EGFR(L858R/T790M) (IC50 = 0.26 nM)

JBJ-04-125-02, which has an IC50 of 0.26 nM for EGFRL858R/T790M, is a strong EGFR inhibitor with mutant structure and EGFR activity [1].

Based on the single agent activity in vitro, we sought to determine whether JBJ-04-125-02 could also be effective in vivo. JBJ-04-125-02 exhibited a moderate half-life of 3h and a high area under the curve of 728577min·ng/mL (AUClast) following 3 mg/kg intravenous (IV) dose. A 20mg/kg oral dose of JBJ-04-125-02 achieved an average maximal plasma concentration of 1.1μM with an oral bioavailability of only 3% (Table S3A). Based on these findings, we performed a pharmacodynamic study whereby EGFR L858R/T790M/C797S genetically engineered mice (GEM), following tumor development, were treated with 3 doses of either vehicle, or 100mg/kg of JBJ-02-112-05, or 50mg/kg or 100mg/kg of JBJ-04-125-02 by oral gavage administered once daily and evaluated the effects on phosphorylation of EGFR and downstream signaling (Figure 2C). Both the 50mg/kg and the 100mg/kg doses effectively inhibited phosphorylation of EGFR, AKT and ERK1/2 (Figure 2C). JBJ-02-112-05 (at 100mg/kg) also inhibited phosphorylation of EGFR and downstream signaling pathways although not as robustly as JBJ-04-125-02 (Figure 2C). In a subsequent efficacy study, we treated EGFR L858R/T790M/C797S GEM mice with vehicle, 100mg/kg of JBJ-02-112-05, or 50mg/kg of JBJ-04-125-02 and followed the change in tumor volume by serial MRI imaging. Despite the better pharmacokinetic profile of JBJ-02-112-05 (Table S3A) compared to JBJ-04-125-02, it was ineffective in the efficacy study and tumor growth was similar to the vehicle control. In contrast, JBJ-04-125-02 treatment led to marked tumor regressions within 4 weeks of treatment (Figure 2D), which were sustained for 15 weeks of treatment (Figure 2E). Despite the poor oral bioavailability of JBJ-04-125-02, long-term treatment led to drug accumulation in plasma and tumor, which likely accounted for its efficacy (Table S3B). Notably, JBJ-04-125-02 treatment was not associated with weight loss or overt signs of toxicity (Figure S2A and data not shown)[1].

HTRF-based EGFR biochemical assays[1]
Biochemical assays with L858R/T790M EGFR were carried out using a homogeneous time-resolved fluorescence (HTRF) KinEASE-TK assay as described previously at the ICCB Longwood Screening Facility at Harvard Medical School. Assays were performed with enzyme concentration of 20pM and 100µM ATP. Inhibitor compounds in DMSO were dispensed directly into 384-well plates with the D300 Digital dispenser followed immediately by the addition of aqueous buffered solutions using the Multidrop Combi Reagent Dispenser. IC50 values were determined with 11- or 23-point inhibition curves in triplicate.

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EGFR protein expression and purification[1]
Constructs spanning residues 696–1022 of the human EGFR (including wild-type, L858R/T790M, L858R/T790M/C797S, and T790M/V948R mutant sequences) were prepared in a His6 and GST-fusion double tagged format using the pTriEX system for expression in Sf9 insect cells essentially as described. EGFR kinase proteins were purified by Ni-NTA and glutathione-affinity chromatography, followed by size-exclusion chromatography after cleavage with TEV to remove the His6-GST fusion partner following established procedures.

Cell viability assays[1]
Ba/F3, H1975, H3255GR and H3255DR cells were treated with increasing concentrations of inhibitors for 72 hours and growth or the inhibition of growth was assessed by MTS assay according to previously established methods. For experiments that investigate the effect of JBJ-04-125-02 in the presence of EGF or cetuximab, 10ng/ml of EGF, 1μg/ml or 10μg/ml of cetuximab added at the same time that cells were treated with inhibitors.

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Biotinylated drug pull-down assay[1]
For biochemical pull-down assays, 10g of free EGFR-L858R/T790M or osimertinib-labeled EGFR-L858R/T790M were incubated with streptavidin-conjugated agarose beads and 4-fold molar excess of biotinylated JBJ-04-125-02 compound for an hour at 4°C. Streptavidin beads were recovered by centrifugation, washed three times with 10 bed volumes of TBS buffer, and bound proteins were analyzed by SDS-PAGE. Osimertinib-labeled EGFR-L858R/T790M was prepared by purification of EGFR-L858R/T790M in the presence osimertinib; 100M osimertinib was added to insect cell lysates, and 2M osimertinib was maintained in purification buffers prior to the final size exclusion step. Stoichiometric labeling with osimertinib was confirmed by mass spectrometry.
For in vitro pull-down assays, cells were treated with dose-escalated WZ-4002, osimertinib or afatinib for two hours before they were subjected to lysis and protein quantification. 500 g of protein was incubated with either biotinylated-linker (control) or with biotinylated JBJ-04-125-02 for two hours before 50% NeutrAvidin agarose beads slurry was added for an hour to precipitate the EGFR that was associated to the biotinylated allosteric inhibitor in the presence of increasing doses of inhibitors. The beads were washed with PBS containing 1% IGEPAL and extraneous buffer was removed before the samples were suspended in 2X SDS sample preparation buffer for Western blotting analyses.

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Crosslinking Assay[1]
H1975 and H3255GR cells were washed with ice-cold PBS twice followed by incubation with 1mM of BS3 in PBS for 30 minutes before the reaction was quenched with 20mM of Tris-HCl pH 7.4 for 15 minutes. Cells were then washed with ice cold PBS twice again before they were lysed in NP40 lysis buffer and processed for Western blotting analyses.

Pharmacokinetic studies[1]
All procedures described are covered under existing protocols and have been approved by the Scripps Florida IACUC to be conducted in the Scripps vivarium, which is fully AAALAC accredited. Pharmacokinetics were determined in n=3 male C57Bl/6 mice. Compounds were dosed as indicated in the text via intravenous tail vein injection or by oral gavage. Blood was collected using minimal sampling techniques where ~25 µL blood is collected from a small nick in the tail using Li-heparin-coated hematocrit tubes at 5min, 15min, 30min, 1h, 2h, 4h, 6h, and 8h. Plasma was generated via centrifugation using a hematocrit rotor. Plasma concentration was determined via LC-MS/MS by comparison of the analyte/IS peak area using a nine-point standard curve between 0.4ng/mL and 2000ng/mL prepared in mouse plasma. Pharmacokinetic analysis was done with WinNonlin, Centara inc. using a noncompartmental model.

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In vivo studies[1]
The EGFR L858R/T790M/C797S mutant mouse cohort was established and reported previously. These mice were monitored by MRI to quantify lung tumor burden before being assigned to various treatment study cohorts. All the treatment mice had equal initial tumor burden before starting treatment. H1975 and DFCI282 cells were grown subcutaneously in Nu/Nu mice purchased from Charles River Laboratories International Inc. Mice were randomly grouped, and treatment initiated when tumor size reached 100 to 200 mm3. Each cohort included at least 5 mice. For pharmacodynamic (PD) studies, tumors were harvested at 3 hours after the last dose. For single agent efficacy studies, GEM and H1975 xenograft mice were treated for up to 15 weeks daily and 30 days respectively and monitored daily. For combination efficacy studies, H1975 and DFCI282 xenograft mice were treated for 28 days. Drugs were then withdrawn and mice were monitored daily for up to 101 days and 31 days respectively. Tumor size of all mice were monitored, and volumes were calculated using the following formula: (mm3) = length × width × width × 0.5. All mice were sacrificed when tumor volume reached approximately 2000mm3. [1]
Both JBJ-02-112-05 and JBJ-04-125-02 were dissolved in 5% NMP (5% 1-methyl-2-pyrrolidinone: 95% PEG-300). Osimertinib was dissolved in 0.5% HMPC (0.5% Hydroxypropyl methylcellulose: 99.5% 0.05N hydrogen chloride). Mice received 2.5mg/kg or 25mg/kg osimertinib once daily orally.

[1]. Single and Dual Targeting of Mutant EGFR with an Allosteric Inhibitor. Cancer Discov. 2019 Jul;9(7):926-943.

Allosteric kinase inhibitors offer a potentially complementary therapeutic strategy to ATP-competitive kinase inhibitors due to their distinct sites of target binding. In this study, we identify and study a mutant-selective EGFR allosteric inhibitor, JBJ-04-125-02, which as a single agent can inhibit cell proliferation and EGFRL858R/T790M/C797S signaling in vitro and in vivo. However, increased EGFR dimer formation limits treatment efficacy and leads to drug resistance. Remarkably, osimertinib, an ATP-competitive covalent EGFR inhibitor, uniquely and significantly enhances the binding of JBJ-04-125-02 for mutant EGFR. The combination of osimertinib and JBJ-04-125-02 results in an increase in apoptosis, a more effective inhibition of cellular growth, and an increased efficacy in vitro and in vivo compared with either single agent alone. Collectively, our findings suggest that the combination of a covalent mutant-selective ATP-competitive inhibitor and an allosteric EGFR inhibitor may be an effective therapeutic approach for patients with EGFR-mutant lung cancer. SIGNIFICANCE: The clinical efficacy of EGFR tyrosine kinase inhibitors (TKI) in EGFR-mutant lung cancer is limited by acquired drug resistance, thus highlighting the need for alternative strategies to inhibit EGFR. Here, we identify a mutant EGFR allosteric inhibitor that is effective as a single agent and in combination with the EGFR TKI osimertinib.[1]

C29H26FN5O3S

543.61184835434

543.17

C, 64.07; H, 4.82; F, 3.49; N, 12.88; O, 8.83; S, 5.90

2140807-05-0

JBJ-04-125-02;2060610-53-7; 2140807-05-0 (racemic) ; 2060610-53-7 (R-isomer)

132020316

White to off-white solid powder

3.8

3

8

6

39

870

0

VHQVOTINPRYDAO-UHFFFAOYSA-N

InChI=1S/C29H26FN5O3S/c30-21-5-8-25(36)24(16-21)26(27(37)33-29-32-11-14-39-29)35-17-20-2-1-19(15-23(20)28(35)38)18-3-6-22(7-4-18)34-12-9-31-10-13-34/h1-8,11,14-16,26,31,36H,9-10,12-13,17H2,(H,32,33,37)

2-(5-fluoro-2-hydroxyphenyl)-2-[3-oxo-5-(4-piperazin-1-ylphenyl)-1H-isoindol-2-yl]-N-(1,3-thiazol-2-yl)acetamide

JBJ-04-125-02 racemate; JBJ0412502; JBJ-04-125-02; JBJ04-125-02; JBJ 0412502; JBJ 04-125-02; JBJ-0412502

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)

DMSO: 100~250 mg/mL (184.0~459.9 mM)

Solubility in Formulation 1: 2.08 mg/mL (3.83 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% 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 20.8 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: 2.08 mg/mL (3.83 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 20.8 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: ≥ 2.08 mg/mL (3.83 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 20.8 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.)