Interrupting specific hydrogen bonds between ELF3 and MED23 as an alternative drug resistance-free strategy for HER2-overexpressing cancers.

[Display omitted] • ELF3-MED23 hotspot was efficiently deciphered by using customized small molecules. • ELF3-MED23 PPI depends on specific H-bondings to upregulate HER2 expression. • PPI inhibitory activity of YK1 was specific to ELF3-MED23. • YK1-mediated specific H-bond interruption induces promising anticancer activities. • Anticancer effect of YK1 was even significant against trastuzumab-refractory clones. • YK1 held highly drug-like properties showing favorable PK profile. HER2 overexpression induces cancer aggression and frequent recurrences in many solid tumors. Because HER2 overproduction is generally followed by gene amplification, inhibition of protein–protein interaction (PPI) between transcriptional factor ELF3 and its coactivator MED23 has been considered an effective but challenging strategy. This study aimed to determine the hotspot of ELF3-MED23 PPI and further specify the essential residues and their key interactions in the hotspot which are controllable by small molecules with significant anticancer activity. Intensive biological evaluation methods including SEAP, fluorescence polarization, LC-MS/MS-based quantitative, biosensor, GST-pull down assays, and in silico structural analysis were performed to determine hotspot of ELF3-MED23 PPI and to elicit YK1, a novel small molecule PPI inhibitor. The effects of YK1 on possible PPIs of MED23 and the efficacy of trastuzumab were assessed using cell culture and tumor xenograft mouse models. ELF3-MED23 PPI was found to be specifically dependent on H-bondings between D400, H449 of MED23 and W138, I140 of ELF3 for upregulating HER2 gene transcription. Employing YK1, we confirmed that interruption on these H-bondings significantly attenuated the HER2-mediated oncogenic signaling cascades and exhibited significant in vitro and in vivo anticancer activity against HER2-overexpressing breast and gastric cancers even in their trastuzumab refractory clones. Our approach to develop specific ELF3-MED23 PPI inhibitor without interfering other PPIs of MED23 can finally lead to successful development of a drug resistance-free compound to interrogate HER2 biology in diverse conditions of cancers overexpressing HER2. [ABSTRACT FROM AUTHOR]