Selectivity profiling of irreversible EGFR inhibitors using COVALfinder®

Designing selective irreversible inhibitors

Irreversible inhibition involves an initial binding step driven by affinity and a time-dependent inactivation step driven by covalent bond formation1-2. A recent study demonstrated how IC50 inadequately reflects the selectivity profile of the BTK irreversible inhibitors ibrutinib and acalabrutinib. Although acalabrutinib seems to be more selective than ibrutinib when comparing IC50 alone, the assessment of selectivity using efficiency of inactivation (Kinact/KI) ratios corroborate clinical data demonstrating similar safety profiles between the therapies3.

Selectivity profiling of irreversible EGFR inhibitors using COVALfinder®

EGFR is a receptor tyrosine kinase, member of the ErbB family together with HER2, HER3 and HER4. EGFR overexpression is frequent in many types of human malignancies, including non-small cell lung cancer (NSCLC). Afatinib, Dacomitinib and Neratinib are quinazoline based irreversible pan-ErbB drugs. Afatinib and Dacomitinib have been approved for the treatment of EGFR mutated NSCLC and Neratinib for the treatment of HER2-positive breast cancer.

Here we study the selectivity profile of three irreversible EGFR inhibitors using COVALfinder®.

  • The binding profiles for all the irreversible inhibitors showed a slow onset of EGFR, HER2 and HER4 inhibition (representative data for HER2 inactivation by Dacomitinib is shown in Fig.1, A).
  • Each inhibitor with the three kinases tested also displayed the characteristic time-dependent reduction in IC50 due to the formation of the covalent complex over time (Fig.1, C and D).
  • The dependence of kobs on inhibitor concentration displayed a two-step inhibition (Fig.1, B). This behavior is consistent with either slow onset reversible inhibition or irreversible inhibition. However, in all cases the y-intercept was zero, which is only consistent with irreversible binding.
Figure 1. Characterization of the kinetics of HER2 inactivation by the irreversible inhibitor Dacomitinib. (A) Progress curve of HER2 incubated with increasing compound concentrations. (B) Dependence of kobs on Dacomitinib concentration. (C) Dose-response curves over time. (D) IC50 values over time.

Inactivation kinetics profiling is crucial for selectivity assessment

  • If we take into account the IC50 values to calculate the selectivity ratio, all the inhibitors are pan-ErbB drugs.
  • However, using kinact/KI the selectivity profile of Afatinib and Dacomitinib dramatically changes (Table 1).
  • Afatinib and Dacomitinib bind preferentially to EGFR and HER4.
  • Neratinib is a pan-ErbB inhibitor.
  • The covalency quadrant (Fig.2) clearly shows that the selectivity of Afatinib and Dacomitinib towards HER2 is due to a lower affinity (KI) and reactivity (kinact).  
  • These results explain the efficiency of Afatinib and Dacomitinib in the treatment of EGFR mutated NSCLC and the efficiency of Neratinib in HER2-positive breast tumors.
Figure 2. Covalency quadrant scale. Classification of covalent binders by target comparing the rate of covalent bond formation (kinact) against affinity (KI).
Table 1. Kinetic analysis of irreversible inhibition of ErbB family. Calculation of the selectivity ratio is based on the IC50 values reported in the literature and kinact/KI values obtained with COVALfinder® for off-targets relative to EGFR. NA: no data available in the literature. Empty fields indicate that the selectivity ratio cannot be calculated.


  1. Ayah A. et al. (2020) Advances in covalent kinase inhibitors. Chem Soc Rev. 49(9):2617-2687.
  2. Strelow J.M. et al. (2017) A Perspective on the Kinetics of Covalent and Irreversible Inhibition. SLAS Discov. 22(1):3-20.
  3. Hopper M. et al. (2020) Relative Selectivity of Covalent Inhibitors Requires Assessment of Inactivation Kinetics and Cellular Occupancy: A Case Study of Ibrutinib and Acalabrutinib. J Pharmacol Exp Ther. 372(3):331-338.
  4. Engelman J.A. et al. (2007) PF00299804, an irreversible pan-ERBB inhibitor, is effective in lung cancer models with EGFR and ERBB2 mutations that are resistant to gefitinib. Cancer Res. 67(24):11924-32.
  5. Rabindran S.K. et al. (2004) Antitumor activity of HKI-272, an orally active, irreversible inhibitor of the HER-2 tyrosine kinase. Cancer Res. 64(11):3958-65.
  6. Li D. et al. (2008) BIBW2992, an irreversible EGFR/HER2 inhibitor highly effective in preclinical lung cancer models. Oncogene. 27(34):4702-11.