The Binding Mode of Side Chain‐ and C3‐Modified Epothilones to Tubulin

Abstract

The tubulin‐binding mode of C3‐ and C15‐modified analogues of epothilone A (Epo A) was determined by NMR spectroscopy and computational methods and compared with the existing structural models of tubulin‐bound natural Epo A. Only minor differences were observed in the conformation of the macrocycle between Epo A and the C3‐modified analogues investigated. In particular, 3‐deoxy‐ (compound 2) and 3‐deoxy‐2,3‐didehydro‐Epo A (3) were found to adopt similar conformations in the tubulin‐binding cleft as Epo A, thus indicating that the 3‐OH group is not essential for epothilones to assume their bioactive conformation. None of the available models of the tubulin–epothilone complex is able to fully recapitulate the differences in tubulin‐polymerizing activity and microtubule‐binding affinity between C20‐modified epothilones 6 (C20‐propyl), 7 (C20‐butyl), and 8 (C20‐hydroxypropyl). Based on the results of transferred NOE experiments in the presence of tubulin, the isomeric C15 quinoline‐based Epo B analogues 4 and 5 show very similar orientations of the side chain, irrespective of the position of the nitrogen atom in the quinoline ring. The quinoline side chain stacks on the imidazole moiety of β‐His227 with equal efficiency in both cases, thus suggesting that the aromatic side chain moiety in epothilones contributes to tubulin binding through strong van der Waals interactions with the protein rather than hydrogen bonding involving the heteroaromatic nitrogen atom. These conclusions are in line with existing tubulin polymerization and microtubule‐binding data for 4, 5, and Epo B.

Recent advances in microtubule-stabilizing agents

“…In 1996, Höfle et al [42][43] made a comprehensive report on the production, biological properties, crystal structure, physical and spectroscopic data, as well as conformations in a solution of epothilone A and B, which provided basic data for the further study of epothilones. In 2010, Altmann et al [44] investigated the interactions Waals interactions with the protein. In order to identify the molecular factors involved in the emergence of drug resistance induced by four tubulin mutations, Navarrete et al [45] investigated the structure and dynamic properties of epothilone-β-tubulin complexes with wild-type and mutated tubulin by using molecular dynamic simulations, which is helpful to conduct in-depth understanding on the epothilones' action mechanism and SAR.…”

Epothilones

“…In a second study, they also showed that the NMR-derived tubulinbound conformation of Epo A corresponds to a low energy conformation of the free ligand in aqueous solution. 155 Recent modeling studies by Botta and co-workers, 156 which were based on a previously developed pseudoreceptor model for b-tubulin, 148,149 also support the validity of the NMRderived bioactive conformation of epothilones (and, in addition, suggest the existence of a common pharmacophore between epothilones and taxol).…”

On the Importance of the Thiazole Nitrogen in Epothilones: Semisynthesis and Microtubule-Binding Affinity of Deaza-Epothilone C

“…On the other hand, we had been successful ourselves in the elaboration of ketone 2 into epothilone A analogs bearing modified thiazole, pyrimidine or pyridine moieties in place of the natural thiazole heterocycle by means of HWE chemistry (Hauenstein & Altmann, unpublished experiments; see also refs. [26,27]). Unfortunately, the attempted HWE coupling of 2 with phosphonate 4 did not yield any of the desired olefin (Scheme 1).…”