title: Interactions of halichondrin B and eribulin with tubulin.
abstract: Compounds that modulate microtubule dynamics include highly effective anticancer drugs, leading to continuing efforts to identify new agents and improve the activity of established ones. Here, we demonstrate that [(3)H]-labeled halichondrin B (HB), a complex, sponge-derived natural product, is bound to and dissociated from tubulin rapidly at one binding site per αβ-heterodimer, with an apparent K(d) of 0.31 μM. We found no HB-induced aggregation of tubulin by high-performance liquid chromatography, even following column equilibration with HB. Binding of [(3)H]HB was competitively inhibited by a newly approved clinical agent, the truncated HB analogue eribulin (apparent K(i), 0.80 μM) and noncompetitively by dolastatin 10 and vincristine (apparent K(i)'s, 0.35 and 5.4 μM, respectively). Our earlier studies demonstrated that HB inhibits nucleotide exchange on β-tubulin, and this, together with the results presented here, indicated the HB site is located on β-tubulin. Using molecular dynamics simulations, we determined complementary conformations of HB and β-tubulin that delineated in atomic detail binding interactions of HB with only β-tubulin, with no involvement of the α-subunit in the binding interaction. Moreover, the HB model served as a template for an eribulin binding model that furthered our understanding of the properties of eribulin as a drug. Overall, these results established a mechanistic basis for the antimitotic activity of the halichondrin class of compounds.
authors.affiliation: Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute at Frederick, National Institutes of Health, Frederick, Maryland 21702, United States.
authors.forename: Ruoli
authors.initials: R
authors.lastname: Bai
authors.forename: Tam Luong
authors.initials: TL
authors.lastname: Nguyen
authors.forename: James C
authors.initials: JC
authors.lastname: Burnett
authors.forename: Onur
authors.initials: O
authors.lastname: Atasoylu
authors.forename: Murray H G
authors.initials: MH
authors.lastname: Munro
authors.forename: George R
authors.initials: GR
authors.lastname: Pettit
authors.forename: Amos B
authors.initials: AB
authors.lastname: Smith
authors.forename: Rick
authors.initials: R
authors.lastname: Gussio
authors.forename: Ernest
authors.initials: E
authors.lastname: Hamel
mesh_terms: D000818:Animals; D050256:Antimitotic Agents / Q000378:metabolism*; D001665:Binding Sites; D002417:Cattle; D004988:Ethers, Cyclic / Q000378:metabolism*; D005663:Furans / Q000378:metabolism*; D007659:Ketones / Q000378:metabolism*; D018942:Macrolides; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D011161:Porifera; D011485:Protein Binding; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism*; D000095662:Polyether Polyketides
chemical_list: D050256:Antimitotic Agents; D004988:Ethers, Cyclic; D005663:Furans; D007659:Ketones; D018942:Macrolides; D014404:Tubulin; C070519:halichondrin B; D000095662:Polyether Polyketides; C490954:eribulin