A Membrane-Disruptive Action of VBIT-4 Challenges Its Role as a Widely Used VDAC1 Oligomerization Inhibitor

VDAC, the most abundant protein in the outer mitochondrial membrane, plays a central role in mitochondrial physiology. Its oligomerization has been considered to be involved in critical processes, such as mitochondrial DNA release and apoptosis; yet, the underlying molecular mechanisms remain poorly defined. VBIT-4, a small molecule widely regarded as a VDAC1 oligomerization inhibitor, has seen extensive applications over the past five years without proper mechanistic characterization. Using high-speed atomic force microscopy, we directly visualized VDAC1 oligomerization in planar lipid membranes and examined the effects of VBIT-4. Unexpectedly, we discovered that VBIT-4 partitioned into lipid bilayers at micromolar concentrations and disrupted membrane structure irrespective of VDAC1’s presence. Complementary approaches—including single-channel electrophysiology, microscale thermophoresis, and coarse-grained molecular dynamics—corroborated VBIT-4 strong membrane partitioning and destabilizing activity, while showing no detectable effect on VDAC1 or its clustering. Consistent with this behavior, VBIT-4 induced VDAC1-independent cytotoxicity in HeLa cells at concentrations above 10 µM. Together, these findings demonstrate that VBIT-4 acts by perturbing lipid bilayers and promoting membrane defects rather than by inhibiting VDAC1 oligomerization. Thus, providing an alternative mechanism of action for interpreting studies using VBIT-4 underscores the need to consider its potent, nonspecific membrane activity in cells.