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. 2020 Nov 5;11(12):2484–2490. doi: 10.1021/acsmedchemlett.0c00472

Complex Crystal Structures of EGFR with Third-Generation Kinase Inhibitors and Simultaneously Bound Allosteric Ligands

Janina Niggenaber 1, Leonie Heyden 1, Tobias Grabe 1, Matthias P Müller 1, Jonas Lategahn 1, Daniel Rauh 1,*
PMCID: PMC7734789  PMID: 33335671

Abstract

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Osimertinib is a third-generation tyrosine kinase inhibitor (TKI) and currently the gold-standard for the treatment of patients suffering from non-small cell lung cancer (NSCLC) harboring T790M-mutated epidermal growth factor receptor (EGFR). The outcome of the treatment, however, is limited by the emergence of the C797S resistance mutation. Allosteric inhibitors have a different mode of action and were developed to overcome this limitation. However, most of these innovative molecules are not effective as a single agent. Recently, mutated EGFR was successfully addressed with osimertinib combined with the allosteric inhibitor JBJ-04-125-02, but surprisingly, structural insights into their binding mode were lacking. Here, we present the first complex crystal structures of mutant EGFR in complex with third-generation inhibitors such as osimertinib and mavelertinib in the presence of simultaneously bound allosteric inhibitors. These structures highlight the possibility of further combinations targeting EGFR and lay the foundation for hybrid inhibitors as next-generation TKIs.

Keywords: Cancer, Non-small cell lung cancer, structural biology, kinase, epidermal growth factor receptor

Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all cases of lung cancer, which is the leading cause of cancer-related deaths worldwide.1 But activating epidermal growth factor receptor (EGFR) mutations such as L858R or exon19 deletions can be successfully treated with erlotinib,2 gefitinib,3,4 and afatinib5,6 (Figure 1A), which are approved treatments for patients with NSCLC. However, the acquired T790M resistance mutation within the ATP-binding site of EGFR rapidly emerges during treatment.7−9 Therefore, the pyrimidine-based inhibitor osimertinib10−12 (Figure 1A) was developed and revolutionized the field of NSCLC treatment. This covalent third-generation inhibitor showed clinical success in the treatment of T790M-positive patients13 as well as in first-line settings14 due to its wild type-sparing properties.12 Mavelertinib15 (Figure 1A) is another third-generation covalent inhibitor based on the purine scaffold for the treatment of EGFR-driven NSCLC. Despite the initial efficacy of the third-generation inhibitors, resistance mutations emerged, with C797S being most prevalent.16 The Cys797 is the site for covalent binding for all irreversible EGFR-inhibitors,11 and therefore, the covalent inhibitors become less effective upon emergence of the C797S mutation.17,18 To date, no therapeutic agent is available to overcome this drug-resistance in patients.19 A promising approach, however, is the treatment with allosteric inhibitors, such as EAI001,20 EAI045,20,21 and JBJ-04-125-02,22 or the dibenzodiazepinone-based inhibitors EAI002, DDC400223 and compound 3 (Figure 1B) since these inhibitors bind to the allosteric pocket of the inactive kinase with an outward displacement of the αC-helix. These inhibitors exhibit mutant selectivity over wild type EGFR, which arises from the direct contact of the aminothiazole or benzyl moiety to the gatekeeper methionine (Met790).20 Moreover, the allosteric inhibitors cannot bind the inactive state of wild type EGFR because of steric clashes with the side chains of Leu858 and Leu861. These two leucine residues lie in a short helical area of the activation loop. Therefore, the L858R mutation, which disrupts this helical element, is required within the activation loop (Figure S6).20,24 However, this treatment approach is limited due to an increased asymmetric EGFR dimer formation which disrupts the allosteric binding site.20,25 Therefore, most allosteric inhibitors must be combined with antibodies such as cetuximab, which hamper the dimer formation. However, the clinical application is limited due to missing mutant-specificity of cetuximab.26,27 In the past, a dual targeting approach has been reported to overcome resistance in the Bcr-Abl kinase. Therein, the ATP-competitive inhibitors imatinib or nilotinib were successfully combined with allosteric inhibitors, which bound to an alternative site of the kinase (PDB-ID: 3K5V). Remarkably, the dual targeting of Bcr-Abl overcame resistance to either agent alone.28 Faber et al. recently published another promising approach of dual targeting of kinases. They discovered an 8-anilino-1-naphthalene sulfonic acid (ANS) that binds an allosteric pocket in cyclin-dependent kinase 2 (Cdk2). Together with the orthosteric Cdk2 inhibitors dinaciclib and roscovitine the affinity of ANS toward Cdk2 increased drastically.29 Very recently, studies revealed that the optimized allosteric EGFR inhibitor JBJ-04-125-02, which shows single-agent activity in some models, can successfully bind together with osimertinib and provoke an increase in potency as compared to monotherapy. Therefore, the inhibitor combination constitutes a potential therapeutic approach for EGFR-driven NSCLC patients.22 However, structural insights into the simultaneous binding of osimertinib and allosteric inhibitors to EGFR have been lacking to date.

Figure 1.

Figure 1

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Overview of the chemical structures and structural alignment studies of active site-directed inhibitors together with allosteric inhibitors. (A) Chemical structures of the inhibitors osimertinib, afatinib, WZ4002, mavelertinib, and spebrutinib. (B) Chemical structures of the allosteric inhibitors EAI001, EAI045, JBJ-04-125-02, EAI002, DDC4002 and compound 3. (C) Structural alignment studies of the crystal structure of EAI045 bound to EGFR-T790M/V948R (PDB-ID: 6P1L) together with osimertinib (modified from PDB-ID: 4LI5), afatinib (PDB-ID: 4G5J), WZ4002 (PDB-ID: 3IKA), and mavelertinib (PDB-ID: 5UGC). Steric clashes are marked with red circles.

Against this background, we set out to solve the crystal structure of EGFR in complex with both an active site inhibitor and an allosteric inhibitor. Here, we present the first X-ray structure of osimertinib and EAI045 bound to EGFR-T790M. Moreover, we employed structural alignment studies which revealed further potential combinations. Ultimately, these studies resulted in two more complex crystal structures of mavelertinib and EAI001 as well as the Bruton’s tyrosine kinase (BTK) inhibitor spebrutinib30 (Figure 1A) and EAI001. These three unique X-ray structures can support structure-based developments to overcome EGFR mutant NSCLC.

Results

To predict the binding mode of allosteric inhibitors and osimertinib together in mutated EGFR, we performed structural alignment studies (Figure 1C). The comparison of published structures utilizing allosteric inhibitors showed a similar orientation and localization of the aminothiazole moiety or the benzyl ring of the dibenzodiazepinone-based inhibitors toward the ATP-binding pocket (Figure S1A). The alignment was thus carried out with EAI045 bound to EGFR-T790M/V948R (PDB-ID: 6P1L) and osimertinib (modified from PDB-ID: 4LI5). In line with the studies by To et al.,22 it was revealed that both compounds fit perfectly side by side into the ATP- and the allosteric pockets of the inactive mutant. In order to further verify these findings and to gain detailed structural insights into EGFR-T790M bound with this compound combination, we set out to solve the crystal structures. To create the essential inactive conformation of the kinase with an outward displacement of the αC-helix, which creates the pocket that is occupied by allosteric inhibitors,20 we utilized the V948R31 mutation. This mutation prohibits the dimerization of EGFR31 and, therefore, the allosteric sites are accessible, as mentioned in previous studies.22 The purified protein was incubated simultaneously with osimertinib and EAI045, while the covalent bond formation was monitored via protein mass spectrometry (Figure 2A). Next, we employed a crystallization experiment to gain complex crystal structures of EGFR-T790M/V948R. To our delight, these efforts resulted in protein crystals, which nicely diffracted at the Swiss Light Source (SLS), and the resulting data set could be refined to a resolution of 2.5 Å. This structure showed clear and unambiguous electron density for the inhibitors EAI045 and osimertinib within the previously described pockets of mutated EGFR (Figure 2B–D). In addition, the covalent bond of the Michael acceptor with Cys797 could be resolved in the electron density map (Figure 2C). Two typical hydrogen bonds between the aminopyrimidine scaffold and Met793 of the hinge region are formed. Moreover, osimertinib is involved in hydrophobic interactions with the hydrophobic clamp (Leu718, Val726, and Leu844) and Ala743 and Leu792 (Figure 2B and S2B). The aminothiazole ring of EAI045 is inserted between the gatekeeper methionine (Met790) and the catalytic lysine (Lys745). The isoindolin-1-one ring extends to a hydrophobic cleft at the end of the allosteric pocket, formed by Leu747, Ile759, Ala763, Met766, Leu777, Leu788, and Leu862. The side chains of Leu858 and Leu861 are not resolved in this structure. Furthermore, the carbonyl oxygen of the isoindolin-1-one moiety frames a hydrogen bond to the side chain of Lys745. The hydroxyl group of the para-fluorophenol and the amide-NH of EAI045 form hydrogen bonds to the main chain carbonyl of Phe856 and the Asp855 side chain within the DFG motif, respectively (Figure 2B and S2A). Altogether, the crystal structure confirmed the modeling by To et al. with the highly similar JBJ-04-125-02.22

Figure 2.

Figure 2

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Mass spectra, complex crystal structures, and electron density maps of inhibitors bound to EGFR-T790M/V948R. (A) Mass spectra of apo EGFR-T790M/V948R (gray) and EGFR-T790M/V948R incubated with osimertinib and EAI045 (blue). (B) Complex crystal structure of osimertinib (gray) bound together with EAI045 (blue) to EGFR-T790M/V948R (PDB-ID: 6Z4B) with a resolution of 2.5 Å. (C) 2Fo-Fc maps of osimertinib (gray) and EAI045 (blue) contoured at a r.m.s.d. of 1. (D) Fo-Fc stimulated annealing omit maps of osimertinib and EAI045 (green) contoured at a r.m.s.d. of 2.8. (E) Mass spectra of apo EGFR-T790M/V948R (gray) and EGFR-T790M/V948R incubated with mavelertinib and EAI001 (blue). (F) Complex crystal structure of mavelertinib (gray) bound together with EAI001 (blue) to EGFR-T790M/V948R (PDB-ID: 6Z4D) with a resolution of 2.0 Å. (G) 2Fo-Fc maps of mavelertinib (gray) and EAI001 (blue) contoured at a r.m.s.d. of 1. (H) Fo-Fc stimulated annealing omit maps of mavelertinib and EAI001 (green) contoured at a r.m.s.d. of 2.8. (I) Mass spectra of apo EGFR-T790M/V948R (gray) and EGFR-T790M/V948R incubated with spebrutinib and EAI001 (blue). (J) Complex crystal structure of spebrutinib (gray) bound together with EAI001 (blue) to EGFR-T790M/V948R (PDB-ID: 7A2A) with a resolution of 1.9 Å. (K) 2Fo-Fc maps of spebrutinib (gray) and EAI001 (blue) contoured at a r.m.s.d. of 1. (L) Fo-Fc stimulated annealing omit maps of spebrutinib and EAI001 (green) contoured at a r.m.s.d. of 2.8.

Recently, a panel of new complex structures of osimertinib bound to diverse EGFR mutants was published in the PDB (PDB-IDs: 6JX0, 6JX4, 6JWL, and 6JXT). The binding mode of osimertinib resembled the one reported herein with the exception of cocrystallized EGFR-T790M (PDB-ID: 6JX0) which revealed a different orientation of the indole ring of osimertinib based on unique interactions between the protein and the indole ring (Figure S1B–C).32 However, this different orientation would not be compatible with a simultaneous binding of allosteric inhibitors. In summary, we presented the first X-ray structure of EGFR-T790M/V948R with simultaneously cocrystallized osimertinib and EAI045.

This result prompted us to identify additional possible combinations of allosteric inhibitors with ATP site-directed inhibitors. Therefore, we extended our studies by alignment of EAI045 (PDB-ID: 6P1L) with other literature known inhibitors such as afatinib (PDB-ID: 4G5J), WZ4002 (PDB-ID: 3IKA), and mavelertinib (PDB-ID: 5UGC). In line with previously reported modeling studies,22 the alignment of the anilinoquinazoline-based afatinib with the cocrystal structure of EAI045 revealed a steric clash (Figure 1C, red circle) of the aminothiazole ring of the allosteric inhibitor and the halogenated aniline ring of afatinib. The pyrimidine-based inhibitor WZ4002 does not exhibit this moiety, however, the structural alignment illustrated a steric clash of the chlorine and the aminothiazole moiety of EAI045 (Figure 1C, red circle). Of note, mavelertinib was not included in previous studies but was found to spare the region around the gatekeeper similar to osimertinib and can, therefore, potentially be combined with allosteric inhibitors (Figure 1C).

Based on this finding, we set up further crystallization experiments, as described above. Fortunately, we achieved the growth of crystals in the presence of mavelertinib and EAI001, which exhibited clear diffraction patterns in subsequent X-ray analysis at the synchrotron. The resulting protein structure with a resolution of 2.0 Å offers insights into the binding modes with the respective binding sites in EGFR-T790M/V948R by well-defined electron densities. The covalent bond formation of mavelertinib and Cys797 was again tracked using MS (Figure 2E) and also revealed by clear electron density (Figure 2F–H). Additionally, mavelertinib exhibits two hydrogen bonds to Met793 of the hinge region and is involved in hydrophobic interactions, including the amino acids Leu718, Val726, Ala743, Leu792, and Leu844 (Figure 2F and S3B). Due to the resolution of the structure, a water-mediated network around the warhead of mavelertinib, including Asp800 and Arg841, was identified (not shown). The binding mode of EAI001 resembles that of EAI045 except the lack of a hydrogen bond due to the absence of the phenol ring in contrast to EAI045, as described above. Briefly, polar contacts are formed with the catalytic Lys745 and Asp855, as well as nonpolar interactions with Leu747, Ile759, Ala763, Met766, Leu777, Leu788, Met790, and Leu858. Of note, Leu861 and Leu862 are not resolved in this structure, while Leu858 could be observed with clear electron density, in contrast to the complex structure with EAI045 (Figure 2F and S3A). As mentioned before, the L858R mutation, which disrupts the short helical element of the activation loop of the inactive kinase conformation, is required for the binding of allosteric inhibitors. However, this mutation is not present in the crystallization construct used. Therefore, we observed EAI001 to induce a repositioning of Leu858, resulting in a structural change of the helical element of the activation loop (Figure S6). This structural change might result in a binding energy penalty, which leads to selectivity for EGFR-L858R. Interestingly, the unit cell contains two copies of EGFR. However, the allosteric inhibitor is only bound in one of the two chains. The chain without EAI001 reveals that Phe723 within the glycine-rich loop is flipped toward mavelertinib and with a distance of 4.2 Å contributing to the hydrophobic framework described above. In addition, the gatekeeper methionine exhibits a different conformation because of a missing contact with the allosteric inhibitor (Figure S3C). However, the unoccupied allosteric site gives rise to a water-mediated network of mavelertinib with Arg841 and Thr854 next to the DFG motif (not shown).

Inspection of the structural alignment of WZ4002 and EAI045 in EGFR-T790M/V948R (Figure 1C) revealed a steric clash due to the chlorine moiety of WZ4002 as mentioned above. We came across spebrutinib, which is a structurally similar ATP site-directed inhibitor of BTK with a fluorine moiety instead of the chlorine of WZ4002 (Figure 1A). Therefore, further crystallization experiments with allosteric inhibitors and spebrutinib were performed. Also, in this setup, we obtained crystals in the presence of EAI001 and spebrutinib, which diffracted well at the synchrotron and the obtained structure could be refined to a resolution of 1.9 Å. The covalent bond formation of the Michael acceptor with the side chain of Cys797 was confirmed by mass spectrometry (Figure 2I) and well-defined electron density (Figure 2J-L). Spebrutinib also forms two hydrogen bonds to Met793, and the hydrophobic interactions include the amino acids Leu718, Val726, Ala743, Leu792, Leu844, and Phe723 (Figure 2J and S4B). Interestingly, Phe723 is flipped toward spebrutinib, although the allosteric inhibitor is bound. This finding differs from the complex crystal structures with osimertinib or mavelertinib and the allosteric inhibitors but can be explained by the lower steric demand of spebrutinib in the ATP-binding pocket (Figure S7). In this structure polar contacts are formed between EAI001 and the catalytic Lys745 and, interestingly, via a water-mediated hydrogen bond to Thr854 and Asp855 (not shown). Moreover, a second water-mediated hydrogen bond is formed between the aminothiazole ring of EAI001 and the secondary amine of spebrutinib (not shown). Hydrophobic interactions are formed with Leu747, Ile759, Ala763, Met766, Leu777, Leu788, Met790, and Leu858 (Figure S4A).

These structures of EGFR complexed with mavelertinib or spebrutinib and simultaneously bound EAI001 show that allosteric inhibitors can also bind with other active site-directed small molecule inhibitors not based on the scaffold of osimertinib.

Discussion and Conclusions

Together, we have presented the successful crystallization and structure determination of three different active site inhibitors binding simultaneously with allosteric inhibitors in EGFR-T790M/V948R. These three unique structures provide detailed insights into the binding modes of both types of inhibitors and provide the basis for structure-based design approaches toward novel inhibitors, which can probably be used in combination in order to prevent and overcome the resistance mutation C797S. Dual administration of two independent inhibitors might represent a promising approach with respect to individual pharmacokinetic properties. Moreover, the resistance spectrum toward the combination treatment can be different in contrast to mono administration. We, therefore, believe that the search for further inhibitor combinations constitutes a potential therapeutic approach for EGFR-driven NSCLC patients. The three novel complex structures, presented herein, will guide this process in the future and lay the foundation for hybrid inhibitors as next-generation tyrosine kinase inhibitors.

Acknowledgments

This work was cofunded by the German Federal Ministry for Education and Research (NGFNPlus and e:Med) (Grant No. BMBF 01GS08104, 01ZX1303C), the Deutsche Forschungsgemeinschaft (DFG), the German federal state North Rhine-Westphalia (NRW) and the European Union (Europe-an Regional Development Fund: Investing In Your Future) (EFRE-800400), NEGECA (PerMed NRW), EMODI and Drug Discovery Hub Dortmund (DDHD). We thank the staff at the PXII X10SA beamline for the possibility to collect diffraction data.

Glossary

Abbreviations

ANS

8-anilino-1-naphthalene sulfonic acid

BTK

Bruton’s tyrosine kinase

Cdk2

cyclin-dependent kinase 2

EGFR

epidermal growth factor receptor

MS

mass spectrometry

NSCLC

non-small cell lung cancer

SLS

Swiss Light Source

TKI

tyrosine kinase inhibitor.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00472.

  • Experimental procedures; supplementary figures and tables (PDF)

Author Present Address

† (J.L.) PearlRiver Bio GmbH, Otto-Hahn-Strasse 15, 44227 Dortmund, Germany.

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

The authors declare the following competing financial interest(s): J.L. is shareholder and full-time employee of PearlRiver Bio GmbH. D.R. is shareholder and consultant of PearlRiver Bio GmbH.

Supplementary Material

ml0c00472_si_001.pdf (1.8MB, pdf)

References

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