Endothelial barrier integrity is important for vascular homeostasis, and 
hyperpermeability participates in the progression of many pathological states, 
such as diabetic retinopathy, ischemic stroke, chronic bowel disease, and 
inflammatory disease. Here, using drug repositioning, we discovered that 
primaquine diphosphate (PD), previously known as an antimalarial drug, was a 
potential blocker of vascular leakage. PD inhibited the linear pattern of 
vascular endothelial growth factors (VEGF)-induced disruption at the cell 
boundaries, blocked the formation of VEGF-induced actin stress fibers, and 
stabilized the cortactin actin rings in endothelial cells. PD significantly 
reduced leakage in the Miles assay and mouse model of streptozotocin 
(STZ)-induced diabetic retinopathy. Targeted prediction programs and 
deubiquitinating enzyme activity assays identified a potential mechanism of 
action for PD and demonstrated that this operates via ubiquitin specific 
protease 1 (USP1). USP1 inhibition demonstrated a conserved barrier function by 
inhibiting VEGF-induced leakage in endothelial permeability assays. Taken 
together, these findings suggest that PD could be used as a novel drug for 
vascular leakage by maintaining endothelial integrity.