Cevipabulin-tubulin complex reveals a novel agent binding site on α-tubulin with tubulin degradation effect

Abstract Microtubule, composed of αβ-tubulin heterodimers, remains as one of the most popular anticancer targets for decades. To date, anti-microtubule drugs are mainly functionally divided into microtubule-destabilizing and microtubule-stabilizing agents while microtubule- or tubulin-degradation agents are rarely reported. Six known binding sites on tubulin dimer are identified with five sites on β-tubulin and only one site on α-tubulin (pironetin site), hinting compounds binding to α-tubulin are less well characterized. Cevipabulin, a microtubule-active antitumor clinical candidate, is widely accepted as a microtubule-stabilizing agent by binding to the vinblastine site. Our X-ray crystallography study reveals that, in addition binding to the vinblastine site, cevipabulin also binds to a novel site on α-tubulin (named the seventh site) which located near the nonexchangeable GTP. Interestingly, we find the binding of cevipabulin to the seventh site induces tubulin degradation. As the non-exchangeable GTP has structural role and is important for the stability of tubulin dimers, we propose and confirm the tubulin degradation mechanism as: Cevipabulin at the seventh site puts the αT5 loop outward to make the non-exchangeable GTP exchangeable, which reduces the stability of tubulin and results in its destabilization and degradation. Our results confirm a novel agent binding site on α-tubulin and shed light on the development of tubulin degraders as a new generation of anti-microtubule drugs targeting this novel site.