Structure-based virtual screening for selective connexin hemichannel blockers: a historical perspective on the discovery of a small organic inhibitor

Introduction: Connexin hemichannels are critical mediators of cellular signaling in both physiological and pathological states, yet their selective pharmacological modulation has remained elusive because of the close structural similarity between hemichannels and gap junction channels formed by the same connexins. Methods: We combined structure-based virtual screening of a connexin hemichannel model with experimental validation - ethidium/DAPI dye-uptake assays, single-channel and whole-cell electrophysiology, and Lucifer yellow gap-junctional coupling assays - to identify and characterise D4, a small chiral organic molecule, and we synthesised and tested both of its enantiomers. Results: ≈ 5 nM) without inhibiting gap junction channels formed by the same connexin, and it does not block C×36 hemichannels. The R-enantiomer is fully active whereas the S-enantiomer is inactive, demonstrating stereospecific molecular recognition. Electrophysiology confirms complete blockade of discrete C×43 hemichannel currents at 10 nM, with no effect on pannexin-1, CALHM1 or TRPV2 channels. Discussion: These findings establish D4(R) as a first-in-class, gap-junction-sparing hemichannel blocker and provide a validated molecular scaffold for next-generation connexin therapeutics, addressing a long-standing challenge in connexin pharmacology and opening new avenues for dissecting hemichannel function under physiological and pathophysiological conditions.