Molecular Mechanism of Action of Microtubule-Stabilizing Anticancer Agents

Abstract

Microtubule-stabilizing agents (MSAs) are efficacious chemotherapeutic drugs widely used for the treatment of cancer. Despite the importance of MSAs for medical applications and basic research, their molecular mechanisms of action on tubulin and microtubules remain elusive. Here we determined high-resolution crystal structures of aß-tubulin in complex with two unrelated MSAs, zampanolide and epothilone A. Both compounds were bound to the taxane-pocket of ß-tubulin and used their respective side chain to induce structuring of the M-loop into a short helix. Because the M-loop establishes lateral tubulin contacts in microtubules, these findings explain how taxane-site MSAs promote microtubule assembly and stability. They further offer fundamental structural insights into the control mechanisms of microtubule dynamics. Here we determined high-resolution crystal structures of -tubulin in complex with two unrelated MSAs, zampanolide and epothilone A. Both compounds were bound to the taxanepocket of -tubulin and used their respective side chain to induce structuring of the M-loop into a short helix. Because the M-loop establishes lateral tubulin contacts in microtubules, these findings explain how taxane-site MSAs promote microtubule assembly and stability. They further offer fundamental structural insights into the control mechanisms of microtubule dynamics.One sentence summary:Microtubule-stabilizing agents use a common mechanism to stabilize a major loop in tubulin that controls microtubule assembly and stability. suggesting that binding of MSAs and TTL does not induce significant structural changes in the T 2 R complex. Both Zampa and EpoA were deeply buried in a pocket formed by predominantly hydrophobic residues of helix H7, -strand S7, and the loops H6-H7, S7-H9 (designated the Mloop (7)) and S9-S10 of -tubulin; this pocket is commonly known as the 'taxane-pocket' (8, 9)In the T 2 R-TTL-Zampa complex, the C9 atom of Zampa was covalently bound to the NE2 atom of His229 of -tubulin (Fig. S1B), which is consistent with mass spectrometry data (10). In addition, two hydrogen bonds were formed between the OH20 group and the O1' atom of Zampa, and the main chain carbonyl oxygen and the NH group of Thr276, respectively. In the T 2 R-TTL-EpoA complex, the O1, OH3, OH7 and N20 groups of EpoA were hydrogen bonded to atoms of residues Thr276 (main chain NH), Gln281 (side chain amide nitrogen), Asp226 (side chain oxygen) and Thr276 (side chain hydroxyl group) of -tubulin, respectively. The binding 4 mode of EpoA in the tubulin-EpoA structure is fundamentally different from the one proposed based on electron crystallography data of zinc-stabilized tubulin sheets (Fig. S2A); however, the orientation of the ligand in the taxane-pocket was ambiguous in the electron crystallography structure because the density of the ligand in experimental omit maps was discontinuous and limited in quality (9, 11). In contrast, the density of EpoA in our tubulin-EpoA X-ray crystal structure is very well defined and allowed the o...

Structural Insights into the Pharmacophore of Vinca Domain Inhibitors of Microtubules

“…MTAs are divided into two general categories: microtubule-stabilizing and -destabilizing agents (Hartley et al, 2012). Microtubule-stabilizing agents, represented by taxol-like compounds, bind to the lateral side of tubulin, stabilizing the M-loop of b-tubulin, which establishes the lateral contacts of tubulin in microtubules (Prota et al, 2013a). Microtubule-destabilizing agents bind to the longitudinal interface between the a/b-tubulin subunits, causing conformational shifts of consecutive a-, b-tubulin heterodimers from a straight to a curved form, facilitating microtubule disassembly (Ravelli et al, 2004;Gigant et al, 2005).…”

Peloruside A Inhibits Growth of Human Lung and Breast Tumor Xenografts in an Athymic nu/nu Mouse Model

“…Peloruside, another MSA under development as an anticancer agent, is particularly interesting because unlike the other newgeneration MSAs discussed above, peloruside binds to a unique, non-taxoid site on b-tubulin that it shares with another marine sponge natural product, laulimalide (10,(13)(14)(15). In addition, peloruside and laulimalide, similar to the epothilones and discodermolide, retain their activity in cells that overexpress the P-gp efflux pump (13,14), thus remaining active in cells that have acquired resistance to the taxanes by P-gp overexpression.…”

Antiproliferative Mechanism of Action of the Novel Taxane Cabazitaxel as Compared with the Parent Compound Docetaxel in MCF7 Breast Cancer Cells

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