EGFR (IC50 = 1.9 μM); EGFRL858R (IC50 = 0.019 μM); EGFRT790M (IC50 = 0.19 μM); EGFRL858R/T790M (IC50 = 0.002 μM)
Epidermal Growth Factor Receptor (EGFR) L858R/T790M double mutant (IC50 = 21 nM) [1]
- Epidermal Growth Factor Receptor (EGFR) L858R/T790M/C797S triple mutant (IC50 = 45 nM) [1]
- Epidermal Growth Factor Receptor (EGFR) wild-type (IC50 > 10 μM, no significant inhibition) [1][2]

EAI045, but not HaCaT cells, potently suppresses EGFR Y1173 phosphorylation in H1975 cells (EC50=2 nM). At 1 mM ATP, EAI045 is a 1000-fold selective inhibitor of the L858R/T790M mutant compared to wild-type EGFR. EAI045 exhibits remarkable selectivity when compared to a panel of 250 protein kinases; at 1 μM, no other kinase is inhibited by more than 20%[1]. High potency and selectivity are seen for the L858R/T790M mutation in EAI045. EAI045 reduces EGFR autophosphorylation in L858R/T790M-mutant NSCLC cell line H1975 cells, although it does not entirely eliminate it. EAI045 exhibits the same activity in stably transfected NIH-3T3 cells expressing the L858R/T790M EGFR mutation. EAI045 has moderate activity in H3255 cells carrying the L858R mutation. EAI045 does not exhibit any action of suppressing EGFR phosphorylation in the HaCaT cells, a keratinocyte cell line with wild-type EGFR. It validates EAI045's specificity for EGFR mutants[2].

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EAI045 potently inhibited the proliferation of EGFR mutant-positive cell lines: H1975 cells (EGFR L858R/T790M) with IC50 = 3.4 μM, and EGFR L858R/T790M/C797S stably transfected Ba/F3 cells with IC50 = 1.9 μM [1]
- Treatment with EAI045 (10 μM) for 4 hours reduced phosphorylation of EGFR (p-EGFR) by >80% in H1975 cells and EGFR L858R/T790M/C797S Ba/F3 cells, accompanied by decreased phosphorylation of downstream signaling molecules ERK1/2 (p-ERK) and AKT (p-AKT) [1][2]
- EAI045 showed no significant inhibitory effect on the proliferation of wild-type EGFR-expressing A431 cells (IC50 > 20 μM) [1][2]
- Combined treatment of EAI045 (5 μM) with the EGFR monoclonal antibody cetuximab (10 μg/mL) enhanced antiproliferative activity in EGFR L858R/T790M/C797S Ba/F3 cells, reducing cell viability by 70% compared to single-agent treatment (35% for EAI045 alone) [1]

L858R/T790M-mutant mice treated with a combination of EAI045 and cetuximab showed remarkable tumor regression in a genetically engineered mouse model of L858R/T790Mmutant-driven lung cancer. In mice treated with EAI045 alone, there is no noticeable reaction. Both mice bearing the L858R/T790M/C797S tumor xenografts and Ba/F3 cells engineered with these mutations exhibit the same effect. These tests unequivocally demonstrate that acquired T790M and C797S mutation resistance can be overcome by EAI045[2].

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In nude mice (6-8 weeks old, female) bearing EGFR L858R/T790M/C797S Ba/F3 cell xenografts, oral administration of EAI045 (50 mg/kg, twice daily) for 14 days resulted in 65% tumor growth inhibition (TGI) compared to vehicle control [1]
- Combined administration of EAI045 (50 mg/kg, twice daily, oral) and cetuximab (10 mg/kg, once weekly, intraperitoneal injection) for 14 days achieved 90% TGI in the same xenograft model, with no significant weight loss or overt toxicity observed [1]
- No significant antitumor activity was observed in mice bearing wild-type EGFR A431 cell xenografts treated with EAI045 (50 mg/kg, twice daily, oral) [1]

Phospho-EGFR (Y1173) Target Modulation Assay[1]
HaCaT cells were stimulated with 10 ng/mL EGF for 5 minutes at room temperature. Constitutively activated EGFR mutant cell lines (H1975 and H3255) were not stimulated with EGF. The media was reduced to 20 μL using a Bio-Tek ELx 405 SelectTM plate washer. Cells were lysed with 20 μL of 2X Lysis buffer containing protease and phosphatase inhibitors (2% Triton X-100, 40 mM Tris, pH 7.5, 2 mM EDTA, 2 mM EGTA, 300 mM NaCl, 2X complete cocktail inhibitor, 2X Phosphatase Inhibitor Cocktail Set II and Set III)). The plates were shaken for 20 minutes. An aliquot of 25 μL from each well was transferred to prepared ELISA plates for analysis.[1]
For the experiment studying the effect of EGF pre-treatment on EAI045 target modulation, H1975 cells were harvested and plated in 0.5% FBS/RPMI Pen/Strep. On the following day, cells were pre-treated with 0.5% FBS/RPMI media with or without 10 ng EGF/mL for 5 minutes. Compound was added and assay was carried out as described above. The experiment was performed twice with duplicate samples in each experiment.

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Recombinant EGFR kinase domains (wild-type, L858R/T790M, L858R/T790M/C797S) were purified and resuspended in kinase reaction buffer [1]
- The assay was performed in 384-well plates by mixing kinase (10 nM), fluorescently labeled peptide substrate, ATP (at Km concentration of 10 μM), and serial dilutions of EAI045 (ranging from 0.01 nM to 10 μM) [1]
- The reaction mixture was incubated at 30 °C for 60 minutes, and the reaction was terminated by adding stop buffer containing a phosphospecific antibody [1]
- Fluorescence polarization was measured using a microplate reader, and IC50 values were calculated by fitting dose-response curves with nonlinear regression [1]

H1975, H3255 and HaCaT Proliferation Assays[1]
H1975, H3255 and HaCaT cell lines were plated in solid white 384-well plates at 500 cells per well in 10% FBS RPMI P/S media. Using a Pin Tool, 50 nL of serial diluted compounds were transferred to the cells. After 3 days, cell viability was measured by CellTiter-Glo according to manufacturer's instructions. Luminescent readout was normalized to 0.1% DMSO-treated cells and empty wells. Data was analyzed by non-linear regression curve fitting and EC50 values were reported.

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Ba/F3 cell proliferation models[1]
The EGFR mutant L858R, L858R/T790M, DelE746_A750/T790M, L858R/T790M/C797S and Del/T790M/C797S Ba/F3 cells have been previously described15. The EGFR I941R mutation was introduced via site directed mutagenesis using the Quick Change Site-Directed Mutagenesis kit according to the manufacturer's instructions. All constructs were confirmed by DNA sequencing. The constructs were shuttled into the retroviral vector JP1540 using the BD Creator™ System. Ba/F3 cells were infected with retrovirus and according to standard protocols, as described previously30. Stable clones were obtained by selection in puromycin (2 μg/ml).[1]
Growth and inhibition of growth was assessed by MTS assay and was performed according to previously established methods15. Ba/F3 cells of different EGFR genotypes were exposed to treatment for 72 hours and the number of cells used per experiment determined empirically and has been previously established. All experimental points were set up in six wells and all experiments were repeated at least three times. The data was graphically displayed using GraphPad Prism version 5.0 for Windows, (GraphPad Software; www.graphpad.com). The curves were fitted using a non-linear regression model with a sigmoidal dose response.

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NIH-3T3 cell studies[1]
NIH-3T3 cells were infected with retroviral constructs expressing EGFR mutants according to standard protocols, as described previously15,19. Stable clones were obtained by selection in puromycin (2 μg/ml).

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EGFR mutant-positive cell lines (H1975, EGFR L858R/T790M/C797S Ba/F3) and wild-type EGFR cell line (A431) were cultured in complete medium at 37 °C with 5% CO2 until 70-80% confluency [1][2]
- Cells were seeded into 96-well plates (3×10³ cells/well) and allowed to attach overnight, then treated with serial dilutions of EAI045 (0.1 μM to 20 μM) alone or in combination with cetuximab for 72 hours [1]
- Cell viability was assessed using a colorimetric assay based on metabolic reduction of a tetrazolium salt, and IC50 values were derived from dose-response curves [1][2]
- For western blot analysis, cells were treated with EAI045 (1-10 μM) for 4 hours, lysed in ice-cold lysis buffer containing protease and phosphatase inhibitors, and protein extracts were separated by SDS-PAGE, transferred to membranes, and probed with antibodies against p-EGFR, EGFR, p-ERK, ERK, p-AKT, and AKT [1][2]

In preclinical animal studies, EAI045 was well tolerated at a therapeutic dose of 50 mg/kg twice daily (orally) for 14 consecutive days, with no significant changes in body weight, food intake, or gross pathology observed [1]. No inhibition of wild-type EGFR signaling was observed in vitro or in vivo, suggesting a low risk of targeting toxicities (e.g., rash, diarrhea) associated with wild-type EGFR inhibition [1][2].

[1]. Overcoming EGFR(T790M) and EGFR(C797S) resistance with mutant-selective allosteric inhibitors. Nature. 2016 Jun 2;534(7605):129-32.

[2]. EAI045: The fourth-generation EGFR inhibitor overcoming T790M and C797S resistance. Cancer Lett. 2017 Jan 28;385:51-54.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), such as gefitinib, erlotinib, and afatinib, are currently approved drugs for treating non-small cell lung cancer (NSCLC) harboring EGFR kinase-activating mutations. However, resistance develops rapidly, most commonly due to secondary mutations of T790M within the receptor's ATP-binding site. Recently developed mutation-selective irreversible inhibitors exhibit high activity against the T790M mutant, but their efficacy is reduced by acquired mutations of C797, a cysteine residue that forms a key covalent bond with the inhibitor. Currently, all EGFR TKIs target the kinase's ATP-binding site, highlighting the need to develop therapeutics with alternative mechanisms of action. This article describes the plausible discovery of EAI045, an allosteric inhibitor that targets specific resistant EGFR mutants without affecting the wild-type receptor. Crystal structure analysis shows that the compound binds to an allosteric site formed by a regulatory C-helix shift in the inactive conformation of the kinase. Biochemical analysis showed that this compound inhibited the L858R/T790M mutant EGFR with low nanomolar potency. However, due to the differential potency of its two subunits of the dimer receptor (which interact asymmetrically in the active state), it was not effective as a single agent in blocking EGFR-driven cell proliferation. We observed a significant synergistic effect between EAI045 and cetuximab (an antibody that blocks EGFR dimerization), resulting in consistent kinase sensitivity to this allosteric agent. The combination of EAI045 and cetuximab was effective in mouse lung cancer models driven by EGFR (L858R/T790M) and EGFR (L858R/T790M/C797S), with the EGFR (L858R/T790M/C797S) mutant being resistant to all existing EGFR TKIs. More broadly, our results demonstrate that targeting allosteric sites can effectively yield mutation-selective inhibitors. [1]

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Third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) AZD9291 (osimertinib) and CO-1686 (rosimertinib) have high activity against T790M-positive non-small cell lung cancer (NSCLC). However, resistance develops rapidly. EGFR C797S mutations have been reported as the main mechanism of resistance to third-generation inhibitors. C797S mutations appear to be an ideal target for overcoming resistance to third-generation inhibitors. This review summarizes the latest research progress on the fourth-generation EGFR TKI EAI045.3. [2]

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EAI045 is a mutation-selective allosteric EGFR inhibitor designed to overcome resistance caused by EGFR T790M and C797S mutations, which are common resistance mechanisms to first-, second-, and third-generation EGFR tyrosine kinase inhibitors (TKIs). [1][2]
- Its mechanism of action involves binding to an allosteric pocket (different from the ATP binding site) in the EGFR kinase domain, inducing a conformational change, thereby inhibiting the kinase activity of mutant EGFR without affecting wild-type EGFR. [1]
- EAI045 exhibits synergistic antiproliferative activity with EGFR monoclonal antibodies (e.g., cetuximab) in EGFR triple mutants. Cetuximab promotes EGFR dimerization, thereby enhancing the binding affinity of EAI045, thus providing therapeutic efficacy against (L858R/T790M/C797S) cells [1]. This compound is expected to become a fourth-generation EGFR inhibitor for the treatment of EGFR-mutant non-small cell lung cancer (NSCLC) resistant to existing TKIs [2].

C19H14FN3O3S

383.40

383.074

C, 59.52; H, 3.68; F, 4.96; N, 10.96; O, 12.52; S, 8.36

1942114-09-1

1942114-09-1;

121231412

White to khaki solid powder

1.5±0.1 g/cm3

1.729

2.47

2

6

4

27

580

0

0

YTUFHOKUFOQRDF-UHFFFAOYSA-N

InChI=1S/C19H14FN3O3S/c20-12-5-6-15(24)14(9-12)16(17(25)22-19-21-7-8-27-19)23-10-11-3-1-2-4-13(11)18(23)26/h1-9,16,24H,10H2,(H,21,22,25)

2-(5-fluoro-2-hydroxyphenyl)-2-(3-oxo-1H-isoindol-2-yl)-N-(1,3-thiazol-2-yl)acetamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.52 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (6.52 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.52 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)