BCI inhibits MKP3 by targeting the kinase-binding domain and disrupting ERK2 interaction.

Mitogen-activated protein kinase phosphatase 3 (MKP3), also known as dual-specificity phosphatase 6 (DUSP6), is a critical regulator of ERK signaling, and its dysregulation is implicated in diseases such as cancer. The small-molecule inhibitor BCI ((E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one) has been reported to inhibit MKP3, thereby enhancing ERK signaling and promoting selective cytotoxicity in cancer cells. However, the molecular mechanism underlying BCI-mediated MKP3 inhibition remains unclear. In this research, we characterized the interaction between BCI and MKP3 using NMR titration, microscale thermophoresis (MST), enzymatic assays, and AlphaFold 3 (AF3) modeling. Our results demon-strate that BCI selectively binds to the kinase-binding domain (KBD) of MKP3, rather than its catalytic domain (CD), thereby disrupting the MKP3-ERK2 interaction and impairing MKP3 activation. Enzymatic assays further reveal that BCI significantly reduces ERK2-mediated MKP3 activity without directly interfering with substrate binding at the active site. AF3 structural modeling suggests that BCI binding induces local conformational changes, notably an outward shift of the α4-helix, which exposes a hydrophobic pocket essential for BCI accommodation. Moreover, BCI exhibits differential bind-ing affinities across the MKP family, showing significant interactions with the KBDs of MKPX and MKP5, but markedly weaker or negligible binding to those of MKP1, MKP2, and MKP4. Together, these findings uncover a novel KBD-targeting mechanism of MKP3 inhibition by BCI and highlight the potential of selectively modulating MAPK phosphatases through allosteric disruption of kinase-phosphatase interactions. This strategy may offer a new avenue for the design and optimization of targeted phosphatase inhibitors.