Structure-based discovery of acridone alkaloids as disruptors of the HEG1-KRIT1 protein-protein interaction in cerebral cavernous malformation signaling.

Abstract

The HEG1-KRIT1 interaction is essential for endothelial cerebral cavernous malformation complex localization and signaling, making this interface a relevant target for chemical disruption. This study aimed to identify acridone alkaloids capable of inhibiting HEG1 binding to KRIT1 through integrated virtual screening, molecular simulations, binding-energy analysis, drug-likeness prediction, and experimental validation. Screening of 2,449 acridone alkaloids prioritized five candidates: 5-hydroxynoracronycine, acronycine, arborinine, glycocitrine I, and rutacridone. Computational analyses showed stable binding within the HEG1-recognition pocket of KRIT1 and predicted interface weakening, with rutacridone and 5-hydroxynoracronycine producing the strongest destabilizing effects. A bead-based flow-cytometry competition assay confirmed concentration-dependent inhibition, with half-maximal inhibitory concentration values of 2.21 ± 0.31 µM for rutacridone, 2.30 ± 0.30 µM for arborinine, and 2.90 ± 0.35 µM for 5-hydroxynoracronycine. These findings identify rutacridone, arborinine, and 5-hydroxynoracronycine as promising acridone-based starting points for developing HEG1-KRIT1 interaction disruptors. The image explains the difference between normal and disrupted HEG1-KRIT1 protein interactions affecting CCM signaling. On the left, the homeostatic HEG1-KRIT1 complex forms at the endothelial cell membrane, where HEG1 and VE-cadherin proteins connect with KRIT1 (FERM domain) and CCM2 and CCM3 proteins. This complex activates a signaling cascade through MEKK3, MEK5, ERK5, MEF2, and KLF2/4, resulting in regulated CCM signaling. On the right, a small-molecule protein-protein interaction disruptor blocks the binding between HEG1 and KRIT1, shown by a red cross. This disruption prevents the normal complex formation, leading to dysregulated CCM signaling and an altered KLF2/4 transcriptional response. A predicted binding model illustrates the small molecule at the HEG1-KRIT1 interface, with HEG1 cytoplasmic tail and KRIT1 FERM domain labeled.