Tubulin photoaffinity labeling study with a plinabulin chemical probe possessing a biotin tag at the oxazole.

A new bioactive photoaffinity probe KPU-252-B1 (4) possessing a biotin tag on the oxazole ring of a potent plinabulin derivative KPU-244 (2) was synthesized via the Cu(I)-catalyzed Huisgen's cycloaddition reaction to understand the precise binding mode of the diketopiperazine-based anti-microtubule agent plinabulin on tubulin. Probe 4 showed significant binding affinity toward tubulin and cytotoxicity against an HT-29 cells. A photoaffinity labeling study suggested that probe 4 specifically recognizes tubulin at a binding site that binds plinabulin or colchicine, most likely near or at the colchicine binding site, which is located at the interfacial region formed by α-and β-tubulin association. The results also demonstrated that probe 4 may serve as a useful plinabulin chemical probe to investigate the molecular mechanism by which anti-microtubule diketopiperazine derivatives operate.