Abstract
It has been proposed that one of the mechanisms of taxane-site ligand-mediated tubulin activation is modulation of the structure of a switch element (the M-loop) from a disordered form in dimeric tubulin to a folded helical structure in microtubules. Here, we used covalent taxane-site ligands, including cyclostreptin, to gain further insight into this mechanism. The crystal structure of cyclostreptin-bound tubulin reveals covalent binding to βHis229, but no stabilization of the M-loop. The capacity of cyclostreptin to induce microtubule assembly compared to other covalent taxane-site agents demonstrates that the induction of tubulin assembly is not strictly dependent on M-loop stabilization. We further demonstrate that most covalent taxane-site ligands are able to partially overcome drug resistance mediated by βIII-tubulin (βIII) overexpression in HeLa cells, and compare their activities to pironetin, an interfacial covalent inhibitor of tubulin assembly that displays invariant growth inhibition in these cells. Our findings suggest a relationship between a diminished interaction of taxane-site ligands with βIII-tubulin and βIII tubulin-mediated drug resistance. This supports the idea that overexpression of βIII increases microtubule dynamicity by counteracting the enhanced microtubule stability promoted by covalent taxane-site binding ligands.
Highlights
Microtubule-based chemotherapy is one of the most effective treatment options for both solid tumors and hematological malignancies [1,2] and continues to be used even in combination with newInt
Those microtubules were subsequently depolymerized yielding tubulin dimers bound to cyclostreptin
We found that βHis229 is is modified by cyclostreptin in the β-tubulin-derived tryptic peptide containing the sequence modified by cyclostreptin in the β-tubulin-derived tryptic peptide containing the sequence
Summary
Microtubule-based chemotherapy is one of the most effective treatment options for both solid tumors and hematological malignancies [1,2] and continues to be used even in combination with new. Binding to the taxane-, the laulimalide-, or in the case of triazolopyrimidines, the vinca-site these sites are targeted by cancer chemotherapies: the maytansine It could be speculated that compounds targeting the taxane-site agents are extremely effective anticancer drugs when used as the warheads of tumor-targeting delivered with an antibody could be a promising ADC strategy with a higher therapeutic antibodies. Compounds that bind tubulin of action that can effectively engage the target, in spite of the lower concentrations delivered to the covalently are not susceptible to efflux by P-glycoprotein or other membrane efflux pumps due to tumor by this method. In the work described in this manuscript we have employed a multidisciplinary approach involving biochemical techniques and structural biology to understand how tubulin assembles into stable microtubules as a result of the binding of small molecules to the taxane-site. We utilize the different biochemical and structural properties of the covalent microtubule binders zampanolide, taccalonolide AJ, and cyclostreptin to reveal the structural basis for drug-induced microtubule assembly
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