Zampanolide Binding to Tubulin Indicates Cross-Talk of Taxane Site with Colchicine and Nucleotide Sites

Abstract

The marine natural product zampanolide and analogues thereof constitute a new chemotype of taxoid site microtubule-stabilizing agents with a covalent mechanism of action. Zampanolide-ligated tubulin has the switch-activation loop (M-loop) in the assembly prone form and, thus, represents an assembly activated state of the protein. In this study, we have characterized the biochemical properties of the covalently modified, activated tubulin dimer, and we have determined the effect of zampanolide on tubulin association and the binding of tubulin ligands at other binding sites. Tubulin activation by zampanolide does not affect its longitudinal oligomerization but does alter its lateral association properties. The covalent binding of zampanolide to β-tubulin affects both the colchicine site, causing a change of the quantum yield of the bound ligand, and the exchangeable nucleotide binding site, reducing the affinity for the nucleotide. While these global effects do not change the binding affinity of 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one (MTC) (a reversible binder of the colchicine site), the binding affinity of a fluorescent analogue of GTP (Mant-GTP) at the nucleotide E-site is reduced from 12 ± 2 × 10 M in the case of unmodified tubulin to 1.4 ± 0.3 × 10 M in the case of the zampanolide tubulin adduct, indicating signal transmission between the taxane site and the colchicine and nucleotide sites of β-tubulin.

Crystal Structure of the Cyclostreptin-Tubulin Adduct: Implications for Tubulin Activation by Taxane-Site Ligands

“…Our high-resolution structure of cyclostreptin-bound tubulin together with new MS-HPLC experiments performed at a higher resolution challenge our previous hypothesis of cyclostreptin binding to βAsn228 and βThr220 [6] and instead demonstrates that cyclostreptin unequivocally interacts with βHis229, a residue which is known to be responsible for the lack of sensitivity of yeast tubulin to paclitaxel [30]. In fact, given the key role of βAsn228 in stabilizing the guanine nucleotide at the E-site with two hydrogen bonds, a cyclostreptin interaction with this residue would severely affect nucleotide binding, which is not the case [31]. There is also no evidence supporting the βThr220 adduct we observed previously [6].…”

Beyond the Paclitaxel and Vinca Alkaloids: Next Generation of Plant-Derived Microtubule-Targeting Agents with Potential Anticancer Activity

“…Extensive studies showed that taccalanolides AF and AJ covalently bind to the taxane-site on β-tubulin [25,27]. Notably, to date, only three other microtubule stabilizing agents, zampanolide, dactylolide, and cyclostreptin, have been reported to react covalently with tubulin [28,29]. Taccalonolide AJ covalently interacted with tubulin in a similar manner to cyclostreptin [27].…”

Antitubulin sulfonamides: The successful combination of an established drug class and a multifaceted target

“…Apart from the taxanes (paclitaxel, docetaxel, and cabazitaxel, and an albumin bound paclitaxel nano‐droplet formulation [Abraxane®]) only the aza‐epothilone B analog ixabepilone has been approved . Recently, drugs covalently binding to the taxoid site of tubulin (in particular to aminoacids H229β, N228β, and D226β lining one side of H7) such as zampanolide, dactylolide, and taccalonolide AJ have also appeared, but despite the potential benefits of a different mechanism of action, longer lifetimes, and less clearance, they have not yet reached the clinic.…”