An engineered lipocalin that tightly complexes the plant poison colchicine for use as antidote and in bioanalytical applications.

摘要: Colchicine is a toxic alkaloid prevalent in autumn crocus (Colchicum autumnale) that binds to tubulin and inhibits polymerization of microtubules. Using combinatorial rational protein design, we have developed an artificial binding based on the human lipocalin 2 colchicine with dissociation constant 120 pm, i.e. 10000-fold stronger than tubulin. Crystallographic analysis engineered lipocalin, dubbed Colchicalin, revealed major structural changes flexible loop region forms ligand pocket at open end eight-stranded β-barrel, resulting lid-like structure over deeply buried colchicine. A cis-peptide bond between residues Phe71 Pro72 #2 constitutes peculiar feature allows intimate contact tricyclic ligand. directed evolution, achieved extraordinary half-life more 9 h for Colchicalin-colchicine complex. Together chemical robustness availability activated derivatives, this also opens applications as general-purpose affinity reagent, including facile quantification biological samples. Given lipocalins, known Anticalin® proteins, represent class clinically validated biopharmaceuticals, Colchicalin may offer therapeutic antidote scavenge reverse its poisoning effect situations acute intoxication.