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 C 2 -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. (-)-Lomaiviticin A (1) possesses half-maximal inhibitory potencies (IC 50 s) in the low nanomolar-picomolar range against several cultured cancer cell lines (1, 2). (-)-Lomaiviticin C (3) and (-)-kinamycin C (4) are several orders of magnitude less potent, whereas (-)-lomaiviticin B (2) is ∼10-100-fold less potent. The cytotoxicity of 1 derives from the induction of double-strand breaks (DSBs) in DNA (18,19). K562 cells exposed to 5 nM of 1 for 30 min accumulated DSB levels that were comparable to 40 Gy of ionizing radiation. This DNA cleavage activity is not recapitulated by 3 or 4, suggesting both diazofluorenes of 1 are essential for cleavage activity. DNA DSBs are exceedingly cytotoxic (20), and these data provide an explanation for the remarkable potency of 1.The molecular mechanism of DNA DSB induction by (-)-lomaiviticin A (1) has been studied (18). Cell-free deuteriumlabeling experiments are consistent with a pathway comprising reductive activation of one diazofluorene (Fig. 1B) to generate the vinyl radical intermediate 1•, followed by hydrogen atom abstraction from the deoxyribose chain, a process known to lead to singlestrand breaks (SSBs) (21). Reductive activation of the remaining diazofluorene, followed by hydrogen atom abstraction, is believed to cleave the complementary DNA (cDNA) strand, leading to the observed DSB.However, the mode of interaction of (-)-lomaiviticin A (1) with DNA has not been elucidated. As C(sp 2 ) radicals are high-energy species, the mechanistic model outlined above pr...

Synergistic potentiation of (−)-lomaiviticin A cytotoxicity by the ATR inhibitor VE-821

“…We monitored the production and colocalization of phosphor-SER139 H2AX (γH2AX) 31 and 53BP1 32 foci as an indicator of DSB formation, as previously described. 2a,2b,8d In the presence of 1 alone (up to 250 pM) γH2AX and 53BP1 foci were observed, as expected. However, in the presence of 7 (10 μM), pan-nuclear staining of nuclear γH2AX and the formation of apoptotic bodies (also displaying pan-staining) was observed, which is indicative of apoptosis.…”

“…1 The exquisite cytotoxicity of 1 derives from induction of DNA SSBs and DSBs in tissue culture. 2 The kinetics of DNA double-strand break induction and PI3KKs pathways-dependent phosphorylation of histone H2AX by 1 have been analyzed. 2b Reduction of histone H2AX phosphorylation induced by 1 was observed in K562 cells pretreated with inhibitors (KU55933, 3 caffeine, 4 and NU-7441 5 ) of the major PI3KKs ATM, ATR, and DNA, respectively.…”

“…2 The kinetics of DNA double-strand break induction and PI3KKs pathways-dependent phosphorylation of histone H2AX by 1 have been analyzed. 2b Reduction of histone H2AX phosphorylation induced by 1 was observed in K562 cells pretreated with inhibitors (KU55933, 3 caffeine, 4 and NU-7441 5 ) of the major PI3KKs ATM, ATR, and DNA, respectively. In addition, 1 displayed selective toxicity toward BRCA2-, PTEN-, KU80-, DNA-PKcs, and ATM-deficient cell lines, all of which have been implicated in DSB repair.…”

“…In addition, 1 displayed selective toxicity toward BRCA2-, PTEN-, KU80-, DNA-PKcs, and ATM-deficient cell lines, all of which have been implicated in DSB repair. 2a BRCA2-deficient VC8 and PTEN-deficient U251 cell lines are particularly sensitive to 1 (LC 50 = 1.5 ± 0.5 and 2.0 ± 0.6 pM, respectively) with selectivities of >11.6 versus the isogenic cell lines transfected with and expressing functional BRCA2 and PTEN genes. Collectively, these results indicate activation of non-homologous end joining (NHEJ) 6 and homologous recombination (HR) 7 repair, the two canonical pathways by which DNA DSBs are ameliorated, in cells treated with 1 and suggest acceptable therapeutic indices may be attainable when 1 is applied toward NHEJ- or HR-deficient tumors.…”

“…As the cytotoxicity of 1 has been shown to derive from induction of DSBs in DNA, 2 we conducted a neutral comet unwinding assay 30 to probe for an increase in DSBs in cells treated with 1 and VE-821 ( 7 ). Consistent with the cell viability studies above (Figure 2B), treatment of K562 cells with 50–500 pM 1 alone did not lead to measurable levels of DSB production, as compared to control (Figure 4).…”