Oxygenated 9H-carbazole-3-carbaldehyde scaffolds as cerebral cavernous malformation-relevant protein-protein interaction disruptors of the HEG1-KRIT1 interface.

The HEG1-KRIT1 protein-protein interaction is a central component of endothelial CCM-complex organization, linking junctional KRIT1 recruitment to signaling pathways implicated in cerebral cavernous malformation pathobiology, and represents a structurally defined target for small-molecule disruption. In this study, a focused library of 127 oxygenated 9H-carbazole-3-carbaldehyde derivatives was screened to identify candidate inhibitors of the HEG1-KRIT1 interface. Structure-based virtual screening against the HEG1/HKi2-recognition pocket of the KRIT1 FERM domain prioritized heptaphylline, mukoenine B, and murrayacine for further evaluation. Docking analysis showed that all three candidates occupied the KRIT1 interfacial pocket, while 1000 ns molecular dynamics simulations revealed compound-specific effects on ligand retention, interface stability, and KRIT1 conformational dynamics. MM/PBSA-based PPI disruption analysis showed that murrayacine was the only carbazole candidate with a positive ΔΔGPPI value (+ 3.07 kcal/mol), indicating predicted weakening of the HEG1-KRIT1 interface, whereas heptaphylline and mukoenine B showed negative ΔΔGPPI values consistent with non-disruptive or stabilizing behavior. Protein-ligand MM/PBSA analysis identified mukoenine B as the most energetically stable KRIT1 binder, while dynamic interface metrics and free energy landscape analysis supported murrayacine as the strongest functional disruptor. Experimental validation using a bead-based flow-cytometry competition assay confirmed dose-dependent inhibition of HEG1-KRIT1 binding, with murrayacine showing HKi2-like potency (IC50 = 3.85 ± 0.30 µM) and outperforming mukoenine B and heptaphylline. In silico ADMET profiling further supported murrayacine as the most drug-like candidate. Collectively, these findings identify murrayacine as a promising functional carbazole-based HEG1-KRIT1 competition inhibitor. Because noncovalent MD showed substantial ligand-pose displacement for HKi2 and murrayacine, the computational results are interpreted as exploratory and pose-dependent, and direct KRIT1-binding validation will be required in future studies.