Structural basis for DNA cleavage by the potent antiproliferative agent (-)-lomaiviticin A

Abstract

(-)-Lomaiviticin A (1) is a complex antiproliferative metabolite that inhibits the growth of many cultured cancer cell lines at low nanomolar-picomolar concentrations. (-)-Lomaiviticin A (1) possesses a C2-symmetric structure that contains two unusual diazotetrahydrobenzo[b]fluorene (diazofluorene) functional groups. Nucleophilic activation of each diazofluorene within 1 produces vinyl radical intermediates that affect hydrogen atom abstraction from DNA, leading to the formation of DNA double-strand breaks (DSBs). Certain DNA DSB repair-deficient cell lines are sensitized toward 1, and 1 is under evaluation in preclinical models of these tumor types. However, the mode of binding of 1 to DNA had not been determined. Here we elucidate the structure of a 1:1 complex between 1 and the duplex d(GCTATAGC)2 by NMR spectroscopy and computational modeling. Unexpectedly, we show that both diazofluorene residues of 1 penetrate the duplex. This binding disrupts base pairing leading to ejection of the central AT bases, while placing the proreactive centers of 1 in close proximity to each strand. DNA binding may also enhance the reactivity of 1 toward nucleophilic activation through steric compression and conformational restriction (an example of shape-dependent catalysis). This study provides a structural basis for the DNA cleavage activity of 1, will guide the design of synthetic DNA-activated DNA cleavage agents, and underscores the utility of natural products to reveal novel modes of small molecule-DNA association.

Keywords: DNA; NMR; double-strand break; natural product.

Fig. 1.

(A) Structures of (–)-lomaiviticins A–C (1–3), (–)-kinamycin C (4), and positional numbering of 1. (B) Reductive activation of lomaiviticin A (1) generates the vinyl radical 1•. Hydrogen atom abstraction is believed to initiate formation of an SSB. Reductive activation of the remaining diazofluorene, followed by hydrogen atom abstraction from the cDNA strand, induces DSB formation.

Fig. 2.

Equimolar FID assays using 1–4 and thiazole orange as the intercalator probe. Conditions: [1–4] = 0.88 μM, [thiazole orange] = 1.25 μM, [base pairs] = 0.88 μM, 1 h, 24 °C. Mean percent displacement from three experiments and error bars representing 1 SD from the mean are shown.

Fig. 3.

Selected regions of the ¹H NMR spectrum of d(GCTATAGC)₂ and (−)-lomaiviticin A (1; 0, 0.5, or 1 equiv, bottom to top). Green bars denote the site of binding. The 14.0–10.5-ppm spectra were obtained in 10% D₂O–90% H₂O using the Watergate scheme. The 8.7–6.5-ppm spectra were obtained in phosphate-buffered D₂O. Experimental conditions: 10 mM phosphate buffer (pH 7.5), 25 mM sodium chloride, 800 MHz, 24 °C. For additional spectroscopic data, see SI Appendix, Fig. S1.

Fig. 4.

NOE walk of the A/G H8→H1′(n-1) or T/C H6→H1′(n-1) region for (A) d(GCTATAGC)₂ only and (B) the complex between (−)-lomaiviticin A (1) and d(GCTATAGC)₂ (1:1). Breaks in the walk are indicated by circular nodes.

Fig. 5.

Solution structure of (–)-lomaiviticin A (1) complexed to 5′-G₁C₂T₃A₄T₅A₆G₇C₈-3′. (A) Full stereoview. Bases flipped out of the duplex are shown in green. (B) Intercalation site, front view. (C) Intercalation site, top view. A6 H5′, A6 H4′, A14 H1′, and T13 H4′ are shown as white balls in A and as green balls in B and C and are located 4.2, 4.3, 4.2, and 4.9 Å, respectively, from the nearest diazo carbon atom.