Discovery of a Novel Mutant-Selective Epidermal Growth Factor Receptor Inhibitor Using in silico Enabled Drug Discovery Platform

21 June 2024, Version 2


Despite the success of first, second and third generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in treatment of non-small cell lung cancer (NSCLC) with classical EGFR mutations (L858R or Exon 19 deletions), disease progression often occurs due to the acquisition of additional mutations in the EGFR kinase domain that confer TKI resistance. Specifically, acquisition of both T790M and C797S resistance mutations results in an EGFR variant that is resistant to all approved EGFR TKIs. Herein, we report a physics-based computationally-driven lead identification approach which successfully identified structurally-unique imidazo[3.2-b]pyrazole derivatives as reversible inhibitors of EGFR classical mutations bearing both T790M and C797S. Importantly, they spare EGFR WT to avoid known EGFR WT-driven cutaneous toxicities. During profiling of imidazo[3.2-b]pyrazole derivatives, we elucidated the bioactivation mechanism causing CYP3A4/5 time-dependent inhibition (TDI) and found key modifications to suppress bioactivation and mitigate the TDI risk. Representative lead compound 31 inhibited EGFR L858R/T790M/C797S in biochemical assays with a Ki = 2.1 nM, and EGFR del19/T790M/C797S in a Ba/F3 cellular assay with a IC50 = 56.9 nM. Deuterated analog of 31 (38) demonstrated dose-dependent tumor growth inhibition in a Ba/F3 EGFR del19/T790M/C797S CDX model by 47% at 50 mg/kg BID and 92% at 100 mg/kg BID.


Free energy perturbation
hit identification
CYP3A4/5 time-dependent inhibition
epidermal growth factor receptor
EGFR triple mutant inhibitor
non-small cell lung cancer

Supplementary materials

Supporting information
1. Compound property space of the AutoDesigner Core Design output from compound 1 and 2 2. Kinase selectivity profile of 16, 18, and 38 Tested at 1 3. CYP3A4 TDI of EGFR TKIs 4. Single crystal X-ray diffraction data of 55 5. X-ray data collection and refinement statistics for compound 3 and 16 6. NMR, LC-MS, and HPLC spectra of final compounds


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