Figure 1.
KAI, a specific inhibitor of KIF13B-mediated VEGFR2 trafficking, inhibits neovascularization in wet AMD. (A) Schematic showing the domains of KIF13B, binding sites of other cargoes, and peptides derived from KIF13B. (B) Pull-down assay showing the interaction of VEGFR2 with KAI but not with ctrl. Biotin-KAI and ctrl were immobilized on the streptavidin beads and incubated with the lysate of HUVEC. The proteins bound on the beads and input in the cell lysate were detected with the antibodies against VEGFR2 and CentA. VEGFR2 interacted with KAI but not with ctrl. Both peptides did not interact with CentA. (C) Binding affinity (KD) of KAI to RTKs analyzed by SPR. Biotin-tagged KAI and ctrl were immobilized on the streptavidin-chip and tested for affinity with recombinantly expressed cytosolic kinase domains of VEGFR2, PDGFRα, PDGFRβ, and EGFR (Fisher Scientific). VEGFR2 showed a high affinity to KAI (KD = 6.8 ± 0.6 nM), whereas other RTKs had negligible (KD > 200 nM) or no binding. All RTKs did not interact with ctrl. (D, E, F) The dose-dependent effect of KAI was tested by intravitreal injection of KAI 0.5 µg, 2 µg, and 10 µg after laser photocoagulation. Represent images of OCT and staining of the flat-mount of choroid/sclera with DyLight594-ILB4 were shown. Bars: 100 µm in OCT, 200 µm in ILB4 staining. N = 5 mice were used for PBS, KAI 0.5 µg, 2 µg, and 10 µg groups. Four laser burns were induced in each mouse, and the average neovascularization area per mouse was shown in graphs E and F. The cross-section of the CNV area in OCT (orange dashed line) was measured and plotted in graph E. *P = 0.045 one-way ANOVA, compared to PBS. (N = 4, 5, 3, 4 mice for PBS, 0.5 µg, 2 µg, 10 µg KAI, respectively. Mice with mild cataract due to anesthesia were unable to be monitored by OCT and were omitted from the measurement.) CNV surface area of the flat-mount was assessed by the area of ILB4-staining and plotted in graph F. *P = 0.034, one-way ANOVA. (N = 4, 4, 5, 5 mice in PBS, 0.5 µg, 2 µg, 10 µg KAI, respectively. Two choroidal tissues were damaged during the preparation of flat mount, and we were unable to assess the area of CNV.)