Pitfalls and considerations in determining the potency and mutant selectivity of covalent epidermal growth factor receptor inhibitors

Pursuing enzyme inhibitors with molecules that form covalent bonds with the desired target is an attractive focus in drug development that is increasing in prevalence. However, challenges arise when carrying out assessments of their time-dependent inhibitory properties as well as making correlations with values reported in the literature. Given the prominent focus on the Epidermal Growth Factor Receptor (EGFR) tyrosine kinase in oncology, and the diverse structures and binding modes of covalent EGFR inhibitors, this perspective seeks to explore various broadly relevant factors that arise in the measurement of kinetic parameters within this class of drugs. A review of several studies indicates that variable literature potency values require investigators to include appropriate reference molecules and consistent substrate conditions for experimental consistency and proper benchmarks. The impact on covalent inhibitor potency with respect to common buffer conditions and compound liquid handling is surveyed highlighting the importance of multiple experimental variables when conducting these assays. Additionally, when assessing the potency for inhibitor selectivity in targeting EGFR mutants over wild-type (WT), it is ideal to consider ratios of true potency due to the variable ATP substrate binding affinities. The overview presented here, although most directly applicable to the tyrosine kinase inhibitor field, serves inhibitor assessments broadly by providing guided insights into conducting biochemical assays for designing and validating next-generation covalent inhibitors

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

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

Linking ATP and allosteric sites to achieve superadditive binding with bivalent EGFR kinase inhibitors

Bivalent molecules consisting of groups connected through bridging linkers often exhibit strong target binding and unique biological effects. However, developing bivalent inhibitors with the desired activity is challenging due to the dual motif architecture of these molecules and the variability that can be introduced through differing linker structures and geometries. We report a set of alternatively linked bivalent EGFR inhibitors that simultaneously occupy the ATP substrate and allosteric pockets. Crystal structures show that initial and redesigned linkers bridging a trisubstituted imidazole ATP-site inhibitor and dibenzodiazepinone allosteric-site inhibitor proved successful in spanning these sites. The re-engineered linker yielded a compound that exhibited significantly higher potency (~60 pM) against the drug-resistant EGFR L858R/T790M and L858R/T790M/C797S, which was superadditive as compared with the parent molecules. The enhanced potency is attributed to factors stemming from the linker connection to the allosteric-site group and informs strategies to engineer linkers in bivalent agent design