Figure 6.

AOH1996 binds to tubulin and disrupts PCNA–γ-tubulin interactions. A, Tubulin polymerization assay performed in vitro using purified tubulin. AOH1996 partially inhibits polymerization relative to DMSO and paclitaxel (positive control) but more effectively than the inert analog AOH1996-8Nq and the microtubule-depolymerizing agent colcemid (negative control). B, SPR analysis of AOH1996 and AOH1996-8Nq binding to purified tubulin. Sensorgrams (top) and fitted binding curves (bottom) demonstrate that AOH1996 binds tubulin with higher affinity than AOH1996-8Nq. C, Apparent dissociation constants (Kd) derived from SPR data. AOH1996 binds tubulin with a mean Kd of ∼30 μmol/L, whereas AOH1996-8Nq shows modest, yet significantly weaker, binding with a mean Kd of ∼70 μmol/L. Graphs represent the mean and range of nine independent experiments. D, Co-immunoprecipitation (co-IP) of endogenous PCNA from HeLa lysates treated with or without AOH1996 or AOH1996-8Nq, followed by immunoblotting for α-, β-, or γ-tubulin. AOH1996 treatment reduces the interaction between PCNA and γ-tubulin by ∼50%, whereas AOH1996-8Nq has no effect. Normal rabbit IgG was used as a negative IP control. Right, quantification of γ-tubulin co-IP signal normalized to DMSO. Bar graphs represent the mean and range from at least two biological replicates. E, Dose–response curve from MTT assay shows reduced impact of AOH1996-8Nq (IC₅₀ value not reached) on HeLa viability compared with AOH1996-treated (IC₅₀ = 125 nmol/L) cells. Statistical comparisons were made using unpaired two-tailed t tests. **, P < 0.01.