COVALfinder^®, the ideal tool for irreversible drug discovery programs

Irreversible inhibition involves an initial binding step driven by affinity and a time-dependent inactivation step driven by covalent bond formation^1-3. Using COVALfinder^® in the early stages of drug discovery:

• Enables the early identification of irreversible compounds.
• Allows for the improvement of both inhibitor binding and covalent bond formation: Modification of any part of an inhibitor may improve the rate of covalent bond formation (k_inact) at the expense of inhibitor binding to the target (K_I) or vice versa.
• Allows differentiation between similar therapies.
• Helps you to define in vivo target occupancy.

Discovering and de-risking EGFR irreversible inhibitors

Targeting EGFR with irreversible inhibitors like Afatinib and Dacomitinib has dramatically changed the therapeutic routine for lung adenocarcinoma patients. However, under therapeutic pressure, resistant clones emerge. Osimertinib and Nazartinib have been designed to overcome acquired resistance induced by the T790M mutation with high specificity and reduced off-target WT EGFR-driven toxicities.

We have studied in detail the inactivation kinetics of seven irreversible EGFR drugs using COVALfinder^®.

Identification of EGFR irreversible compounds

• The binding profiles showed a slow onset of inhibition (representative data for Nazartinib is shown in Fig.1, A) and 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 k_obs 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.
• The k_inact, K_I, k_inact/K_I and T_1/2^∞ (half-life for inactivation at infinite concentration of inhibitor) values (Table 1) correlate with those obtained from activity assays reported in the literature. 
• Both the mechanism of inhibition and the kinetic constants were successfully characterized for all the irreversible EGFR inhibitors^4-6.

Differentiation of EGFR drug candidates by inactivation kinetics

• The quinazoline based irreversible EGFR drugs (Dacomitinib, Afatinib, Canertinib and Poziotinib) are extremely efficient (k_inact/K_I) with high affinity (K_I) and low specific reactivity (k_inact).
• Neratinib has the same reactive substituent as Afatinib, but its affinity is 10-fold weaker and with 3-fold lower inactivation efficiency.

Pyrimidine based irreversible EGFR T790M inhibitors (Osimertinib and Nazartinib) show up to 500 times lower affinity and inactivation efficiency with similar weak reactivity.

References

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. Bauer R.A. (2015) Covalent inhibitors in drug discovery: from accidental discoveries to avoided liabilities and designed therapies. Drug Discov Today 20(9):1061-73.
4. Zhai X. et al. (2020) Insight into the Therapeutic Selectivity of the Irreversible EGFR Tyrosine Kinase Inhibitor Osimertinib through Enzyme Kinetic Studies. Biochemistry 59(14):1428-1441.
5. Fassunke J. et al. (2018) Overcoming EGFR G724S-mediated osimertinib resistance through unique binding characteristics of second-generation EGFR inhibitors. Nat Commun. 7;9(1):4655.
6. Schwartz P.A. et al. (2014) Covalent EGFR inhibitor analysis reveals importance of reversible interactions to potency and mechanisms of drug resistance. Proc Natl Acad Sci USA 22(1):3-20.