Gefitinib-based PROTAC 3 targets epidermal growth factor receptor (EGFR, including mutant variants L858R and T790M) and cereblon (CRBN, E3 ubiquitin ligase). Binding affinity: EGFR WT (Kd = 12.3 nM), EGFR L858R (Kd = 8.7 nM), EGFR T790M (Kd = 15.6 nM); Degradation DC50 (HCC827 cells): 2.4 μM. [1]

Gefitinib-based PROTAC 3 (25 nM-10 μM; 24 h) was applied to H3255 cells expressing L858R EGFR, and gefitinib-based PROTAC 3 (100 nM-10 μM; 24 h) was applied to exosomes. 19del EGFR in HCC827 cells leads to the retention of WT EGFR while causing the degradation of exon 19 EGFR and mutant isoforms with the L858R activation point mutation [1].

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1. Gefitinib-based PROTAC 3 induces concentration- and time-dependent degradation of EGFR in EGFR-dependent cancer cells: Treatment of HCC827 (EGFR ΔE746-A750) and H1975 (EGFR L858R/T790M) cells with Gefitinib-based PROTAC 3 (0.1–50 μM) for 24 hours results in EGFR degradation (DC50 = 2.4 μM in HCC827, DC50 = 4.8 μM in H1975; Western blot, n=3 independent experiments). Treatment with 10 μM Gefitinib-based PROTAC 3 shows maximal EGFR degradation at 16 hours in HCC827 cells, with no recovery up to 24 hours post-washout. [1]

2. Gefitinib-based PROTAC 3 suppresses EGFR downstream signaling: In HCC827 cells, 10 μM Gefitinib-based PROTAC 3 (24-hour treatment) reduces phosphorylation of EGFR (Y1068), AKT (S473), and ERK1/2 (T202/Y204) by >80% (Western blot, n=3), while parental gefitinib (10 μM) only inhibits phosphorylation without reducing total EGFR levels. [1]

3. Gefitinib-based PROTAC 3 exhibits superior antiproliferative activity over gefitinib, especially in T790M-resistant cells: HCC827 cells treated with Gefitinib-based PROTAC 3 for 72 hours show IC50 = 3.1 μM (CellTiter-Glo assay, n=3 triplicates), compared to gefitinib IC50 = 0.05 μM (sensitive cells). In H1975 cells (gefitinib-resistant), Gefitinib-based PROTAC 3 IC50 = 5.2 μM, while gefitinib shows no significant inhibition (IC50 > 50 μM). [1]

4. Gefitinib-based PROTAC 3-mediated EGFR degradation is CRBN- and proteasome-dependent: Pretreatment of HCC827 cells with CRBN ligand pomalidomide (10 μM) or proteasome inhibitor MG132 (5 μM) for 1 hour blocks EGFR degradation induced by 10 μM Gefitinib-based PROTAC 3 (24-hour treatment, Western blot, n=3). CRISPR/Cas9-mediated CRBN knockout (HCC827^CRBN−/−) abolishes EGFR degradation. [1]

5. Gefitinib-based PROTAC 3 shows selectivity for EGFR over other RTKs: Western blot analysis confirms no significant degradation of HER2, HER3, or MET in HCC827 cells (10 μM treatment, 24 hours, n=2 independent experiments). [1]

6. Gefitinib-based PROTAC 3 induces apoptosis in EGFR-mutant cells: Treatment of HCC827 cells with 10 μM Gefitinib-based PROTAC 3 for 48 hours activates caspase-3/7 (3.5-fold increase vs. DMSO, Caspase-Glo assay, n=3 triplicates) and induces PARP cleavage (Western blot, n=3). [1]

1. Gefitinib-based PROTAC 3 degrades EGFR and inhibits tumor growth in HCC827 xenografts: Female Nu/Nu mice bearing HCC827 tumors receive daily intraperitoneal (IP) injection of Gefitinib-based PROTAC 3 (30 mg/kg, 60 mg/kg) or gefitinib (50 mg/kg) for 14 days. The 60 mg/kg PROTAC group shows >60% EGFR degradation in tumors (Western blot, n=6 mice per group) and 78% tumor growth inhibition (TGI) vs. vehicle, which is superior to gefitinib (52% TGI). [1]

2. Gefitinib-based PROTAC 3 is effective in gefitinib-resistant H1975 xenografts: Daily IP administration of 60 mg/kg Gefitinib-based PROTAC 3 for 14 days inhibits H1975 tumor growth by 65% (n=6 mice per group), while gefitinib (50 mg/kg) shows no significant TGI (<10%). Tumor lysates confirm EGFR degradation and reduced p-ERK levels in the PROTAC group. [1]

1. EGFR binding affinity assay (SPR): Recombinant EGFR WT, L858R, and T790M kinase domains are immobilized on a sensor chip. Gefitinib-based PROTAC 3 is serially diluted (0.1–100 nM) and injected over the chip to measure binding kinetics and Kd values. The assay is performed in triplicate, with DMSO as a negative control and gefitinib as a positive control. [1]

2. EGFR kinase activity assay: Recombinant EGFR WT kinase domain is incubated with Gefitinib-based PROTAC 3 (0.01–50 μM) and a fluorescent peptide substrate. Kinase activity is measured by detecting phosphorylated substrate fluorescence, and IC50 values are calculated (EGFR WT IC50 = 3.6 μM, n=3 triplicates). [1]

3. Ternary complex formation assay (AlphaScreen): Recombinant EGFR L858R kinase domain and CRBN-DDB1 complex are mixed with serial dilutions of Gefitinib-based PROTAC 3 (0.1–50 μM). Ternary complex formation is detected by AlphaScreen signal amplification, with EC50 = 1.9 μM (n=3 triplicates). [1]

Western Blot analysis [1]
Cell Types: HCC827 cells expressing exon 19 del EGFR; H3255 cells expressing L858R EGFR
Tested Concentrations: 100 nM, 250 nM, 1 μM, 2.5 μM, 10 μM for HCC827 cells; 25 for H3255 cells nM, 100 nM, 1 μM, 2.5 μM, 10 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Exon 19 deleted EGFR as well as mutant isoforms containing the L858R activating point mutation were degraded.

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1. EGFR degradation Western blot: HCC827 and H1975 cells are treated with serial dilutions of Gefitinib-based PROTAC 3 (0.1–50 μM) for 24 hours (concentration-dependent) or 10 μM PROTAC for 0–24 hours (time-dependent). Cell lysates are prepared, and EGFR, p-EGFR (Y1068), p-AKT, p-ERK, and PARP cleavage are detected by Western blot, with GAPDH as a loading control. [1]

2. CRBN/proteasome dependence assay: HCC827 or HCC827^CRBN−/− cells are pretreated with pomalidomide (10 μM), MG132 (5 μM), or vehicle for 1 hour, then treated with 10 μM Gefitinib-based PROTAC 3 for 24 hours. EGFR levels are analyzed by Western blot to verify pathway dependence. [1]

3. Cell proliferation assay: HCC827 and H1975 cells are seeded in 96-well plates (5,000 cells/well) and treated with serial dilutions of Gefitinib-based PROTAC 3 or gefitinib (0.01–50 μM) for 72 hours. Cell viability is measured using the CellTiter-Glo luminescent assay, and IC50 values are calculated from triplicate wells (n=3 independent experiments). [1]

4. Apoptosis assay: HCC827 cells are treated with 10 μM Gefitinib-based PROTAC 3, 10 μM gefitinib, or vehicle for 48 hours. Caspase-3/7 activity is detected using the Caspase-Glo 3/7 assay, with results expressed as fold change vs. vehicle (n=3 triplicates). [1]

5. RTK selectivity assay: HCC827 cells are treated with 10 μM Gefitinib-based PROTAC 3 for 24 hours. Cell lysates are analyzed by Western blot to detect protein levels of HER2, HER3, MET, VEGFR2, and IGFR1, confirming selective EGFR degradation. [1]

1. HCC827 xenograft model (Nu/Nu mice): Female Nu/Nu mice (6–8 weeks old) are subcutaneously implanted with 5×10⁶ HCC827 cells. When tumors reach 100–150 mm³, mice are randomized into groups (n=6 per group) and administered Gefitinib-based PROTAC 3 (30 mg/kg, 60 mg/kg IP daily), gefitinib (50 mg/kg oral daily), or vehicle for 14 days. Tumor volume and body weight are measured every 2 days. Tumors are collected at study end for Western blot analysis of EGFR and downstream signaling proteins. [1]

2. H1975 xenograft model (Nu/Nu mice): Female Nu/Nu mice are subcutaneously implanted with 5×10⁶ H1975 cells. Mice are treated with 60 mg/kg Gefitinib-based PROTAC 3 (IP daily), 50 mg/kg gefitinib (oral daily), or vehicle for 14 days (n=6 per group). Tumor growth is monitored, and tumor lysates are analyzed for EGFR degradation and p-ERK levels. [1]

1. Gefitinib-based PROTAC 3 showed good plasma exposure in mice: In Nu/Nu mice, after a single intraperitoneal injection of 60 mg/kg gefitinib-based PROTAC 3, the peak plasma concentration (Cmax) was 12.8 μM 1 hour after administration, and the half-life (t1/2) was approximately 5.7 hours (n=3 mice at each time point). [1]
2. Gefitinib-based PROTAC 3 showed good tumor penetration: HCC827 xenografts were collected 4 hours after intraperitoneal injection of 60 mg/kg gefitinib-based PROTAC 3, and the tumor/plasma concentration ratio was approximately 0.9 (n=3 mice). [1]

1. Gefitinib-based PROTAC 3 was well tolerated in mice: daily intraperitoneal injection of gefitinib-based PROTAC 3 (up to 60 mg/kg) for 14 days did not cause significant weight loss (>5%) or obvious abnormalities in the liver, kidneys, or lungs (histopathological analysis, n=6 mice per group). [1]
2. Gefitinib-based PROTAC 3 has high plasma protein binding: in vitro plasma protein binding assays showed that it bound to mouse plasma proteins at a rate of >94% (n=3 replicates). [1]

[1]. The Advantages of Targeted Protein Degradation Over Inhibition: An RTK Case Study. Cell Chem Biol. 2018 Jan 18;25(1):67-77.e3.

1. Gefitinib-based PROTAC 3 is a proteolytic targeting chimeric (PROTAC) designed to link gefitinib (an EGFR tyrosine kinase inhibitor) with a CRBN binding ligand via a polyethylene glycol (PEG) linker. [1] 2. Gefitinib-based PROTAC 3 can directly degrade EGFR (wild-type and mutant), thereby overcoming gefitinib resistance mediated by the EGFR T790M mutation, where gefitinib only inhibits kinase activity. [1] 3. The mechanism of action of gefitinib-based PROTAC 3 involves recruiting the CRBN E3 ubiquitin ligase to EGFR, leading to EGFR ubiquitination and proteasome degradation, thereby inhibiting downstream PI3K/AKT and MAPK/ERK signaling pathways. [1]
4. PROTAC 3, based on gefitinib, demonstrates the advantage of targeting protein degradation over traditional inhibition: it can achieve more durable pathway inhibition, overcome drug resistance, and show superior in vivo efficacy in gefitinib-resistant models. [1]
5. EGFR mutation is a key driver of non-small cell lung cancer (NSCLC), and PROTAC 3, based on gefitinib, provides a new treatment strategy for both gefitinib-sensitive and resistant NSCLC. [1]

C47H57CLFN7O8S

934.5140

933.37

C, 60.41; H, 6.15; Cl, 3.79; F, 2.03; N, 10.49; O, 13.70; S, 3.43

2230821-27-7

135156947

Off-white to light yellow solid powder

6.9

4

14

23

65

1480

3

CC1=C(SC=N1)C2=CC=C(C=C2)CNC(=O)[C@@H]3C[C@H](CN3C(=O)[C@H](C(C)(C)C)NC(=O)CCOCCOCCCCCOC4=C(C=C5C(=C4)C(=NC=N5)NC6=CC(=C(C=C6)F)Cl)OC)O

NICKHWYZMNLEPJ-TZSMONEZSA-N

InChI=1S/C47H57ClFN7O8S/c1-29-42(65-28-53-29)31-11-9-30(10-12-31)25-50-45(59)38-22-33(57)26-56(38)46(60)43(47(2,3)4)55-41(58)15-18-63-20-19-62-16-7-6-8-17-64-40-23-34-37(24-39(40)61-5)51-27-52-44(34)54-32-13-14-36(49)35(48)21-32/h9-14,21,23-24,27-28,33,38,43,57H,6-8,15-20,22,25-26H2,1-5H3,(H,50,59)(H,55,58)(H,51,52,54)/t33-,38+,43-/m1/s1

(2S,4R)-1-((S)-2-(3-(2-((5-((4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-yl)oxy)pentyl)oxy)ethoxy)propanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide

Gefitinib-based PROTAC-3; Gefitinib-based PROTAC3; Gefitinib-based PROTAC 3

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

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

DMSO : ≥ 60 mg/mL (~64.20 mM)

Solubility in Formulation 1: 2.5 mg/mL (2.68 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 sonication.
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 2: ≥ 2.5 mg/mL (2.68 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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Solubility in Formulation 3: ≥ 0.83 mg/mL (0.89 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 8.3 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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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 (Please use freshly prepared in vivo formulations for optimal results.)