Correction: A high-throughput effector screen identifies a novel small molecule scaffold for inhibition of ten-eleven translocation dioxygenase 2.

[This corrects the article DOI: 10.1039/D2MD00186A.].

Addressing the Osimertinib Resistance Mutation EGFR-L858R/C797S with Reversible Aminopyrimidines.

Drug resistance mutations emerging during the treatment of non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) inhibitors represent a major challenge in personalized cancer treatment and require constant development of new inhibitors. For the covalent irreversible EGFR inhibitor osimertinib, the predominant resistance mechanism is the acquired C797S mutation, which abolishes the covalent anchor point and thus results in a dramatic loss in potency. In this study, we present next-generation reversible EGFR inhibitors with the potential to overcome this EGFR-C797S resistance mutation. For this, we combined the reversible methylindole-aminopyrimidine scaffold known from osimertinib with the affinity driving isopropyl ester of mobocertinib. By occupying the hydrophobic back pocket, we were able to generate reversible inhibitors with subnanomolar activity against EGFR-L858R/C797S and EGFR-L858R/T790M/C797S with cellular activity on EGFR-L858R/C797S dependent Ba/F3 cells. Additionally, we were able to resolve cocrystal structures of these reversible aminopyrimidines, which will guide further inhibitor design toward C797S-mutated EGFR.

A high-throughput effector screen identifies a novel small molecule scaffold for inhibition of ten-eleven translocation dioxygenase 2.

Ten-eleven translocation dioxygenases (TETs) are the erasers of 5-methylcytosine (mC), the central epigenetic regulator of mammalian DNA. TETs convert mC to three oxidized derivatives with unique physicochemical properties and inherent regulatory potential, and it initializes active demethylation by the base excision repair pathway. Potent small molecule inhibitors would be useful tools to study TET functions by conditional control. To facilitate the discovery of such tools, we here report a high-throughput screening pipeline and its application to screen and validate 31.5k compounds for inhibition of TET2. Using a homogenous fluorescence assay, we discover a novel quinoline-based scaffold that we further validate with an orthogonal semi-high throughput MALDI-MS assay for direct monitoring of substrate turnover. Structure-activity relationship (SAR) studies involving >20 derivatives of this scaffold led to the identification of optimized inhibitors, and together with computational studies suggested a plausible model for its mode of action.

Optimization of Covalent MKK7 Inhibitors via Crude Nanomole-Scale Libraries.

High-throughput nanomole-scale synthesis allows for late-stage functionalization (LSF) of compounds in an efficient and economical manner. Here, we demonstrated that copper-catalyzed azide-alkyne cycloaddition could be used for the LSF of covalent kinase inhibitors at the nanoscale, enabling the synthesis of hundreds of compounds that did not require purification for biological assay screening, thus reducing experimental time drastically. We generated crude libraries of inhibitors for the kinase MKK7, derived from two different parental precursors, and analyzed them via the high-throughput In-Cell Western assay. Select inhibitors were resynthesized, validated via conventional biological and biochemical methods such as western blots and liquid chromatography-mass spectrometry (LC-MS) labeling, and successfully co-crystallized. Two of these compounds showed over 20-fold increased inhibitory activity compared to the parental compound. This study demonstrates that high-throughput LSF of covalent inhibitors at the nanomole-scale level can be an auspicious approach in improving the properties of lead chemical matter.