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. 2025 Apr 9;8(1):111.
doi: 10.1038/s42004-025-01501-6.

Factors affecting irreversible inhibition of EGFR and influence of chirality on covalent binding

Pasquale A Morese  1 , Ayaz Ahmad  2 , Mathew P Martin  3 , Richard A Noble  3 , Sara Pintar  3 , Lan Z Wang  3 , Shangze Xu  2 , Andrew Lister  4 , Richard A Ward  4 , Agnieszka K Bronowska  2 , Martin E M Noble  3 , Hannah L Stewart  5 , Michael J Waring  6
Affiliations

Factors affecting irreversible inhibition of EGFR and influence of chirality on covalent binding

Pasquale A Morese et al. Commun Chem. .

Erratum in

Abstract

The discovery of targeted covalent inhibitors is of increasing importance in drug discovery. Finding efficient covalent binders requires modulation of warhead reactivity and optimisation of warhead geometry and non-covalent interactions. Uncoupling the contributions that these factors make to potency is difficult and best practice for a testing cascade that is pragmatic and informative is yet to be fully established. We studied the structure-reactivity-activity relationships of a series of analogues of the EGFR inhibitor poziotinib with point changes in two substructural regions as well as variations in warhead reactivity and geometry. This showed that a simple probe displacement assay that is appropriately tuned in respect of timing and reagent concentrations can reveal structural effects on all three factors: non-covalent affinity, warhead reactivity and geometry. These effects include the detection of potency differences between an enantiomeric pair that differ greatly in their activity and their capacity to form a covalent bond. This difference is rationalised by X-ray crystallography and computational studies and the effect translates quantitatively into cellular mechanistic and phenotypic effects.

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Conflict of interest statement

Competing interests: The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Structures of prominent TCIs: inhibitors of BTK (ibrutinib, 1), EGFR (osimertinib, 2) and mutant kRAS (sotorasib, 3).
Fig. 2
Fig. 2
Structures of poziotinib 4 and gefitinib 5, covalent and non covalent anilinoquinazoline-based EGFR inhibitors respectively.
Fig. 3
Fig. 3. Development of the TR FRET based probe displacement assay.
a Gefitinib derived fluorescent probe 6; (b) Schematic overview of the assay.
Fig. 4
Fig. 4
Deconstruction of the poziotinib structure to design analogues varying the warhead (red), cyclic amine spacer (blue), quinazoline substituent (R) and aniline (green).
Scheme 1
Scheme 1. Synthesis of compounds.
7-15. Reagents and conditions: a) Acryloyl chloride, NaHCO3, THF, 0 °C – r.t., 12 h (7-9, 14: 9–71%) or propiolic acid, HATU, DIPEA, DMA, rt, 2 h (10-12, 15: 37–75%); (b) NH2Ar, DMF, 75 °C, 1.5 h, 44%; (c) i. TFA, DCM, rt, 1 h, ii. propiolic acid, HATU, DIPEA, DMA, rt, 2 h, (13: 91%).
Scheme 2
Scheme 2. Synthesis of compounds 16-21.
Reagents and conditions: (a) N-Boc-mesylate, K2CO3, DMF, 80 °C, 12 h (24–65%) or propiolic acid, HATU, DIPEA, DMA, rt, 2 h (10-12, 15: 37–75%); (b). i. TFA, DCM, rt, 1 h, ii. acryloyl chloride, NaHCO3, THF, 0 °C – r.t., 12 h (16-18: 13-42%) or propiolic acid, HATU, DIPEA, DMA, rt, 2 h, (19-21: 60–76%).
Scheme 3
Scheme 3. Synthesis of compounds 22 and 23.
Reagents and conditions: (a) N-Boc-mesylate, K2CO3, DMF, 80 °C, 12 h (24-65%) or propiolic acid, HATU, DIPEA, DMA, rt, 2 h (10-12, 15: 37-75%); (b). i. TFA, DCM, rt, 1 h, ii. acetyl chloride, NaHCO3, THF, 0 °C – r.t., 12 h (22-23: 17-21%).
Fig. 5
Fig. 5. Matched molecular pair analysis of DM and WT EGFR potency comparing the warheads, aniline, and cyclic amine spacers.
Blue denotes the acrylamides, and red denotes propiolamides, line connections show matched pairs.
Fig. 6
Fig. 6. Characterisation of 16 and 17 by mass spectrometry and cellular pharmacology.
a Intact protein mass spectrometry analysis comparing the degree of modification between 16 and 17; (b) Cellular pEGFR inhibition determined by HTRF in A431 and H1975 cells (n = 1); (c) Concentration response curves of the A431 cell viability in response to inhibitors (72 h) (n = 1, error = SD).
Fig. 7
Fig. 7. X-ray crystal structures of 16 and 17 in complex with WT EGFR.
a 16 (R-enantiomer) and b 17 (Senantiomer). Continuous electron density 2Fo-Fc refinement map (blue mesh contoured to contoured to 0.15e/Å3 (1.0 r.m.s.d)) indicating the presence of a covalent bond with Cys797 (bond length 1.79Å, bond angle 113˚) for 17 but not 16. The difference map (Fo-Fc) with 17 omitted for refinement showing clear evidence of adduct formation (Fig. S4). Secondary structure shown in white ribbon, and electrostatic surface shown for active site (PDB: 9FZS 16, 9FZR 17).
Fig. 8
Fig. 8. Covalent docking of 16 an 17 to EGFR.
a 16 and b 17. Spheres around atoms denote their calculated affinity contributions (green – favourable, red – unfavourable).
See this image and copyright information in PMC

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