The Application of REDOR NMR to Understand the Conformation of Epothilone B

Abstract

The structural information of small therapeutic compounds complexed in biological matrices is important for drug developments. However, structural studies on ligands bound to such a large and dynamic system as microtubules are still challenging. This article reports an application of the solid-state NMR technique to investigating the bioactive conformation of epothilone B, a microtubule stabilizing agent, whose analog ixabepilone was approved by the U.S. Food and Drug Administration (FDA) as an anticancer drug. First, an analog of epothilone B was designed and successfully synthesized with deuterium and fluorine labels while keeping the high potency of the drug; Second, a lyophilization protocol was developed to enhance the low sensitivity of solid-state NMR; Third, molecular dynamics information of microtubule-bound epothilone B was revealed by high-resolution NMR spectra in comparison to the non-bound epothilone B; Last, information for the macrolide conformation of microtubule-bound epothilone B was obtained from rotational-echo double-resonance (REDOR) NMR data, suggesting the X-ray crystal structure of the ligand in the P450epoK complex as a possible candidate for the conformation. Our results are important as the first demonstration of using REDOR for studying epothilones.

Keywords: REDOR; bioactive conformation; epothilone B; microtubules; solid-state NMR.

Figure 1

(A) The chemical structures of epothilone A and B; (B) The binding modes of epothilone A (PDB 4I50, blue) and B (PDB 1Q5D, magenta), the spatial coordinates being arbitrary and three-dimensionally rotated for the two conformations to overlap.

Figure 2

Retrosynthesis of analog 3.

Figure 3

Solid-state magic-angle spinning (MAS) NMR spectra of analog 3, microtubule-bound (A,C) or dispersed in PIPES buffer (B,D). (A) Experimental (black) and simulated (red; shifted left for clarity) ²H spectra with the calculated quadrupolar parameters displayed, 81,910 scans at 0.5 s repetition; (B) Experimental ²H spectrum; (C) Experimental ¹⁹F spectrum, 2560 scans at 50 s repetition; (D) Experimental ¹⁹F spectrum. All the spectra were acquired at ambient temperature with Hahn-Echo without proton decoupling and the isotropic resonances are marked on the spectra.

Figure 4

(A) The ²H{¹⁹F} REDOR spectra (S₀ and ΔS) of 2-fluoro-2-methyl-d₃-malonic acid ([2-F,2-Me-d₃]MA) with the dephasing times marked on the corresponding spectra; (B) ²H{¹⁹F} REDOR curves (ΔS/S₀), where the experimental dephasing (•) is consistent with the SIMPSON calculation for a single distance of 3.6 (±0.2) Å. The NMR spectra were acquired at ambient temperature.

Figure 5

²H{¹⁹F} REDOR spectra of the microtubule-bound analog 3 as a function of dipolar evolution with magic-angle spinning at 13,000 Hz, full-echo (S₀), dephased-echo (S), and difference (ΔS) spectra. Dipolar evolution times are marked in multiples of the rotor period (T_r). The acquisition scans for the 2, 8, 16, and 24T_r experiments were 81,920, 163,840, 163,840, and 368,640, respectively. The NMR experiments were performed at ambient temperature.

Figure 6

Comparison of the experimental REDOR data with protein-bound conformations of epothilones in the literature. (A) Cytochrome P450epoK-bound epothilone B (PDB 1Q5D); (B) T₂R-TTL-EpoA complex (PDB 4I50); (C) Tubulin-bound epothilone A [20]; (D) Tubulin-bound epothilone A in zinc-stabilized 2D sheets (PDB 1TVK); and (E) The SIMPSON curves of ²H{¹⁹F} REDOR dephasing calculated as a function of the dipolar evolution time for the above four conformers. For a systematic comparison, 26-methyl was added to the known structures of epothilone A (B–D). A shorter distance between the 26-fluoromethyl to the germinal trideuteriomethyl groups (C-22 and C-23) is displayed by dotted lines with the marked value. The error bars shown in the difference (ΔS) spectra (E) represent one standard deviation of the noise levels from the peak maxima.

Figure 7

Hydrogen bond interactions of the epothilone B conformer epoB(TUB)_1q5d with amino residues in tubulin dimer. Oxygen, nitrogen, sulfur, and hydrogen are indicated in red, blue, yellow, and white, respectively. Carbon in the drug molecule and tubulin protein are indicated magenta and grey, respectively. Hydrogen bonds are indicated by dotted lines, and their lengths are given.