A Unique Mode of Microtubule Stabilization Induced by Peloruside A

Abstract

Microtubules are significant therapeutic targets for the treatment of cancer, where suppression of microtubule dynamicity by drugs such as paclitaxel forms the basis of clinical efficacy. Peloruside A, a macrolide isolated from New Zealand marine sponge Mycale hentscheli, is a microtubule-stabilizing agent that synergizes with taxoid drugs through a unique site and is an attractive lead compound in the development of combination therapies. We report here unique allosteric properties of microtubule stabilization via peloruside A and present a structural model of the peloruside-binding site. Using a strategy involving comparative hydrogen-deuterium exchange mass spectrometry of different microtubule-stabilizing agents, we suggest that taxoid-site ligands epothilone A and docetaxel stabilize microtubules primarily through improved longitudinal interactions centered on the interdimer interface, with no observable contributions from lateral interactions between protofilaments. The mode by which peloruside A achieves microtubule stabilization also involves the interdimer interface, but includes contributions from the alpha/beta-tubulin intradimer interface and protofilament contacts, both in the form of destabilizations. Using data-directed molecular docking simulations, we propose that peloruside A binds within a pocket on the exterior of beta-tubulin at a previously unknown ligand site, rather than on alpha-tubulin as suggested in earlier studies.

Hallmarks of Molecular Action of Microtubule Stabilizing Agents

“…This difference appears to be related to the binding modes of the drugs in CET, which are also characterized in the present study, and is, therefore, consistent with the previously observed synergistic activities of different MSA combinations (19 -22). Furthermore, we find a significant deviation between the binding modes and the stabilizing activities of MSAs in CET as compared with those reported in BBT (16). As the major difference between the two sources of tubulin is the isotype composition, our results emphasize the importance of tubulin isotype content on drug binding and MT stabilization.…”

“…Several possibilities have been proposed for where this alternative site may be. Although computational studies based on NMR suggested that the most likely binding site for laulimalide and peloruside A is in ␣-tubulin (23), hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments similar to those utilized in the current study proposed a binding site adjacent to the taxane pocket in the ␤-tubulin subunit (16). The latter work also proposed a distinct mode for MT stabilization for peloruside A, with relaxation of intradimer contacts and ␤-␤ interactions across the lateral interface accompanying binding.…”

Docking and molecular dynamics studies of the binding between Peloruside A and tubulin

“…The binding sites 1 and 2 are located in b-tubulin, while binding site 3 is in a-tubulin. The binding site 1 consists of b-tubulin residues Gln291, Ala296, Lys336 and Asn337, and it is basically in agreement with the alternative site previously reported 13,14 . The binding site 2 is made up of b-tubulin residues Asp224, His227, Thr274, Arg276, Arg282 and Gly360, and it just located in the well-known taxane site.…”

“…In docking mode 1, the binding site is composed of b-tubulin residues Pro287, Thr290, Gln291, Gln292, Phe294, Asp295, Ala296, Pro305, Arg306, Gln329, Asn332, Val333, Lys336, Asn337 and Tyr340. It is worth the whistle, the previous experimental study has reported that the candidate region for PLA-binding site, being called the alternative site 14 , is composed of peptides b294-301 (H9-H9 0 loop), b302-314 (H9 0 -S8) and b332-340 (H10 loop) detected by HDX-MS 13 . It is easy to find that the most key residues (Phe294, Asp295, Ala296, Pro305, Arg306, Asn332, Val333, Lys336, Asn337 and Tyr340) of the binding site 1 predicted by the docking appear in the alternative site.…”

“…The initial studies through molecular docking and TR-NOESY nuclear magnetic resonance (NMR) proposed that the binding site of PLA was on a-tubulin 11,12 . However, Huzil et al 13 proposed a specific PLA-binding site that was adjacent to the taxane-binding site in the b-tubulin by using hydrogen-deuterium exchange mass spectrometry (HDXMS). As a matter of fact, the PLA-binding site proposed by Huzil et al is also called the alternative site.…”