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The clinical efficacy of epidermal growth factor receptor (EGFR) kinase inhibitors in EGFR mutant non-small cell lung cancer (NSCLC) is limited by the development of drug resistance mutations, including the gatekeeper T790M mutation1-3. Strategies aimed at targeting EGFR T790M with irreversible inhibitors have had limited success and are associated with toxicity due to concurrent inhibition of wild type EGFR4,5. All current EGFR inhibitors possess a structurally related quinazoline based core scaffold and were identified as ATP-competitive inhibitors of wild type EGFR. Here we identify a covalent pyrimidine EGFR inhibitor by screening an irreversible kinase inhibitor library specifically against EGFR T790M. These agents are 30-100 fold more potent against EGFR T790M, and up to 100 fold less potent against wild type EGFR, than quinazoline based EGFR inhibitors in vitro and are effective in murine models of lung cancer driven by EGFR T790M. Co-crystallization studies reveal a structural basis for the increased potency and mutant selectivity of these agents. These mutant selective irreversible EGFR kinase inhibitors may be clinically more effective and better tolerated than quinazoline based inhibitors. Our findings demonstrate that functional pharmacological screens against clinically important mutant kinases represent a powerful strategy to identify new classes of mutant selective kinase inhibitors.
EGFR kinase inhibitors, gefitinib and erlotinib, are effective clinical therapies for NSCLCs that harbor activating mutations in the EGFR kinase domain1,6. The most common EGFR mutations, L858R and delE746_A750, impart both an increased affinity for gefitinib or erlotinib and a decreased affinity for ATP relative to wild type (WT) EGFR7,8. The clinical efficacy of gefitinib/erlotinib is however ultimately limited by the development of acquired drug resistance such as by mutation of the gatekeeper T790 residue (T790M) which is detected in 50% of clinically resistant patients2,3. Unlike the analogous T315I mutation in ABL, which introduces a steric impediment for imatinib binding, EGFR T790M only modestly effects gefitinib binding but more importantly restores the affinity for ATP, similar to that of WT EGFR9.
Most EGFR inhibitors are based on a 4-anilinoquinazoline core scaffold and were initially identified as ATP-competitive inhibitors of WT EGFR. This includes irreversible inhibitors that, unlike gefitinib, contain an electrophilic functionality which undergoes a Michael addition reaction with a conserved cysteine residue present in EGFR (Cys797). The covalent nature of these compounds allows them to achieve greater occupancy of the ATP-site relative to reversible inhibitors providing the ability to inhibit EGFR T790M in pre-clinical models, in spite of the increased ATP affinity conferred by this secondary mutation4,10,11. However, all current irreversible inhibitors are less potent in cell line models harboring EGFR T790M compared to those with an EGFR activating mutation alone (Figure S1) and at clinically achievable concentrations, these agents do not inhibit EGFR T790M in vitro10-13. Since the ATP affinity of EGFR T790M is similar to WT EGFR, the concentration of quinazoline-based EGFR inhibitors required to inhibit EGFR T790M, will also effectively inhibit WT EGFR. In patients, this concurrent inhibition of WT EGFR, results in skin rash and diarrhea, and limits the ability to achieve plasma concentrations sufficient to inhibit EGFR T790M. As a consequence the clinical efficacy of the irreversible EGFR inhibitors CI-1033, HKI-272 and PF00299804 has been limited, especially in gefitinib/erlotinib resistant NSCLC patients, and the dose limiting toxicity has been diarrhea and skin rash5,14,15.
We hypothesized that the anilinoquinazoline scaffold may not be the most potent or specific for inhibiting EGFR T790M because it relies on the small size and hydrogen bonding interactions with the gatekeeper threonine of WT EGFR. We prepared a focused library of common kinase inhibitor core scaffolds where one of the side chains was modified with an acrylamide group at a position that molecular modeling predicted to react with Cys 797. This library was screened for compounds that could inhibit the growth of both gefitinib resistant (PC9GR; delE746_A750/T790M) and sensitive (PC9; delE746_A750) cell lines but were not toxic up to 10 μM against A549 (KRAS mutant) or H3122 (EML4-ALK) cells. We compared our findings to both reversible (gefitinib) and irreversible EGFR inhibitors (CL-387,785 and HKI-272). Three closely related pyrimidines, WZ3146, WZ4002, and WZ8040 were identified from the screen that possessed up to a 300-fold lower IC50 against the PC9GR cells compared to clinical stage inhibitors such as HKI-272 (Figures1A, 1B and Table S1). We observed a similar increased potency of the WZ compounds in the H1975 (L858R/T790M) cell line and in Ba/F3 cells harboring EGFR T790M (Figure 1B and Tables S1 and S2). The increased cellular potency correlated with inhibition of EGFR, AKT and ERK1/2 phosphorylation in NSCLC cell lines (Figures 1C and S2) and with the more potent inhibition of EGFR phosphorylation by WZ4002 in NIH-3T3 cells expressing different EGFR T790M mutant alleles (Figures 1D and S3). The profile against ERBB2 was markedly different; the WZ compounds were less potent than CL-387,785 or HKI-272 (Tables S1 and S2) and did not inhibit ERBB2 phosphorylation in 3T3 cells expressing the ERBB2 gatekeeper (T798I) mutation (data not shown). Analysis of recombinant EGFR T790M kinase incubated with WZ3146 by electrospray mass spectrometry revealed stoichiometric addition of one inhibitor molecule to the protein. Analysis of a pepsin digest of the modified protein by tandem MS identified Cys 797 as the site of modification thus verifying covalent bond formation between WZ3146 and EGFR (Figure S4).
We profiled WZ3146 and WZ4002 against a panel of 400 kinases using the Ambit kinome screening platform (Table S3 and Figure S5). For WZ4002, kinases that exhibited greater than 95% inhibition relative to the DMSO control (Ambit score < 5) at 10 μM were selected for measurement of their dissociation constants (Table S3). In addition to EGFR, we observed potent inhibition of several of the ten kinases that possess a cysteine at the same position as EGFR including a subset of the TEC-family kinases (Figure S6). Cross-reactivity with BMX has been reported for irreversible quinazoline-derived EGFR inhibitors16. To confirm whether the observed binding activity translated into cellular inhibition, WZ4002 and WZ3146 were profiled against Ba/F3 cells transformed with TEL fusions of BMX, BLK, JAK2 and JAK3. WZ4002, which possesses an ortho-methoxy group at the C2-aniline substituent, is more selective for EGFR compared to WZ3146 (Table S4).
We next determined whether the increased potency of the WZ compounds against mutant EGFR also applied to WT EGFR. We used EGFR WT HN11 cells17 and Ba/F3 cells harboring the EGFR vIII mutation which contains a WT kinase domain (Figure 2A and Table S2). These compounds were 3-100-fold less potent, with WZ4002 being least potent, compared to CL-387,785 and HKI-272 at inhibiting the growth of the EGFR WT cells. Furthermore, WZ4002 was 100-fold less effective at inhibiting phosphorylation of WT EGFR compared to the quinazoline inhibitors (Figure 2B). Similarly, WZ4002 inhibited EGFR kinase activity of recombinant L858R/T790M protein more potently than of WT EGFR, while the opposite was observed with HKI-272 and gefitinib (Figures S7A and S7B).
In order to better understand the potency and relative selectivity for EGFR T790M over WT EGFR, we determined the crystal structure of WZ4002 in complex with EGFR T790M (Figures 3A, 3B and S8 and Table S5). The compound binds within the ATP-binding cleft of the enzyme, forming the expected covalent bond with Cys797. As expected based upon co-structures of related pyrimidine derived inhibitors with CDK218, JNK119, and FAK20, the anilinopyrimidine core of WZ4002 forms a bidentate hydrogen bonding interaction with the ‘hinge’ residue Met 793 (Figure 3B). The chlorine substituent on the pyrimidine ring contacts the mutant gatekeeper residue, Met790. The hydrophobicity conferred by this mutation likely contributes to the potency of these compounds against the T790M mutant. The aniline ring forms a hydrophobic interaction with the α-carbon of Gly796 and its methoxy substituent extends toward Leu792 and Pro794 in the hinge region. The greater selectivity of the WZ4002 compound likely derives from the fact that both JAK3 and TEC-family kinases have a bulkier residue (tyrosine in JAK3, phenylalanine in TEC-family) in the position of Leu792, which would be expected to sterically interfere with the methoxy group in WZ4002. The reactive acrylamide moeity and the linking phenyl ring comprise the other arm of the inhibitor. The “linker” phenyl ring lies roughly perpendicular to the pyrimidine core; this orientation juxtaposes the acrylamide with the thiol of Cys797 for covalent bond formation (Figure 3B).
We further determined whether WZ4002 is effective in vivo using mouse lung cancer models harboring either EGFR L858R/T790M or Del E746_A750/T790M. We chose WZ4002 for the in vivo studies because in vitro it was least potent against WT EGFR and against other cysteine containing kinases (Tables S2 and S4) but was effective against EGFR T790M. A pharmacokinetic study was performed to determine the achievable plasma concentration (429ng/ml), half life (2.5 hours), and the oral bioavailability (24%) of WZ4002 (Tables S6, S7 and S8). In a pharmacodynamic study WZ4002 effectively inhibited EGFR, AKT and ERK1/2 phosphorylation (Figure 4A) which was associated with a significant increase in TUNEL positive and a significant decrease in Ki67 positive cells compared to vehicle alone treated mice (Figures 4B and 4C). To evaluate whether WZ4002 imparted a differential effect on WT EGFR in vivo, we evaluated EGFR phosphorylation in the hair bulb from mouse skin following treatment with either erlotinib or WZ4002 (Figures 2C and 2D). Only erlotinib significantly inhibited EGFR phosphorylation in the hair bulb. In a 2 week efficacy study, WZ4002 treatment resulted in significant tumor regressions compared to vehicle alone in both T790M containing murine models (Figures 4D, 4E and S9). Histological evaluation of the lungs following treatment confirmed significant resolution of the tumor nodules with only few small residual nodules and nodule remnants that had evidence of treatment effect with decreased cellularity and increased fibrosis consistent with remodeling/scarring (Figure 4F). There were no signs of overt toxicity compared with vehicle treated mice during the study as assessed by changes in weight (data not shown), serum creatine and total white blood cell count (Figure S10).
Our studies identify a novel structural class of EGFR kinase inhibitors which are effective in vitro and in vivo models harboring the EGFR T790M mutation. Given the dramatic activity in models with established EGFR T790M, we determined whether WZ4002 treatment could also prevent the development of EGFR T790M using in vitro models harboring EGFR activating mutations. Unlike with gefitinib or HKI-272, which when used at their achievable plasma concentrations lead to development of EGFR T790M in vitro13,21,22, we were unable to isolate any EGFR T790M containing clones from WZ4002 treated Ba/F3 or PC9 NSCLC cells (Table S9). These findings suggest that WZ4002 could also be used as initial therapy for EGFR mutant NSCLC patients and may ultimately lead to a longer time to disease progression than currently achieved with gefitinib1.
Our crystallographic studies provide insight as to why WZ4002 is so much more effective against L858R/T790M than HKI-272. Although both share the irreversible component, the anilinopyrimidine scaffold of WZ4002 is an intrinsically better fit for the mutant gatekeeper methionine (Figure S11). To further test this hypothesis we prepared WZ4003, a reversible analog of WZ4002 which is non-reactive toward Cys797. WZ4003 binds to the L858R/T790M mutant 100-fold more tightly than it does to the WT EGFR (Table S10), confirming that the scaffold per se is indeed specific for the mutant kinase. Importantly, the WZ compounds rely on covalent bond formation for potent cellular inhibition, as evidenced by the 100-fold increase in IC50 of WZ4002 against the EGFR C797S mutants and by the significantly reduced cellular IC50 of WZ4003 against T790M containing Ba/F3 cells (Table S2). These observations highlight the importance of using a library of irreversible kinase inhibitors, as WZ4003 would not have been identified in the initial cellular screen.
Mutations, including at the gatekeeper residue, are a common mechanism of drug resistance to kinase inhibitors. The current approach, using a cellular screen expressing the mutant kinase of interest, can be applied to identify novel agents specifically against drug resistance or oncogenic mutations implicated in human cancers. Such agents may truly be cancer selective and clinically more potent and less toxic than those that also concurrently inhibit the wild type kinase. The agents described here are unique in that they inhibit both the drug sensitizing and resistance mutations but are selective against WT EGFR23,24,25. Further studies are needed to determine whether this class of EGFR inhibitors will be clinically effective in patients with EGFR mutant cancers harboring EGFR T790M mediated acquired drug resistance.
Gefitinib, CL-387,785 and HKI-272 were obtained from commercial sources. The WZ compounds were synthesized using a four step chemical synthesis and described in detail in the Supplementary Methods. The final products were verified by 1H nuclear magnetic resonance (NMR) and LCMS.
EGFR wild type and mutant NSCLC, Ba/F3 cells and NIH-3T3 cells were cultured as previously described10. The PC9GR4 cells were generated as previously described and verified to contain EGFR delE746_A750/T790M by direct sequencing21. Cell proliferation and growth assays were performed using the MTS assay as previously described26. Site directed mutagenesis was performed using the Quick Change Site-Directed Mutagenesis kit (Stratagene; La Jolla, CA) according to the manufacturer's instructions.
In vitro inhibitory enzyme kinetic assays using recombinant EGFR L858R/T790M and WT protein and were performed using the ATP/NADH coupled assay system in a 96-well format as previously described7.
The structure of WZ4002 in complex with EGFR T790M was determined as previously described9.
All studies involving mice were approved by the Dana Farber Cancer Institute Animal Care and Use Committee. EGFR-TL (T790M/L858R) mice were generated as previously described4. EGFR exon19 Deletion-T790M (TD) inducible bitransgenic mice were similarly generated and characterized. Mice were treated either with vehicle (NMP (10% 1-methyl-2-pyrrolidinone: 90% PEG-300) alone or WZ4002 at 25mg/kg gavage daily and followed by MRI scanning as previously described4,27. Histology, immunohistochemistry and immunoblotting analyses were performed according to standard protocols, as previously described4,10.
N-ethyl-N-nitrosourea (ENU) mutagenesis was carried out using EGFR L858R and DelE746_A750 Ba/F3 cells as previously described28. Treated cells were expanded in 100 nM WZ4002, 1 μM WZ4002, 200 nM HKI-272 or 1 μM gefitnib and resistant clones isolated and further characterized.
This study is supported by grants from the National Institutes of Health RO1CA11446 (P.A.J.), R01CA135257 (P.A.J.), R01CA080942 (M.J.E.), R01CA130876-02 (N.S.G.), R01CA116020 (M.J.E.), R01AG2400401 (K.-K.W.), R01 CA122794 (K.-K.W.), R01GM070590 (J.R.E.), National Cancer Institute Lung SPORE P50CA090578 (P.A.J. and K.-K.W.), the Cecily and Robert Harris Foundation (K.-K.W.), Uniting Against Lung Cancer (K.-K.W.), the Flight Attendant Medical Research Institute (K.-K.W.)., the Hazel and Samuel Bellin research fund (P.A.J.) and the Damon Runyon Foundation Cancer Innovation Award (N.S.G.). This work is contribution 948 from the Barnett Institute. Dr. Jänne receives royalties as a co-inventor on a patent awarded for the discovery of EGFR mutations, licensed to Genzyme Genetics, which was not involved in this study. M.J.E. is a consultant for and receives research support from Novartis Institutes for Biomedical Research. N.S.G., M.J.E. and P.A.J. are co-inventors on a provisional patent application covering the inhibitors described in the manuscript which has been licensed to Gatekeeper Pharmaceuticals in which N.S.G., K.-K.W. and P.A.J. are co-founders. The authors would like to thank Sai Advantium Pharma Limited for performing the pharmacokinetic studies.
Author Contributions W.Z., D.E., L.C., C.Y., D.L., M.C. A.B.C. designed experiments, conducted studies and analyzed data. R.E.I. and J.R.E. performed and analyzed the mass spectrometry studies. L.C. and R.P. performed the histologic and immunohistochemistry analyses. M.J.E., K.-K.W., N.S.G. and P.A.J. designed the experiments, analyzed data and wrote the manuscript.
Author Information Structural data have been deposited in the Protein Data Bank under the accession code 3IKA.
Full Methods and any associated references are located in the Supplementary Methods.