bi201380p_si_001.pdf (1.55 MB)
Cyclostreptin Derivatives Specifically Target Cellular Tubulin and Further Map the Paclitaxel Site
journal contribution
posted on 2012-01-10, 00:00 authored by Enrique Calvo, Isabel Barasoain, Ruth Matesanz, Benet Pera, Emilio Camafeita, Oriol Pineda, Ernest Hamel, Christopher
D. Vanderwal, José Manuel Andreu, Juan A. López, José Fernando DíazCyclostreptin is the first microtubule-stabilizing agent
whose mechanism of action was discovered to involve formation of a
covalent bond with tubulin. Treatment of cells with cyclostreptin
irreversibly stabilizes their microtubules because cyclostreptin forms
a covalent bond to β-tubulin at either the T220 or the N228
residue, located at the microtubule pore or luminal taxoid binding
site, respectively. Because of its unique mechanism of action, cyclostreptin
overcomes P-glycoprotein-mediated multidrug resistance in tumor cells.
We used a series of reactive cyclostreptin analogues, 6-chloroacetyl-cyclostreptin,
8-chloroacetyl-cyclostreptin, and [14C-acetyl]-8-acetyl-cyclostreptin, to characterize the cellular target of
the compound and to map the binding site. The three analogues were
cytotoxic and stabilized microtubules in both sensitive and multidrug
resistant tumor cells. In both types of cells, we identified β-tubulin
as the only or the predominantly labeled cellular protein, indicating
that covalent binding to microtubules is sufficient to prevent drug
efflux mediated by P-glycoprotein. 6-Chloroacetyl-cyclostreptin, 8-chloroacetyl-cyclostreptin,
and 8-acetyl-cyclostreptin labeled both microtubules and unassembled
tubulin at a single residue of the same tryptic peptide of β-tubulin
as was labeled by cyclostreptin (219-LTTPTYGDLNHLVSATMSGVTTCLR-243),
but labeling with the analogues occurred at different positions of
the peptide. 8-Acetyl-cyclostreptin reacted with either T220 or N228,
as did the natural product, while 8-chloroacetyl-cyclostreptin formed
a cross-link to C241. Finally, 6-chloroacetyl-cyclostreptin reacted
with any of the three residues, thus labeling the pathway for cyclostreptin-like
compounds, leading from the pore where these compounds enter the microtubule
to the luminal binding pocket.