The origin of potency and mutant-selective inhibition by bivalent ATP-allosteric EGFR inhibitors

26 September 2023, Version 1


Targeted small-molecule therapies in mutant epidermal growth factor receptor (EGFR) non-small cell lung cancer (NSCLC) have undergone several generations of development in response to acquired drug resistance. With the emergence of the highly prevalent T790M and C797S drug-resistant mutations, a diverse arsenal of ATP-competitive molecules has led to the front-line drug AZD9291 (osimertinib) and several in clinical development. Several allosteric inhibitors bind a site adjacent to the ATP-binding site and exhibit synergy when dosed in combination with certain ATP-competitive inhibitors. Structure-guided design of molecules that anchor to both sites simultaneously, namely ATP-allosteric bivalent inhibitors, have been reported as proof-of-concept EGFR mutant-selective compounds, however their properties are underexplored and currently exhibit modest activity in human cancer cell lines. To better understand the structural and functional properties of such molecules, we have carried out structure-activity relationships (SAR) defining the groups of the allosteric pocket that are responsible for enabling mutant selectivity and potency of this series. We find that the back pocket phenol ring enables stronger binding while the methylisoindolinone is responsible for enabling selectivity for the oncogenic mutations. An optimized allosteric site-binding group and a C797-targeting ATP-site scaffold exhibit inhibitory effects in a variety of EGFR mutant cell lines, which is improved over earlier examples. Additionally, a closely related reversible-binding analogue exhibits mutant-selective activity and ~1 nM biochemical potency against L858R/T790M/C797S and promising antiproliferative effects in human cancer cells indicating that ATP-allosteric bivalent kinase inhibitors may serve as tool compounds in understanding overcoming these important resistance mechanisms. These results highlight the utility of bivalent ATP-allosteric compounds in understanding the impact certain functional groups have in the potency and mutant-selectivity enabled by allosteric pocket binding. The results of this study incentivize further investigations of compounds that bind within an exit vector made accessible in the inactive αC-helix “out” conformation as a novel approach for kinase inhibitors.


Kinase inhibitors
non-small cell lung cancer
allosteric inhibitors
mutant selectivity
structure-based design
drug discovery
epidermal growth factor receptor

Supplementary materials

Supporting Information
Supporting biochemical activities, crystallographic data and statistics, graphics comparing various crystal structures, metabolic stability experiments, kinome selectivity screening, binding modes from KLIFS database


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