Sustainable Syntheses of (-)-Jerantinines A & E and Structural Characterisation of the Jerantinine-Tubulin Complex at the Colchicine Binding Site

Abstract

The jerantinine family of Aspidosperma indole alkaloids from Tabernaemontana corymbosa are potent microtubule-targeting agents with broad spectrum anticancer activity. The natural supply of these precious metabolites has been significantly disrupted due to the inclusion of T. corymbosa on the endangered list of threatened species by the International Union for Conservation of Nature. This report describes the asymmetric syntheses of (-)-jerantinines A and E from sustainably sourced (-)-tabersonine, using a straight-forward and robust biomimetic approach. Biological investigations of synthetic (-)-jerantinine A, along with molecular modelling and X-ray crystallography studies of the tubulin-(-)-jerantinine B acetate complex, advocate an anticancer mode of action of the jerantinines operating via microtubule disruption resulting from binding at the colchicine site. This work lays the foundation for accessing useful quantities of enantiomerically pure jerantinine alkaloids for future development.

Figure 1

A selection of Vinca and Aspidosperma alkaloids.

Figure 2

(A) Speculative biosynthetic pathway of jerantinine A and related vinca alkaloids (1) (B) Sustainable biomimetic semi-synthesis of (−)-jerantinine A (1) and (−)-jerantinine E (3), beginning from the proposed biogenetic precursor, (−)-tabersonine (4).

Figure 3

(A) Effect of natural and synthetic 1 on HCT-116 colony formation at GI₅₀ (see Table 1). Mean survival fraction (%) of treated cells as a percentage of the control population for HCT-116. Cells were seeded (400 per well) and allowed 24 h to attach before being challenged with jerantinine A (24 h). The number of colonies forming after 8 d incubation was determined. Mean (±S.E.M.) values are given. Data were generated from ≥3 separate trials; n = 2 per trial; (B) In vitro tubulin polymerisation in the presence of vehicle control, paclitaxel (5 μM; positive control), nocodazole (5 μM; negative control) or 1 (1 µM); (C) Immunofluorescent and DRAQ5 staining of x) untreated MCF-7 cells and y-z) cells exposed to synthetic 1 (1 × GI₅₀ = 0.81 μM; 24 h), showing the effect on microtubules (green) and the cellular DNA (purple). Synthetic (−)-jerantinine A (1) caused multinucleation (i), nuclear fragmentation (ii) and formation of multipolar spindles (iii).

Figure 4

(A) MDA-MB-231 and MCF-7 breast cancer cells grown in 3D culture with vehicle (DMSO), jerantinine A (1) (5.36 µM or 1.22 µM‚ respectively) or colchicine (10 µM or 14.5 µM‚ respectively). Images from 96 h post agent exposure. Scale bar represents 200 µm. (B) Quantification of the area of the MDA-MB-231 3D colonies over a 96 h period post agent addition. Mean (±S.E.M.) values are given. (C) Quantification of the area of the MCF-7 3D colonies over a 96 h period post agent addition. Mean (±S.E.M.) values are given. Data were generated from ≥2 separate trials; n = 2 per trial; *p < 0.05, **p < 0.01, ****p < 0.0001.

Figure 5

Crystal structure of the tubulin-jerantinine B acetate (9) complex. (A) Overall view of the T₂R-TTL-jerantinine B acetate complex structure. The α- and β-tubulin chains are in dark and light grey ribbon representation, respectively. The tubulin-bound 9 and GTP molecules are represented as green and orange spheres, respectively; (B) Close-up view of the interactions observed between 9 (green) and tubulin (grey). Interacting residues of tubulin are shown in stick representation and are labelled. Oxygen and nitrogen atoms are coloured red and blue, respectively; carbon atoms are in green 9 or grey (tubulin). Secondary structural elements of tubulin are labelled in blue. For simplicity, only α-tubulin residues are indicated with an α; (C) Superimposition of the tubulin-9 (green/grey) and the tubulin-colchicine (pink; PDB ID 4O2B; rmsd of 0.204 Å over 351 Cα-atoms) complex structures. The structures were superimposed onto their β1-tubulin chains; (D) Molecular docking of (−)-jerantinine A (1) in the colchicine binding pocket of tubulin; this pocket is formed by residues in the αT5-loop on α-tubulin, and βH7, βH8, βS8, βS9 and the βT7-loop on β-tubulin. For additional experimental details see the Supporting Information.