(3) Stop buffer (100 mM HEPES, pH 7.5, 0.015% Brij-35, 0.2% Coating Reagent #3, 50 mM EDTA);(4) 2.5-fold kinase solutions (to the 1.25-fold kinase buffers were added the corresponding kinases to formulate 2.5-fold EGFR, HER2, HER4 kinase solutions);(5) 2.5-fold peptide solutions (to the 1.25-fold kinase buffers were added FAM labeled peptide and ATP to formulate the peptide solutions);(6) 5-fold compound solutions (using 100% DMSO to formulate 50-fold compound solutions having different concentration gradients, and diluting with water by 10 times to obtain 5-fold compound solutions having different concentration gradients);2. Adding 5 μL of a 5-fold compound solution to a 384-well plate;3. Adding 10 μL of a 2.5-fold kinase solution to incubate for 10 min;4. Then adding 10 μL of a 2.5-fold peptide solution, and reacting at 28° C. for 1 h; and5. Finally, adding 25 μL of stop buffer to terminate the reaction, and reading the data with Caliper.6. Curve fitting to obtain an IC50 value.The calculated inhibition ratio (%)=(the maximum conversion rate−the conversion rate)/(the maximum conversion rate−the minimum conversion rate)×100The curve fitting was conducted with the Xlfit software to obtain IC50 values.