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. 2023 Oct 19;14(12):2731–2737. doi: 10.1039/d3md00439b

A covalent fragment-based strategy targeting a novel cysteine to inhibit activity of mutant EGFR kinase†

Naoki Kuki a, David L Walmsley c, Kazuo Kanai a, Sho Takechi a, Masao Yoshida a, Ryo Murakami a, Kohei Takano a, Yuichi Tominaga a, Mizuki Takahashi b, Shuichiro Ito b, Naoki Nakao b, Hayley Angove c, Lisa M Baker c, Edward Carter c, Pawel Dokurno c, Loic Le Strat c, Alba T Macias c,‡, Carrie-Anne Molyneaux c, James B Murray c, Allan E Surgenor c, Tomoaki Hamada a, Roderick E Hubbard c
PMCID: PMC10718517  PMID: 38107172

Abstract

Several generations of ATP-competitive anti-cancer drugs that inhibit the activity of the intracellular kinase domain of the epidermal growth factor receptor (EGFR) have been developed over the past twenty years. The first-generation of drugs such as gefitinib bind reversibly and were followed by a second-generation such as dacomitinib that harbor an acrylamide moiety that forms a covalent bond with C797 in the ATP binding pocket. Resistance emerges through mutation of the T790 gatekeeper residue to methionine, which introduces steric hindrance to drug binding and increases the Km for ATP. A third generation of drugs, such as osimertinib were developed which were effective against T790M EGFR in which an acrylamide moiety forms a covalent bond with C797, although resistance has emerged by mutation to S797. A fragment-based screen to identify new starting points for an EGFR inhibitor serendipitously identified a fragment that reacted with C775, a previously unexploited residue in the ATP binding pocket for a covalent inhibitor to target. A number of acrylamide containing fragments were identified that selectively reacted with C775. One of these acrylamides was optimized to a highly selective inhibitor with sub-1 μM activity, that is active against T790M, C797S mutant EGFR independent of ATP concentration, providing a potential new strategy for pan-EGFR mutant inhibition.

Covalent fragment strategy for a previously unexploited cysteine of mutant EGFR.graphic file with name d3md00439b-ga.jpg

Introduction

The tyrosine kinase activity of epidermal growth factor receptor (EGFR) modulates many intracellular signaling pathways associated with control of cell proliferation.1 This activity is upregulated in many cancers through a number of different mechanisms.2 For example, 10–50% of non-small cell lung cancers (NSCLC) are driven by constitutive activation through the L858R point mutation and exon19 deletion mutation (del19). Many ATP competitive inhibitors of EGFR tyrosine kinase activity have been approved for clinical use (key compounds in Fig. 1; for recent reviews, see ref. 3 and 4). The first generation of reversible inhibitors such as gefitinib and erlotinib were followed by a second generation of mainly irreversible inhibitors such as afatinib and dacomitinib, still used in various regimes as first or second line therapies, which form a covalent bond with C797 at the edge of the ATP binding pocket. Resistance arises through a variety of mechanisms, such as the T790M mutation of the gatekeeper residue which increases the affinity for ATP, impacting the efficacy of the inhibitors.5 This led to the development of the third-generation inhibitor osimertinib,6,7 (Fig. 2) although again, resistance can arise by mutation (C797S).8 There is therefore an unmet medical need for a therapy that is effective against the various mutant forms of EGFR that have arisen and we initiated a program to identify an inhibitor of the triple mutant of EGFRL858R(del19)/T790M/C797S, hereafter EGFR_TM. The design of such an inhibitor is challenging, as C797 is not available for covalent bond formation and the ATP affinity will remain high due to the T790M mutation.

Fig. 1. Representative EGFR inhibitors.

Fig. 1

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Fig. 2. The structure of osimertinib (stick, pink carbons) bound to EGFR_wtX (PDB code 6JXT) (schematic in white) with the side chain of residues that are mutated in various forms at positions 775, 790, 797, 858, 865, 866 and 867 in Corey–Pauling–Koltun model (CPK). Note that in this crystal structure C775 has two alternate conformers modeled (only conformer A shown) and there are several missing loops, including A865.

Fig. 2

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There have recently been several reports of novel EGFR inhibitors which are potent reversible and selective inhibitors of EGFR_TM, such as CH7233163 and the macrocycle, BI-4020,9,10 and clinical trials are underway for BLU-945 (Clinicaltrials.gov identifier: NCT04862780) and the as yet undisclosed compound BBT-176 (Clinicaltrials.gov identifier: NCT04820023). We set out to use fragment-based discovery methods to identify new reversible inhibitors of EGFR_TM, but serendipitously discovered a compound that reacted with another cysteine in the active site, C775. Herein, we describe the structure-based discovery of the first C775-targeted inhibitors of EGFR to provide an alternate strategy to overcome the high ATP affinity and resistance to the third generation EGFR inhibitors.

The studies reported here used protein material generated from several constructs of the EGFR kinase domain (details in ESI†): wild type (hereafter termed EGFR_wt); single mutant T790M (EGFR_SM); single mutant L858R (EGFR_SM2); double mutant T790M, L858R (EGFR_DM), double mutant L858R, C775S (EGFR_DM2) and triple mutant of T790M, L858R, C797S (EGFR_TM). For crystallographic work, EGFR_wtX, EGFR_DMX and EGFR_TMX contain three additional mutations (E865A, E866A, K867A) in the activation loop to aid crystal formation for structure determination.11,12

Results and discussion

Our initial studies (see ESI†) used the kinomescan assay at DiscoveRx to identify compounds from the 1350-member Vernalis fragment library that bound to each of EGFR_DM and EGFR_wt.13–15 Although more than 50 of the fragments that were hits against these enzymes were confirmed as inhibiting EGFR_TM in a Lance TR-FRET enzyme assay (hereafter the functional assay) with ligand efficiency (defined as ΔG/heavy atom (HA) count) of greater than 0.39,16 limited early efforts on fragment to hit expansion did not build optimism that a selective inhibitor could be optimized from the reversible binding fragments. However, the crystal structure of thione 1 bound to EGFR_DMX revealed a covalent bond formed with the cysteine residue C775 located at the back of the ATP binding pocket (Fig. 3).17 There are no other covalent inhibitors binding to C775 in EGFR deposited in the PDB nor, to our knowledge, reported in the literature.

Fig. 3. Detail from the crystal structure (PDB code: 8HV1) of fragment 1 (in stick with grey carbon atoms) bound to EGFR_DMX. Water molecules and labelled amino acids in ball and stick (grey carbon atoms) with main chain only for Q791 and M793, and hydrogen bonds in dashed lines. Details of all crystal structure determination in this paper are in ESI† with data collection (Table S1†) and refinement statistics (Table S6†).

Fig. 3

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We believed that it was worth further pursuing C775 as an anchoring residue for development of irreversible inhibitors, as covalent bond formation with C797 is abolished in EGFR_TM. In addition, irreversible EGFR inhibitors targeting C775 in the ATP binding pocket could overcome the high ATP affinity of EGFR_TM simply through covalent binding. We therefore set out to identify more common warhead that could react with C775.

The crystal structure of 1 bound to EGFR_DMX shows that the triazole NH and N of the fragment form direct hydrogen bonds with the backbone carbonyl of the hinge residue Q791 and the hydroxy group of T854, respectively. In addition, there is a water-mediated hydrogen bond with the backbone NH of another hinge residue, M793. Of the protein kinases for which a crystal structure is deposited in the PDB, only two others have a cysteine residue in the back pocket, TTBK1 and MELK with methionine and leucine as gatekeeper residue respectively (cf. T790 in EGFR_wt). An overlay of these structures (Fig. S1†) showed small variations in the conformation of the ATP binding pocket with the position of the cysteine residues in these two kinases shifted somewhat from that in EGFR. In addition, a comprehensive sequence alignment of all human kinase domains suggests that only the putatively inactive TBCK and pseudokinases ROR1 and ROR2 additionally have a cysteine in that equivalent position.18 From these observations, we hypothesized that a covalent compound bound to C775, containing groups able to form main chain interactions with Q791 and M793 would make an effective and selective inhibitor of EGFR_TM.

A list of groups used in covalent inhibitors such as acrylamides, α-halo acetamides, vinyl sulfones, epoxides and cyano groups that could react with C775 (warheads) was generated from a survey of the literature (see for example).19 Minimal models of these warheads were docked using the covalent docking protocol into the X-ray crystal structure of EGFR_DMX with thione 1 removed.20 The docked warheads were prioritized by visual inspection based on their interactions with the protein. Acrylamide was chosen as the most promising warhead as it was predicted to make a hydrogen bond with the main chain of Q791 and hydroxyl of T854. A set of 350 acrylamides (MW < 400) containing aromatic rings were selected from the Sigma Aldrich catalogue of the time and docked into the structure of EGFR_DMX. Ten compounds were chosen (Fig. 4) for study based on predicted interactions with the main chain of M793 (2, 3, 4, 6, 7, 8) with others selected as sampling other parts of the binding site (5, 9, 10, 11).

Fig. 4. Acrylamide containing compounds selected from docking to the structure of EGFR_DMX.

Fig. 4

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These 10 fragments were purchased or synthesized (2, 7, 8, 10, 11 were not available commercially) to investigate whether they form a covalent bond under physiological conditions. Samples of EGFR_DMX and EGFR_TMX were analyzed by TOF-MS after incubation for 1 hour with the acrylamide containing compounds at pH 7.4 (results in Table S5†). In summary, these experiments showed that most of the compounds formed covalent adducts with EGFR, but that only pyridine 4, quinoline 8 and pyrazolopyridine 10 bound to the triple mutant (with C797S). Furthermore, pre-incubation of EGFR_TMX with the ATP-competitive EGFR inhibitor nazartinib or reaction of EGFR_wt with maleimide to block cysteine reaction, prevented all three compounds from forming such covalent adducts. These results are consistent with 4, 8 and 10 binding to a cysteine in the ATP binding pocket of EGFR; X-ray crystal structures (Fig. 5) of the compounds bound to EGFR_TMX confirm that the bond is with C775. In each of the ligands, the core heterocycle makes a two-point hinge binding interaction as the 3-thiopropionyl amide linker (anchoring moiety) is bent. The acrylamide NH forms a hydrogen bond with the backbone carbonyl of Q791 and the core heteroaryl nitrogen hydrogen bonds with the backbone NH of M793. In addition, 4 and 8 adopt a binding mode such that the acrylamide carbonyl forms a hydrogen bond with the hydroxy group of T854. The crystal structure of 4 was also determined after incubation with crystals of EGFR_wtX and EGFR_DMX; a covalent bond with C775 was formed in all structures (Fig. S2†) with minor variation in the conformation of the link to the cysteine.

Fig. 5. Details from the crystal structures of EGFR_TMX with bound acrylamide compounds; (legend as Fig. 3) with carbon atoms for A. fragment 4 in light blue (PDB code: 8HV4); B. fragment 8 in green (PDB code 8HV6) and C. fragment 10 in orange (PDB code: 8HV8).

Fig. 5

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A comparison of the binding modes predicted by the docking calculations with the crystal structures (Fig. S3†) confirms the quality of the models with two exceptions. The position of acrylamide 9 is shifted slightly for reasons that are not clear. More striking is that acrylamide 7 binds in a flipped orientation and does not form a covalent bond with C775. Interestingly, the covalent bond is not formed in the first deposited crystal structure of osimertinib (PDB code 4ZAU) as was found for both structures determined in this study (P. Dokurno, personal communication) of osimertinib bound to EGFR_TMX and to EGFR_wtX.21 This suggests there can be issues in forming the acrylamide bond (perhaps not binding with an optimal orientation for covalent bond formation) at the pH of the crystal. However, overall, the modelling has successfully identified a number of covalent starting points for generating an inhibitor capable of binding to EGFR at pH 7.4.

Acrylamide 4 was selected for optimization based on its small size, the vectors available for synthesis and that it showed 52.3% inhibition of EGFR_TM after incubation (for 1.5 hours) at 500 μM. As summarized in Fig. 6, the first step was to investigate substituents at the 4-position of the pyridine ring. Inspection of the crystal structure showed a hydrophobic patch could be accessed. The addition of a phenyl group (12) led to a significant increase in inhibitory activity. The crystal structure of 12 confirmed that a covalent bond with C775 is conserved, and the core and warhead moiety of 12 largely overlap with the fragment hit 4. Fig. 7 shows a comparison of the X-ray structures of 4 and 12 bound to EGFR_TMX with the structure of osimertinib bound to EGFR_wtX (PDB: 6JXT). This emphasizes how the acrylamides occupy a different part of the EGFR binding site but a biaryl moiety overlaps (pyridine-phenyl in 12 and pyrimidine-pyrrole in osimertinib) and the pendant phenyl of 12 is sitting within an extensive hydrophobic region. This provided the idea to replace the phenyl group of 12 by a 1-methylindol-3-yl group in 13 followed by core hopping from pyridine to pyrimidine in 14, giving a compound which inhibits EGFR_TM activity with sub-micromolar IC50. Overall, this structure-guided elaboration resulted in a 500-fold IC50 improvement from the starting acrylamide.

Fig. 6. Optimization of acrylamide 4; IC50 is concentration of compound giving 50% inhibition of activity after incubation for 1.5 hours.

Fig. 6

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Fig. 7. Details from the crystal structure of EGFR_TMX with bound 12 (PDB code: 8HV9, white carbon atoms, legend as in Fig. 3) with the structure of osimertinib with lime green carbons as bound to EGFR_wtX (PDB: 6JXT) showing the side chains of additional selected amino acids labelled in italics.

Fig. 7

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The properties of acrylamide 14 as a covalent inhibitor were characterized in more detail. The crystal structure of 14 bound to EGFR_TMX confirmed the conserved covalent bond formation of the acrylamide warhead with C775 (Fig. 8) and the expected interaction of the indole moiety with a cluster of hydrophobic side chains. The intact masses of EGFR_SM2 and EGFR_DM2 (which has the C775S mutation) were analyzed after incubation with the compound at pH 7.4. Covalent adducts (+277 Da) were observed with EGFR_SM2 but not with EGFR_DM2 (Fig. S4†), consistent with 14 binding covalently and selectively to C775 and not to the other cysteines in the protein; there are 4 other cysteines in EGFR, one of which is surface exposed and at least one other could be accessible with some small conformational change. The effects of compound preincubation with enzyme on the functional assay were also investigated by incubating enzyme samples with 14 for different lengths of time and with both 5 μM (Km) and 1 mM ATP prior to initiating the phosphorylation reaction by adding peptide (note the functional assay is performed with 1 μM ATP). Increasing the pre-incubation time shifted the inhibition curve to lower IC50, reflecting the time taken for the covalent bond to form. In addition, the similar IC50 for inhibition at different ATP concentrations demonstrates that a C775 targeted irreversible inhibitor can overcome the enhanced ATP affinity conferred by the T790M mutation (Table 1).

Fig. 8. Detail of the crystal structure of 14 (magenta carbon atoms) bound to EGFR_TMX (PDB code: 8HV10); legend as in Fig. 3 with the side chain atoms of V726, L718, V726, K746 and L844 also shown.

Fig. 8

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IC50 for inhibition of EGFR_TM with different concentrations of ATP after incubation with 14 for varying times before initiating the phosphorylation reaction.

0 min 20 min 60 min
5 μM ATP 599 nM 239 nM 167 nM
1 mM ATP 1297 nM 251 nM 150 nM
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Finally, the selectivity profile of 14 for the inhibition of other protein kinases was characterized in a biochemical kinase assay which measured the percentage inhibition for each of 68 kinases (Fig. S5†). Except for EGFR_wt and EGFR mutants, only two kinases were inhibited to less than 80% activity at 1 μM compound and 1 mM ATP: JAK3 (74.2%) and MNK1 (26.5%). Although these two kinases do not have a cysteine at the equivalent position of C775 in EGFR, it is possible 14 binds non-covalently, or can bind to another cysteine within the ATP binding pocket.

Conclusion

In summary, guided by structural information, we report the first irreversible EGFR inhibitors such as 14 which covalently modify C775. This compound is highly selective for EGFR and inhibits the kinase activity in an ATP concentration-independent manner and is active against the mutant EGFR which is resistant to third generation irreversible inhibitors such as osimertinib which target C797. To the best of our knowledge, no other irreversible inhibitors which target C775 have been reported. This may be because C775 is in a small pocket deep in the ATP binding region and is thus inaccessible to the larger compounds typically assayed in high throughput screening campaigns. In addition, as C775 is located near the hinge region and the gatekeeper residue, it may have a role in the kinase activity of EGFR. This is suggested by some preliminary experiments which will be published in due course. This could mean that the C775S point mutation is less likely to occur as a mechanism of acquired resistance to irreversible binding compounds which target C775 in EGFR.

This study demonstrates the value of a covalent fragment strategy to identify opportunities in binding sites. Efforts aiming at wild-type EGFR sparing kinase inhibitors with potent cellular activities are the subject of current investigations.

Author contributions

REH, DLW, JBM, KK, TH and YT directed the research; HA, RM and KT developed and performed assays; LMB refined a protein crystal structure, EC and SI produced proteins, PD and MT determined and refined protein crystal structures, LLS performed mass spectrometry, CAM, NK, ST and MY performed chemical synthesis, ATM conducted the modelling studies, and NN supported the compound design. REH and NK drafted the manuscript. All authors approved the final manuscript.

Conflicts of interest

There is no conflict of interest to declare.

Supplementary Material

MD-014-D3MD00439B-s001

Acknowledgments

The research and preparation of the manuscript was funded by Daiichi Sankyo Co., Ltd. and Vernalis (R&D) Ltd. We thank Masami Ohtsuka for helpful discussions on medicinal chemistry. We appreciate Yasuo Ohata, Yoshiharu Takama, Kumiko Hiramoto, Kiyoko Mizumaki and Nozomi Tsuchiya for assay.

†

Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d3md00439b

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Supplementary Materials

MD-014-D3MD00439B-s001
MD-014-D3MD00439B-s001.pdf (1.7MB, pdf)

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