Newly identified antibacterial compounds are topoisomerase poisons in African trypanosomes

Abstract

Human African trypanosomiasis, caused by the Trypanosoma brucei protozoan parasite, is fatal when left untreated. Current therapies are antiquated, and there is a need for new pharmacologic agents against T. brucei targets that have no human ortholog. Trypanosomes have a single mitochondrion with a unique mitochondrial DNA, known as kinetoplast DNA (kDNA), a topologically complex network that contains thousands of interlocking circular DNAs, termed minicircles (approximately 1 kb) and maxicircles (approximately 23 kb). Replication of kDNA depends on topoisomerases, enzymes that catalyze reactions that change DNA topology. T. brucei has an unusual type IA topoisomerase that is dedicated to kDNA metabolism. This enzyme has no ortholog in humans, and RNA interference (RNAi) studies have shown that it is essential for parasite survival, making it an ideal drug target. In a large chemical library screen, two compounds were recently identified as poisons of bacterial topoisomerase IA. We found that these compounds are trypanocidal in the low micromolar range and that they promote the formation of linearized minicircles covalently bound to protein on the 5' end, consistent with the poisoning of mitochondrial topoisomerase IA. Surprisingly, however, band depletion studies showed that it is topoisomerase IImt, and not topoisomerase IAmt, that is trapped. Both compounds are planar aromatic polycyclic structures that intercalate into and unwind DNA. These findings reinforce the utility of topoisomerase IImt as a target for development of new drugs for African sleeping sickness.

FIG. 1.

Molecular structure of 1895 (A), which is N,N-dimethyl-2-(phenanthro[3,4-d][1,3]dioxol-5-yl)ethanamine, and 0020 (B), which is 2-(piperidin-1-ylmethyl)indeno[1,2,3-de]phthalazin-3-(2H)-one. Of interest, 1895 contains a methylenedioxy motif seen also in etoposide and some semisynthetic derivatives of camptothecin, which are topoisomerase poisons.

FIG. 2.

Killing activity against T. brucei in vitro for 1895 (A) and 0020 (B). For each concentration tested (assayed in quadruplicate), the coefficients of variation were less than 10%. Data were fitted to the equation for the E_max model to obtain EC₅₀s; R² values are 0.99 for both graphs.

FIG. 3.

Free minicircle profiles. T. brucei was treated as indicated and lysed with SDS buffer. Lysates were digested (or not) with proteinase K, as indicated, and DNA was separated by electrophoresis prior to Southern blotting for minicircle DNA. (A) Lanes 1 to 4, 0, 4, 20, and 100 μM etoposide, respectively (5 × 10⁶ cells/lane). (B) Lanes 1 to 7, 0, 10, 20, 40, 60, 80, and 100 μM 1895, respectively (1.7 × 10⁶ cells/lane). (C) Lanes 1 to 6, 0, 20, 40, 80, 100, and 120 μM 0020, respectively (1.7 × 10⁶ cells/lane). Samples treated with 10 and 60 μM had incomplete proteolysis and are not shown. The position of two lanes in this blot have been switched to present results in increasing concentrations. N/G, nicked/gapped; L, linear; CC, covalently closed.

FIG. 4.

Exonuclease susceptibility of minicircle DNA from treated T. brucei. Cells were treated with no drug, 25.5 μM 1895, or 63 μM 0020 as indicated and lysed with SDS. Internal control HindIII-linearized pBluescript was added to each sample prior to exonuclease treatment. (A) Southern blot of minicircle DNA. Lanes 1, 4, and 7, no exonuclease. Lanes 2, 5, and 8, λ exonuclease. Lanes 3, 6, and 9, exonuclease III. There were 2.45 × 10⁷ cells/lane. (B) Reaction internal control pBluescript DNA visualized by ethidium bromide fluorescence prior to DNA transfer for minicircle hybridization.

FIG. 5.

Western blots of T. brucei proteins after treatment as indicated and SDS lysis. (A) Free topoisomerase IA_mt signals with enolase controls. (Left) Lanes 1 to 3, 0, 4, and 100 μM etoposide, respectively (1 × 10⁶ cells/lane). (Center) Lanes 1 to 5, 0, 40, 60, 80, and 100 μM 1895, respectively (8.33 × 10⁵ cells/lane). (Right) Lanes 1 to 5, 0, 20, 60, 100, and 120 μM 0020, respectively (8.33 × 10⁵ cells/lane). In the far-right panel these results were quantitated by first correcting each topoisomerase signal with its cognate enolase control and then normalizing treated samples to untreated controls. Etoposide, filled circles; 1895, filled squares; 0020, filled triangles. (B) Free topoisomerase II_mt signals with enolase controls; concentrations as in panel A. Membranes were hybridized with antibodies to c-Myc (α-cMyc) to visualize c-Myc-tagged topoisomerase IA_mt, to topoisomerase II_mt (α-II_mt), or to enolase (α-en). In the far-right panel, quantitation and symbols are as given above. The quantitative data for all three panels were verified in an analysis by an independent investigator.

FIG. 6.

DNA unwinding assay. Lanes 1 to 4, supercoiled or relaxed plasmid DNA (lanes 1) was treated with topoisomerase IB alone (lanes 2) or with topoisomerase IB in the presence of 1.0 or 2.5 μg/ml ethidium bromide (lanes 3 and 4, respectively). Lanes 5 to 10, topoisomerase IB treatment in the presence of 500, 250, 125, 67.5, 33.8, or 16.9 μM 1895, respectively (A and B), or 310, 155, 77.5, 38.8, 19.4, or 9.70 μM 0020, respectively (C and D). There was 100 ng plasmid DNA/lane. EtBr, ethidium bromide; IB, vaccinia virus topoisomerase IB; sc DNA, supercoiled pBluescript plasmid substrate; re DNA, relaxed pBluescript plasmid substrate.