Novel Beta-Tubulin-Immobilized Nanoparticles Affinity Material for Screening β-Tubulin Inhibitors from a Complex...

In order to efficiently screen and isolate β-tubulin inhibitors, β-tubulin was immobilized on core-shell PMMA/CS (poly(methyl methacrylate)/Chitosan) nanoparticles to produce a new type of immobilized affinity material named β-tubulin-immobilized nanoparticles (β-TIN). The selectivity and adsorption performance of β-TIN were characterized using various control drugs. The β-TIN, the paclitaxel molecularly imprinted ploymers (MIP), and the C18 adsorbing material were compared for selectivity and enrichment ratio. Microtubule-targeting antitumor compounds were screened and isolated from a typical Chinese medicine, Chloranthus multistachys, by β-TIN. Three active compounds (curcolnol, zedoarofuran, and codonolactone) in Chloranthus multistachys extract were captured successfully. Microscale thermophoresis demonstrated that these three compounds strongly bind to β-tubulin, and the dissociation constants (K_d) between the three active compounds and β-tubulin were 1820 ± 0.68 nM, 1640 ± 0.52 nM, and 284 ± 1.00 nM, respectively. Moreover, the binding affinity between codonolactone and β-tubulin was greater than that between paclitaxel and β-tubulin. The antitumor activities of the three compounds were confirmed by the microtubule inhibition model, and the results showed a similar antitumor mechanism as paclitaxel. Molecular dynamics simulations were performed to preliminarily investigate the potential binding sites and the structure-activity relationship between the three active molecules and β-tubulin. Our study is the first to report the use of this novel material which is highly efficient in capturing low-content β-tubulin inhibitors from a complex mixture. The three screened compounds exhibited potential antineoplastic activity, and these lead compounds utilize a new mechanism of action with promising development prospects. Because β-TIN is easily prepared, displays excellent adsorption and selectivity for targets, and can effectively maintain the steric conformation and activities of target proteins, it will be very useful in the screening of lead compounds for different drug target proteins.