The epidermal growth factor receptor (EGFR)-directed tyrosine kinase inhibitors (TKIs) gefitinib, erlotinib and afatinib are approved treatments for non-small cell lung cancers harbouring activating mutations in the EGFR kinase1,2, but resistance arises rapidly, most frequently owing to the secondary T790M mutation within the ATP site of the receptor3,4. Recently developed mutant-selective irreversible inhibitors are highly active against the T790M mutant5,6, but their efficacy can be compromised by acquired mutation of C797, the cysteine residue with which they form a key covalent bond7. All current EGFR TKIs target the ATP-site of the kinase, highlighting the need for therapeutic agents with alternative mechanisms of action. Here we describe the rational discovery of EAI045, an allosteric inhibitor that targets selected drug-resistant EGFR mutants but spares the wild-type receptor. The crystal structure shows that the compound binds an allosteric site created by the displacement of the regulatory C-helix in an inactive conformation of the kinase. The compound inhibits L858R/T790M-mutant EGFR with low-nanomolar potency in biochemical assays. However, as a single agent it is not effective in blocking EGFR-driven proliferation in cells owing to differential potency on the two subunits of the dimeric receptor, which interact in an asymmetric manner in the active state8. We observe marked synergy of EAI045 with cetuximab, an antibody therapeutic that blocks EGFR dimerization9,10, rendering the kinase uniformly susceptible to the allosteric agent. EAI045 in combination with cetuximab is effective in mouse models of lung cancer driven by EGFR(L858R/T790M) and by EGFR(L858R/T790M/C797S), a mutant that is resistant to all currently available EGFR TKIs. More generally, our findings illustrate the utility of purposefully targeting allosteric sites to obtain mutant-selective inhibitors.
Diverse activating mutations within the EGFR kinase domain give rise to a subset of non-small cell lung cancers (NSCLCs). The L858R point mutation and small in-frame deletions in the region encoded by exon 19 are the most common mutations, and are among a subset of oncogenic EGFR alterations that confer enhanced sensitivity to EGFR-directed TKIs11,12,13. The dose-limiting toxicity of anilinoquinazoline TKIs such as erlotinib and gefitinib arises from inhibition of wild-type EGFR in the skin and GI tract, thus this enhanced sensitivity relative to wild-type EGFR creates a therapeutic window that allows effective treatment of patients whose tumours are driven by these mutations. The T790M resistance mutation closes this window, in part by increasing the affinity of the mutant receptor for ATP, which in turn diminishes the potency of these ATP-competitive inhibitors14. Mutant-selective irreversible inhibitors, including the tool compound WZ4002 (ref. 15) and the clinical compounds osimertinib (AZD9291)6,16 and rociletinib (CO-1686)5, are based on a pyrimidine scaffold, and also incorporate a Michael acceptor group that forms a covalent bond with Cys797 at the edge of the ATP binding pocket. Because they bind irreversibly, these agents overcome the enhanced ATP affinity conferred by the T790M mutation. Compounds of this class are demonstrating significant efficacy against T790M mutant tumours in ongoing clinical trials17,18, and osimertinib was recently approved by the US Food and Drug Administration for patients with EGFR T790M-positive NSCLC following progression on previous EGFR TKI therapy. However, laboratory studies and early clinical experience indicate that the efficacy of these agents can be compromised by mutation of Cys797, which thwarts formation of the potency-conferring covalent bond7,15,19.
Reasoning that an allosteric inhibitor could also overcome the enhanced ATP affinity conferred by the T790M mutation, we screened an ~2.5 million compound library using purified EGFR(L858R/T790M) kinase. The biochemical screen was carried out using 1 μM ATP, and active compounds were counter-screened at 1 mM ATP and against wild-type EGFR to identify those that were potentially non-ATP-competitive and mutant selective. Among the compounds identified in the screen, EGFR allosteric inhibitor-1 (EAI001, Fig. 1a) was of particular interest owing to its potency and selectivity for mutant EGFR (half maximal inhibitory concentration (IC50) = 0.024 μM for L858R/T790M at 1 mM ATP, IC50 > 50 μM for wild-type EGFR). Further characterization of the mutant-selectivity of EAI001 revealed modest potency against the isolated L858R and T790M mutants (0.75 μM and 1.7 μM, respectively, Extended Data Fig. 1a). Medicinal-chemistry-based optimization of this compound yielded EAI045 (Fig. 1a), a 3 nM inhibitor of the L858R/T790M mutant with ~1000-fold selectivity versus wild-type EGFR at 1 mM ATP (Table 1). Enzyme kinetic characterization confirmed that the mechanism of inhibition was not competitive with respect to ATP (Table 1, Extended Data Fig. 1b). Profiling of EAI045 against a panel of 250 protein kinases revealed pronounced selectivity; no other kinases were inhibited by more than 20% at 1 μM EAI045 (Extended Data Table 1). Evaluation of EAI045 in a safety pharmacology assay panel revealed also excellent selectivity against non-kinase targets (Extended Data Table 2).
The crystal structure of EAI001 bound to T790M-mutant EGFR showed that the compound binds in an allosteric pocket that is created in part by the outward displacement of the C-helix in the inactive conformation of the kinase (Fig. 1b, c, Extended Data Table 3). The compound binds as a ‘three-bladed propeller’ with the aminothiazole moiety inserted between the mutant gatekeeper methionine and active site residue Lys745. The phenyl substituent extends into a hydrophobic cleft at the back of the pocket and is in contact with Leu777 and Phe856. Finally, the 1-oxoisoindolinyl group extends along the C-helix towards the solvent exposed exterior. The compound also forms a hydrogen bond with Asp855 in the DFG motif. In further support of a non-ATP competitive mechanism, the ATP-analogue adenylyl-imidodiphosphate (AMP-PNP) is bound in the expected manner in the active site cleft (Fig. 1c).
Interestingly, the EGFR inhibitors neratinib20 and lapatinib21 extend into the allosteric site and make interactions that resemble those of two of the three blades of the allosteric agents (Fig. 1d, Extended Data Fig. 2a, c). These ATP-competitive inhibitors are not mutant selective, and they span both the ATP and allosteric sites. Additionally, we note that the EGFR allosteric pocket is roughly analogous to a site in MEK1 that is targeted by a number of allosteric inhibitors that are now approved or in clinical trials22. Despite the similar location of the MEK allosteric site, there is no structural correspondence in the binding modes of the respective allosteric inhibitors (Extended Data Fig. 2a, b).
The mutant-specificity of the EGFR allosteric inhibitors arises from at least two effects. Most apparently, the direct contact of the aminothiazole group with the mutant gatekeeper methionine residue can explain the selectivity for the T790M mutant. Second, the compound cannot bind the fully inactive conformation of the wild-type kinase; simple modelling reveals steric clashes of EAI001 with Leu858 and Leu861 in the N-terminal portion of the activation loop (Extended Data Fig. 3). The L858R mutation rearranges this portion of the activation loop23, thereby enlarging the allosteric pocket. EAI045 may also inhibit other mutants with a similar mechanism of activation, such as L861Q, but we do not expect it to inhibit most exon 19 deletion variants. These mutations shorten the loop leading into the C-helix and may therefore prevent opening of the allosteric pocket.
Initial studies of the cellular activity of EAI045 showed that it potently decreased, but did not completely eliminate, EGFR autophosphorylation in H1975 cells, an L858R/T790M-mutant NSCLC cell line (Fig. 2a). A similar effect was observed in NIH-3T3 cells stably transfected with the L858R/T790M mutant (Extended Data Fig. 4a). This inhibition was selective for mutant EGFR; EAI045 potently inhibited EGFR Y1173 phosphorylation in H1975 cells (half maximal effective concentration (EC50) = 2 nM), but not in HaCaT cells, a keratinocyte cell line with wild-type EGFR (Extended Data Table 4). We observed an intermediate level of activity in the L858R-mutant H3255 cells, a pattern consistent with our biochemical inhibition data (Extended Data Table 4). Despite potent inhibition of mutant EGFR, EAI045 showed no anti-proliferative effect in the H1975 and H3255 cell lines with concentrations as high as 10 μM (Extended Data Table 4). Profiling in a panel of EGFR-mutant Ba/F3 cells revealed that EAI045 inhibited proliferation of L858R/T790M and L858R mutant cells, but not the exon19del/T790M or parental Ba/F3 cells, indicative of on-target mutant-selective activity of the allosteric inhibitor (Extended Data Fig. 4b–e). However, half-maximal inhibition required ~10 μM EAI045, a concentration much higher than the biochemical IC50 of the compound.
In light of the incomplete inhibition of EGFR autophosphorylation and the allosteric mechanism of action of EAI045, we wondered to what extent ligand stimulation would affect inhibition of the mutant receptor. We compared inhibition of EGFR Y1173 phosphorylation in H1975 cells in the presence and absence of exogenous EGF (10 ng ml−1) using an ELISA-based assay. EAI045 inhibited EGFR phosphorylation with a similar EC50 irrespective of EGF stimulation, but notably, inhibition plateaued at 50% in the presence of ligand (Fig. 2b). This phenomenon suggests two populations of receptor, one that remains sensitive to the allosteric inhibitor upon ligand stimulation, and another, equal in number, that is rendered insensitive. Ligand-induced dimerization of the EGF receptor is known to induce an asymmetric interaction of the kinase domains8, and is an apparent potential source of two receptor populations with differential inhibitor sensitivity.
In the EGFR asymmetric dimer, the C-lobe of the ‘activator’ subunit impinges on the N-lobe of the ‘receiver’ subunit, inducing an active conformation in the receiver by reorienting the regulatory C-helix to its inward position (Fig. 2c). In wild-type EGFR, only the receiver subunit is activated. By contrast, both subunits in a mutant receptor are expected to be catalytically active, because oncogenic kinase domain mutations induce the active conformation even in the absence of ligand. As explained above, EAI045 binds a ‘C-helix out’ conformation of the kinase. In the receiver subunit but not the activator, outward displacement of the C-helix is impeded by the asymmetric dimer interaction. Therefore, we hypothesized that EAI045 was a potent inhibitor of the activator subunit of the mutant receptor, but a much less potent inhibitor of the receiver subunit, in which the C-helix is captive. Because the mutant receptor favours dimer formation24,25, this effect could explain both the incomplete inhibition of EGFR autophosphorylation and the apparent disconnect in the biochemical and cellular potencies of the allosteric inhibitor. To test this notion, we exploited an I941R point mutation in the C-lobe of the kinase, which is known to block the asymmetric dimer interaction8,26. The activity of the L858R/T790M mutant is dimerization-independent26 and, as expected, transduction of Ba/F3 cells with EGFR(L858R/T790M/I941R) led to factor-independent proliferation. In support of our hypothesis, Ba/F3 cells bearing this dimerization-defective mutant were markedly more sensitive to the allosteric inhibitor (Fig. 2d).
The therapeutic antibody cetuximab targets the extracellular portion of the EGF receptor, blocking ligand binding and preventing dimer formation9,10. The antibody is not effective clinically in EGFR-mutant NSCLC, and in cell-based studies cetuximab alone does not inhibit L858R/T790M or exon19del/T790M mutant EGFR, because their activity is independent of dimerization26. However, we reasoned that cetuximab should synergize with a kinase-targeted allosteric inhibitor, by converting the inhibitor-resistant receiver population into a monomeric form that is remarkably sensitive to EAI045. Notably, in the presence of cetuximab (10 μg ml−1), EAI045 inhibited proliferation of EGFR(L858R/T790M) Ba/F3 cells with an IC50 of approximately 10 nM, similar to its potency against this mutant in biochemical assays (Fig. 2e). In support of an on-target, mutant-selective effect of the allosteric agent, proliferation of Ba/F3 cells bearing EGFR(exon19del/T790M) was not inhibited by this combination (Fig. 2f).
We next tested the in vivo efficacy of EAI045 in genetically engineered mouse model of L858R/T790M-mutant-driven lung cancer27, both alone and in combination with cetuximab. Mouse pharmacokinetic studies with EAI045 revealed a maximal plasma concentration of 0.57 μM, a half-life of 2.15 h, and oral bioavailability of 26% after dosing at 20 mg kg−1. In a 4-week efficacy study, mice were treated with EAI045 at 60 mg kg−1 by oral gavage once daily, either alone or together with cetuximab (1 mg intraperitoneally every other day). We observed marked tumour regressions in the L858R/T790M-mutant mice treated with the combination, whereas those treated with EAI045 alone did not respond (Fig. 3a). Cetuximab alone had a very modest effect in these mice, as previously observed26. Mice bearing EGFR(exon19del/T790M) were treated using the same protocol, but as expected failed to respond to the combination therapy (Fig. 3b). Magnetic resonance imaging (MRI) studies of cohorts of L858R/T790M and exon19del/T790M mice after combination treatment for 1 or 2 weeks are shown in Extended Data Fig. 5.
Mutation of C797 is expected to confer resistance to all third-generation irreversible EGFR inhibitors that are active on the T790M-mutant EGFR, and a preliminary study reported the C797S alteration in 15 out of 67 patients (22%) with acquired resistance to AZD9291 (ref. 28). Mutations in C797 should not affect the efficacy of EAI045, as this residue is remote from the allosteric binding pocket. Consistent with this expectation, EAI045 in combination with cetuximab potently inhibited L858R/T790M/C797S Ba/F3 cells (Extended Data Fig. 5a) and treatment of genetically engineered L858R/T790M/C797S mice with EAI045 and cetuximab induced marked tumour shrinkage, similar to that observed in the L858R/T790M models (Fig. 3c, Extended Data Fig. 5b). Pharmacodynamic studies performed following two doses of treatment demonstrated that EAI045 in combination with cetuximab effectively inhibited phosphorylation of EGFR and downstream signalling proteins in these mice, but not in mice bearing the insensitive exon19del/T790M mutation (Fig. 3d, e).
The compounds we describe here are among the first allosteric TKIs, and to our knowledge, the first targeting any receptor tyrosine kinase in a mutant-selective manner. Further study is required, but our findings suggest that EAI045 or a related compound in combination with an EGFR dimer-disrupting antibody such as cetuximab would be an effective strategy for treating L858R/T790M-mutant-driven lung cancers, as well as those driven by the triple L858R/T790M/C797S mutation, which are resistant to all current EGFR-targeted therapies. EAI045 and cetuximab exhibit mechanistic synergy, a valuable property for combination agents because it lowers the dose required for efficacy. Ideally, chemotherapeutic agents used in combination should also have non-overlapping mechanisms of toxicity and sensitivity to resistance mutations. EAI045 meets these criteria as well; its lack of activity on wild-type EGFR and other kinases suggest that its dose-limiting toxicity is unlikely to be related to that of cetuximab and ATP-competitive EGFR inhibitors. In addition, given its distinct binding site, its sensitivity to resistance-conferring mutations is expected to be divergent from that of both cetuximab and ATP-site inhibitors. For these reasons, we speculate that an allosteric agent like EAI045 could be used in combination with ATP-site-directed inhibitors, with the goal of preventing the emergence of treatment-associated resistance mutations in the receptor itself.
EGFR protein expression and purification
Constructs spanning residues 696–1022 of the human EGFR (including wild type, L858R, L858R/T790M, T790M, and T790M/V948R mutant sequences) were prepared in a GST-fusion format using the pTriEX system (Novagen) for expression in Sf9 insect cells essentially as described14,23. EGFR kinase proteins were purified by glutathione-affinity chromatography followed by size-exclusion chromatography after cleavage with Tomato etch virus (TEV) or thrombin to remove the GST fusion partner following established procedures14,23.
Purified EGFR(L858R/T790M) enzyme was screened against Novartis compound collection of ~2.5 million using homogeneous time-resolved fluorescence (HTRF)-based biochemical assay format. The screening was performed at 1 μM ATP using a single compound concentration (12.5 μM). 1,322 top hits were picked for follow-up IC50 confirmation. IC50 values were determined at both 1 μM and 1 mM ATP to identify both ATP competitive and non-competitive compounds. Hits were also counter-screened against wild-type EGFR to evaluate the mutant selectivity.
HTRF-based EGFR biochemical assays
Biochemical assays for wild-type EGFR and each mutant were carried out using a HTRF assay as described previously29. Assays were optimized for each ATP concentration. Compound IC50 values were determined by 12-point inhibition curves (from 50 to 0.000282 μM) in duplicate.
Before crystallization, 0.1 mM of EGFR(T790M/V948R) was incubated for 1 h with 0.5 mM EAI001, 1 mM adenosine 5′-(β,γ-imido)triphosphate (AMP-PNP) and 10 mM MgCl2 at room temperature. Crystals of EGFR(T790M/V948R) in complex with EAI001 were prepared by hanging-drop vapour diffusion method over a reservoir solution containing 0.1 M Bis-Tris (pH 5.5), 25% PEG 3350, 5 mM tris (2-carboxyethyl)-phosphine (TCEP). Crystals were flash-frozen in liquid nitrogen after rapid immersion in a cryoprotectant solution containing 0.1M Bis-Tris 5.5, 25% PEG3350, 10% ethylene glycol and 5 mM TCEP. Diffraction data were recorded using a Mar343 image plate detector on a rotating anode source at 100 K. Data were processed and merged as described previously14. The structure was determined by molecular replacement with the program PHASER using an inactive EGFR kinase structure (PDB, 2GS7) as the search model. Repeated rounds of manual refitting and crystallographic refinement were performed using COOT and REFMAC. The inhibitor was modelled into the closely fitting positive Fo − Fc electron density and then included in following refinement cycles. Although the EAI001 preparation used in crystallization was racemic, the density clearly corresponded to the R stereoisomer and was modelled accordingly. Topology and parameter files for the inhibitors were generated using PRODRG. Statistics for diffraction data processing and structure refinement are shown in Extended Data Table 3.
Cells were maintained in 10% FBS/RPMI supplemented with 100 μg ml−1 penicillin/streptomycin (Hyclone SH30236.01). The cells were collected with 0.25% trypsin/EDTA (Hyclone SH30042.1), re-suspended in 5% FBS/RPMI penicillin/streptomycin and plated at 7,500 cells per well in 50 μl of media in a 384-well black plate with clear bottoms (Greiner 789068G). The cells were allowed to incubate overnight in a 37 °C, 5% CO2 humidified tissue culture incubator. The 12-point serial diluted test compounds were transferred to the plate containing cells by using a 50 nl Pin Head device (Perkin Elmer) and the cells were placed back in the incubator for 3 h. All cell lines were tested and found negative for mycoplasma contamination using the MycoAlert Mycoplasma Detection Kit (Lonza).
Phospho-EGFR (Y1173) target modulation assay
HaCaT cells were stimulated with 10 ng ml−1 EGF (Peprotech AF-100-15) for 5 min 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 ELx405 Select plate washer. Cells were lysed with 20 μL of 2× 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, 2× complete cocktail inhibitor (Roche 11 697 498 001), 2× phosphatase inhibitor cocktail set II and set III (Sigma P5726 and P0044)). The plates were shaken for 20 min. An aliquot of 25 μl from each well was transferred to prepared ELISA plates for analysis.
For the experiment studying the effect of EGF pre-treatment on EAI045 target modulation, H1975 cells were collected and plated in 0.5% FBS/RPMI penicillin/streptomycin. On the following day, cells were pre-treated with 0.5% FBS/RPMI media with or without 10 ng EGF per ml for 5 min. Compound was added and assay was carried out as described above. The experiment was performed twice with duplicate samples in each experiment.
Phospho-EGFR (Y1173) ELISA
Solid white 384-well high-binding ELISA plates (Greiner 781074) were coated with 5 μg ml−1 goat anti-EGFR capture antibody overnight in 50 mM carbonate/bicarbonate (pH 9.5) buffer. Plates were blocked with 1% BSA (Sigma A7030) in PBS for 1 h at room temperature, and washes were carried out with a Bio-Tek ELx405 Select using four cycles of 100 μl TBS-Tween (20 mM Tris, 137 mM NaCl, 0.05% Tween-20) per well. A 25 μl aliquot of lysed cell was added to each well of the ELISA plate and incubated overnight at 4 °C with gentle shaking. After washing, 1:1,000 anti-phospho-EGFR in 0.2% BSA/TBS-Tween was added and incubated for 2 h at room temperature. After washing, 1:2,000 anti-rabbit-HRP (horseradish peroxidase) in 0.2% BSA/TBS-Tween was added and incubated for 1 h at room temperature. Chemiluminescent detection was carried out with SuperSignal ELISA Pico substrate. Luminescence was read with an EnVision plate reader.
Cell lysates were equalized to protein content determined by Coomassie Plus protein assay reagent (ThermoScientific 1856210) and loaded onto 4–12% NuPAGE Bis-Tris gels with MOPS running buffer with LDS Sample buffer supplemented with DTT. Gel proteins were transferred to PVDF membranes with an iBlot Gel Transfer Device. 1× Casein-blocked membranes were probed with primary antibodies overnight at 4 °C on an end-over-end rotisserie. Membranes were washed with TBS-Tween and HRP-conjugated secondary antibodies were added for 1 h at room temperature. After washing, HRP was detected using Luminata Forte Western HRP Substrate reagent and recorded with a Bio-Rad VersaDoc imager.
H1975, H3255 and HaCaT proliferation assays
H1975, H3255 and HaCaT cell lines were plated in solid white 384-well plates (Greiner) at 500 cells per well in 10% FBS RPMI penicillin/streptomycin 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 (Promega) according to manufacturer’s instructions. Luminescent readout was normalized to 0.1% DMSO-treated cells and empty wells. Data was analysed by nonlinear regression curve fitting and EC50 values were reported.
Ba/F3 cell proliferation models
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 (Stratagene) 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 (BD Biosciences). 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). Ba/F3 cells have not been authenticated as there is no publicly available fingerprint for Ba/F3 cells. All variants used were confirmed to contain the correct EGFR mutation by sequencing. All Ba/F3 cells were tested for mycoplasma contamination and confirmed to be free of contamination.
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 h and the number of cells used per experiment determined empirically and has been previously established15. 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; http://www.graphpad.com). The curves were fitted using a nonlinear regression model with a sigmoidal dose response.
NIH-3T3 cell studies
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−1).
Mouse efficacy studies
EGFR(TL) (bearing L858R/T790M point mutations) and EGFR(TD) (bearing exon19del/T790M point mutations) mice were generated as previously described15,27. The EGFR(L858R/T790M/C797S) (denoted as TLCS hereafter) mutant mouse cohort was established briefly as follows: the full-length human TLCS cDNA was generated by site-directed mutagenesis using the Quickchange site directed mutagenesis kit (Agilent Technologies) and further verified by DNA sequencing. Sequence-verified targeting vectors were co-electroporated with an FLPe recombinase plasmid into v6.5 C57BL/6J (female) × 129/sv (male) embryonic stem cells (Open Biosystems) as described elsewhere31. Resulting hygromycin-resistant embryonic stem clones were evaluated for transgene integration via PCR. Then, transgene-positive embryonic stem clones were injected into C57BL/6 blastocysts, and the resulting chimaeras were mated with BALB/c wild type mice to determine germline transmission of the TLCS transgene. Further detail on the generation and characterization of the TLCS transgenic mice is provided in Supplementary Fig. 3. Progeny of TL, TD and TLCS mice were genotyped by PCR of tail DNA. The TL and TD mice were fed a doxycycline diet at 6 weeks of age to induce EGFR(TL) or EGFR(TD) expression, respectively. The TLCS mice were intranasally instilled with Ad-Cre (University of Iowa viral vector core) at 6 weeks of age to excise the loxP sites, activating EGFR(TLCS) expression.
The EAI045 compound was dissolved in 10% NMP (10% 1-methyl-2-pyrrolidinone: 90% PEG-300), and was dosed at 60 mg kg−1 daily by oral gavage. Cetuximab was administrated at 1 mg mouse−1 every other day by intraperitoneal injection. The TL, TD and TLCS mice were monitored by MRI to quantify lung tumour burden before being assigned to various study treatment cohorts, which were non-blinded and not formally randomized. All treated mice had an equal initial tumour burden. MRI evaluation was repeated every 2 weeks during treatment. The animals were imaged with a rapid acquisition with relaxation enhancement sequence (repetition time = 2000 ms; echo time = 25 ms) in the coronal and axial planes with a 1-mm slice thickness and with respiratory gating. The detailed procedure for MRI scanning has been previously described27. The tumour burden volumes were quantified using 3-dimensional Slicer software. Source data for tumour volume measurements are provided in Supplementary Fig. 2.
All care of experimental animals was in accordance with Harvard Medical School/Dana-Farber Cancer Institute institutional animal care and use committee (IACUC) guidelines. All mice were housed in a pathogen-free environment at a DFCI animal facility and handled in strict accordance with Good Animal Practice as defined by the Office of Laboratory Animal Welfare. None of the tumour efficacy experiments presented in this manuscript exceeded the 2 cm maximal diameter tumour size, as permitted by the Dana-Farber Cancer Institute IACUC.
Synthesis and characterization of EAI045
2-(5-fluoro-2-hydroxyphenyl)-2-(1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-(1,3-thiazol-2-yl)acetamide (EAI045) was prepared from 2-amino-2-(5-fluoro-2-methoxyphenyl)acetic acid using a reaction sequence similar to that previously described32 followed by demethylation with boron tribromide.
1H NMR (400 MHz, DMSO-d6) δ 12.61 (s, 1H), 9.96 (s, 1H), 7.73 (d, J = 7.5 Hz, 1H), 7.66–7.54 (m, 2H), 7.52 (dd, J = 1.0, 7.4 Hz, 1H), 7.49 (d, J = 3.6 Hz, 1H), 7.27 (d, J = 3.5 Hz, 1H), 7.11 (td, J = 3.2, 8.6 Hz, 1H), 6.90 (dd, J = 4.8, 8.9 Hz, 1H), 6.85 (dd, J = 3.1, 9.2 Hz, 1H), 6.31 (s, 1H), 4.61 (d, J = 17.5 Hz, 1H), 3.98 (d, J = 17.5 Hz, 1H); 19F NMR (376 MHz, DMSO-d6) δ −125.15 (s, 1F); LCMS: Rt 1.278 min; ESMS m/z 384.20 (M + H + ).
Protein Data Bank
The crystal structure of EGFR(T790M/V948R) in complex with EAI001 has been deposited in the Protein Data Bank under accession number 5D41.
This work was supported in part by NIH grants CA116020 (M.J.E.), CA154303 (M.J.E., K.-K.W. and P.A.J.), CA120964 (K.-K.W.) and CA135257 (P.A.J.), and by the Gross-Loh Family Fund for Lung Cancer Research (K.-K.W.). We thank N. Gray for helpful comments on the manuscript.
Supplementary Figure 1 shows the source gel data for Figures 2a, 3d, 3e, and Extended Data Figure 4a. Supplementary Figure 3 contains a description of generation and characterization of LSL-EGFR T790M/L858R/C797S conditional transgenic mice.
This file contains the source data for tumor volume measurements in Figure 3a-c.