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 α/β-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 β-tubulin at a previously unknown ligand site, rather than on α-tubulin as suggested in earlier studies.

Introduction

The success of taxanes in cancer treatment demonstrates the relevance of microtubules as a therapeutic target; however, numerous suboptimal pharmacological properties of these compounds have spurred the discovery and development of taxoid mimetics. While the taxoid binding site appears able to accommodate a growing variety of chemical scaffolds,¹ distinct and separately addressable microtubule stabilization sites may have greater potential to overcome clinical challenges such as chemoresistivity and toxicity, in part through combination therapy² and reduced reliance on toxic drug solubilizers3, 4, 5. Peloruside A⁶ and laulimalide⁷ are two compounds that may offer the foundation for a new generation of therapeutics with these characteristics.

The macrolide peloruside A possesses an activity profile similar to that of the taxanes in that cells are arrested in G2/M phase and undergo apoptosis.⁸ It appears to occupy a site distinct from the taxanes⁹ and synergize with other microtubule-stabilizing agents (MSAs) at the level of tubulin assembly10, 11 and function in cell proliferation assays.¹² Most interestingly, peloruside A retains activity in cell lines overexpressing P-glycoprotein and in those with induced resistance to taxol and the taxoid mimic epothilone A.⁹ This is also true for laulimalide.7, 13 The two compounds have been shown to compete in binding assays,⁹ suggesting they bind to the same or an overlapping site.

For these ligands, the existence of a distinct binding site raises the possibility of a unique mechanism of microtubule stabilization. Typically, the α–β tubulin dimer self-assembles into a tubular arrangement of 13 head-to-tail protofilaments in vivo, although this number is dependent on many factors in vitro.14, 15 A dimeric unit binds two molecules of guanosine 5′-triphosphate (GTP), one at a non-exchangeable site between α- and β-tubulin, and the other at an exchangeable site on β-tubulin. In the assembled state, the hydrolysis of GTP at the exchangeable site introduces significant lattice strain, which manifests through stochastic depolymerizations.¹⁶ During these events, the protofilaments peel away, most likely due to the inherent outward curvature in the dimeric unit.¹⁷ The mechanism by which MSAs such as taxol alleviate this strain is thought to involve contributions from improved longitudinal contacts along the protofilament as well as lateral contacts between protofilaments.¹⁸ Thus, a unique MSA binding site may alter the relative importance of these contacts and a comparative study of MSAs should shed light on the mechanisms of stabilization overall.

Much of our molecular-level understanding of MSA-induced stability has derived from cryoelectron microscopy of MSA and zinc-stabilized tubulin sheets, rather than from microtubules themselves.¹⁸ Unfortunately, creating stable zinc sheets has not been successful for laulimalide-treated tubulin and presumably would also fail for peloruside.¹⁹ In any case, it may be advantageous to apply a technique that can offer a differential analysis of microtubules, in both MSA-free and MSA-stabilized forms, to derive a picture of the structural impact of stabilization and identify the peloruside binding site. As an alternative to electron crystallographic data, we propose to use data from hydrogen–deuterium exchange mass spectrometry (HDX-MS). In the HDX-MS method, deuteration levels are determined from enzymatically generated peptides via liquid chromatography–mass spectrometry (LC-MS) technology.²⁰ This technique has been used to study protein polymers such as actin²¹ and, most recently, also microtubules.²² HDX-MS is a very sensitive probe of fluctuations in hydrogen-bonding networks, reflective of protein dynamics around a low-energy structure and commonly described in terms of “tightening” or “loosening”.²³ At a minimum, this method is useful for interrogating similarities and differences in the modes of microtubule stabilization induced by different ligands.²² Furthermore, while it is simplistic to correlate ligand-induced alterations in deuteration with a “footprint” of a binding site, it may be reasonable to expect that the footprint is at least partially represented by the ligand-altered deuteration levels. Non-covalent protein–ligand interactions are characterized by an enthalpy/entropy compensation phenomenon,²⁴ which can be expected to influence labelling at the binding site provided that the underlying secondary structure offers sufficient dynamic range in hydrogen-bond fluctuations. For example, HDX-MS studies of ligand-bound peroxisome proliferator-activated receptor γ have shown that the binding pocket is represented in the full set of structural stabilizations measured by the technique.25, 26 This partial correspondence has encouraged the use of HDX-MS data for scoring simulations of protein–protein interactions,²⁷ but this approach has not yet been applied to the discovery of binding sites for low molecular weight compounds.

In the present study, we discuss the mechanistic basis for microtubule stabilization imparted by epothilone A, docetaxel, and peloruside A. This work demonstrates a previously unknown mechanism that involves reduced reliance on lateral contacts between protofilaments. We then demonstrate a correlation between the HDX-MS data and known taxoid-site molecules (epothilone A and docetaxel), and on the basis of this evidence describe a coarse localization of the peloruside A binding site using HDX-MS data. We then apply a data-directed ligand-docking strategy to suggest a high-resolution model of the peloruside A binding site.