Identification of anziaic acid, a lichen depside from Hypotrachyna sp., as a new topoisomerase poison inhibitor

Abstract

Topoisomerase inhibitors are effective for antibacterial and anticancer therapy because they can lead to the accumulation of the intermediate DNA cleavage complex formed by the topoisomerase enzymes, which trigger cell death. Here we report the application of a novel enzyme-based high-throughput screening assay to identify natural product extracts that can lead to increased accumulation of the DNA cleavage complex formed by recombinant Yersinia pestis topoisomerase I as part of a larger effort to identify new antibacterial compounds. Further characterization and fractionation of the screening positives from the primary assay led to the discovery of a depside, anziaic acid, from the lichen Hypotrachyna sp. as an inhibitor for both Y. pestis and Escherichia coli topoisomerase I. In in vitro assays, anziaic acid exhibits antibacterial activity against Bacillus subtilis and a membrane permeable strain of E. coli. Anziaic acid was also found to act as an inhibitor of human topoisomerase II but had little effect on human topoisomerase I. This is the first report of a depside with activity as a topoisomerase poison inhibitor and demonstrates the potential of this class of natural products as a source for new antibacterial and anticancer compounds.

Figure 1. Isolation tree of anziaic acid.

Figure 2. Inhibition of E. coli topoisomerase I relaxation activity by lichen extracts and fractions.

(a) Secondary assay of natural product hits from HTS using negatively supercoiled DNA plasmid substrate. Lane 1: no enzyme. Relaxation activity of 10 ng of E. coli topoisomerase I was assayed in the presence of DMSO (lane 2) or 80 µg/mL of natural product extracts hits (lane 3–12). Extracts PL2050/C13 and PL2050/D12 prepared from lichen Hypotrachyna sp. samples were present in lanes 7 and 8 respectively. (b) Assay of HP20ss fractionated extracts of lichen Hypotrachynasp. samples. Lane 1: no enzyme control. Lane 2: Enzyme with DMSO control. Lanes 3–20: serial 4-fold dilutions (12, 3, 0.75 µg/mL) of unfractionated total extract (lanes 3–5), Fraction 1 (lanes 6–8), Fraction 2 (lanes 9–11), Fraction 3 (lanes 12–14), Fraction 4 (lanes 15–17), Fraction 5 (lanes 18–20). S: supercoiled plasmid DNA substrate. N: nicked plasmid DNA. Ro: Relaxed closed plasmid DNA. PR: partially relaxed plasmid DNA.

Figure 3. Effect of lichen Hypotrachyna sp. HP20ss fractions and anziaic acid on cleavage product accumulation by E. coli topoisomerase I.

Single-stranded DNA substrate labeled at the 5′-end with ³²P (S) was incubated with enzyme in the presence of 2 mM MgCl₂. No enzyme (lane 1), enzyme with DMSO (lane 2). a. Unfractionated total extract (lanes 3, 4), fraction 3 (lanes 5, 6), fraction 4 (lanes 7, 8), fraction 5 (lanes 9, 10) with the extract or fraction compounds present at 3.1 µg/mL (lanes 3, 5, 7, 9) or 12.5 µg/mL (lanes 4, 6, 8, 10). b. Anziaic acid was present at 0.9 µM (lanes 3), 1.8 µM (lanes 4), 3.7 µM (lanes 5) and 7.2 µM (lanes 6).

Figure 4. Inhibition of Relaxation activity of E. coli topoisomerase I by anziaic acid.

Assay was carried out in the presence of DMSO or the indicated concentration of anziaic acid with (a) 0.5 mM MgCl₂, (b) 0.5 mM MgCl₂+0.0025% Tween 20, (c) 6 mM MgCl₂, (d) 6 mM MgCl₂+0.0025% Tween 20. -: no enzyme control.

Figure 5. Inhibition of Relaxation activity of Y. pestis topoisomerase I by anziaic acid in the presence of different concentrations of Mg²⁺.

Assays were carried out in the presence of 0.0025% Tween 20 and MgCl₂ concentration of 0.5 mM (a) or 6 mM (b). -: no enzyme control. Anziaic acid is present at the concentration indicated.

Figure 6. Effect of anziaic acid on type IB human topoisomerase I activity.

(a) Assay of relaxation activity –1 U of enzyme was used in each relaxation reaction with 250 ng of supercoiled plasmid DNA. (b) DNA cleavage assay with 5 U of enzyme. Gel electrophoresis was carried out in the presence of 0.5 µg/mL ethidium bromide and camptothecin was used as a positive control at 62.5 µM (C1) or 125 µM (C2). -: no enzyme. S: supercoiled plasmid DN substrate. N: nicked plasmid DNA. Ro: Relaxed closed plasmid DNA. PR: partially relaxed plasmid DNA. CC: Covalently closed circular DNA.

Figure 7. Effect of anziaic acid on E. coli DNA gyrase activity.

(a) Assay of supercoiling activity with 1 U of enzyme and 250 ng of relaxed plasmid DNA substrate. (b) DNA cleavage assay with 5 U of enzyme. Gel electrophoresis was carried out in the presence of 0.5 µg/mL ethidium bromide. Positive controls – F: 100 µM levofloxacin; N: 100 µM norfloxacin. S: supercoiled plasmid DNA substrate. N: nicked plasmid DNA. Ro: Relaxed closed plasmid DNA. PR: partially relaxed plasmid DNA. L: linear DNA CC: covalently closed circular DNA.

Figure 8. Effect of anziaic acid on human topoisomerase IIα activity.

(a) Assay of relaxation activity –1 U of enzyme was used in each relaxation reaction with 250 ng of supercoiled plasmid DNA in the presence of 0.0025% Tween 20. (b) DNA cleavage assay with 5 U of enzyme. Gel electrophoresis was carried out in the presence of 0.5 µg/mL ethidium bromide and m-AMSA was used as a positive control at 25 µM (A1) or 8 µM (A2). -: no enzyme. S: supercoiled plasmid DNA substrate. N: nicked plasmid DNA. Ro: Relaxed closed plasmid DNA. PR: partially relaxed plasmid DNA. L: linear DNA. CC: covalently closed circular DNA.