Discovery of a Carbazole-Derived Lead Drug for Human African Trypanosomiasis

Abstract

The protozoan parasite Trypanosoma brucei causes the fatal illness human African trypanosomiasis (HAT). Standard of care medications currently used to treat HAT have severe limitations, and there is a need to find new chemical entities that are active against infections of T. brucei. Following a "drug repurposing" approach, we tested anti-trypanosomal effects of carbazole-derived compounds called "Curaxins". In vitro screening of 26 compounds revealed 22 with nanomolar potency against axenically cultured bloodstream trypanosomes. In a murine model of HAT, oral administration of compound 1 cured the disease. These studies established 1 as a lead for development of drugs against HAT. Pharmacological time-course studies revealed the primary effect of 1 to be concurrent inhibition of mitosis coupled with aberrant licensing of S-phase entry. Consequently, polyploid trypanosomes containing 8C equivalent of DNA per nucleus and three or four kinetoplasts were produced. These effects of 1 on the trypanosome are reminiscent of "mitotic slippage" or endoreplication observed in some other eukaryotes.

Figure 1. Inhibition of T. brucei proliferation: Exploratory Structure-Activity Relationship (SAR).

T. brucei (4 × 10³cells/mL) in 24-well or 96-well plates were incubated with DMSO or compound (various concentrations) for 48 h. The amount of drug that inhibits trypanosome proliferation 50% (GI₅₀) was determined for each compound. Mean GI₅₀ were determined from two independent experiments (totaling four separate biological replicates) within +/− standard deviation. (a) SAR of Class 1 (R¹ and R² = acetyl groups), Class 2 (R¹ = acetyl group, R² = cyclopentanone), and Class 3 (R¹ and R² = cyclopentanone). (b) Class 1, 2 or 3 structures with hydroxyls at R⁴ and/or R⁵. (c) Other compounds not qualified as Class 1, 2 or 3. *For CBL0209 we do not know which E/Z isomers were tested.

Figure 2. Compounds 2 and 3 reduce trypanosome proliferation in mice.

Mice (n = 4 per group) were infected intraperitoneally with 1 × 10⁴ bloodstream T. brucei. Compound 2, 3 and vehicle were administered orally as indicated in graphs. Doses administered of compound 2: 5 mg/kg, 10 mg/kg and 20 mg/kg. Doses administered of compound 3: 6.25 mg/kg, 12.5 mg/kg and 25 mg/kg (a) Mean survival of vehicle and compound treated mice. Horizontal lines indicate mean survival (days). Student’s t-test was used to compare survival of vehicle to drug-treated mice. *P < 0.0007. (b,c) Parasitemia of 2 (b) or 3 (c) treated mice compared to vehicle. Individual mice are represented by different symbol shapes. Horizontal lines indicate median parasitemia.

Figure 3. Compound 1 cures T. b. brucei infection in a mouse model of HAT.

Mice (n = 4 per group) were infected intraperitoneally with 1 × 10⁴ bloodstream T. brucei. Compound 1 (30 mg/kg or 40 mg/kg) and vehicle were administered orally once per day of treatment for a total of 14 doses. (a) Number of mice alive post-infection. *All remaining mice were cured of trypanosome infection. (b,c) Parasitemia in mice dosed with vehicle or 30 mg/kg of compound 1 (b) or 40 mg/kg of compound 1 (c). Individual mice are represented by different symbol shapes. Horizontal lines indicate median parasitemia. UND = parasitemia undetectable. Mouse (♦) died on day 16 post-infection, no parasitemia observed prior to death (data not presented). *All remaining mice were cured of trypanosome infection.

Figure 4. Summary of effects of compound 1 and ethidium bromide on trypanosome nucleus and kinetoplast copy number.

Results summarized from Supplementary Fig. S5. (a) DMSO-treated, (b) H₂O-treated, (c) 1-treated, (d) ethidium bromide-treated. Mean percentage of cells +/− standard deviation were determined from three independent experiments.

Figure 5. Effects of 24 h compound 1 treatment on trypanosome nucleus and kinetoplast copy number.

T. brucei (1 × 10⁵cells/mL) were treated with 1 (200 nM), ethidium bromide (200 nM), H₂O, or DMSO (0.1% vol/vol) for 24 h as part of a 30 h time-course in HMI-9 medium (Supplementary Fig. S5). Cells were fixed with paraformaldehyde (4% in PBS) and DNA was stained with DAPI (1.5 μM). (a,c) Representative images of 24 h treated cells. Panels = Left: DIC (differential interference contrast), middle: DAPI (red), right: Merge. Bar = 10 μm. (b,d) Nuclei (N) and kinetoplasts (K) were counted from 150 cells for each sample. 1K1N, trypanosomes with one kinetoplast/one nucleus; 2K1N, trypanosomes with two kinetoplasts/one nucleus; 2K2N, trypanosomes with two kinetoplasts/two nuclei; 0K1N, trypanosomes without visible kinetoplasts/one nucleus; XK1N, trypanosomes with more than two kinetoplasts/one nucleus. Mean percentage of cells +/− standard deviation were determined from three independent experiments. Student’s t-test was used to compare organelle copy number distribution of vehicle (DMSO or H₂O) to drug-treated trypanosomes. *P < 0.05, determined by Student’s t-test.

Figure 6. Summary of effects of compound 1 and ethidium bromide on nuclear DNA content.

Results summarized from Supplementary Figs S7 and S8. (a) DMSO-treated, (b) H₂O-treated, (c) 1-treated, (d) ethidium bromide-treated. Mean percentage of cells +/− standard deviation were determined from three independent experiments.

Figure 7. Effects of 24 h compound 1 treatment on nuclear DNA content.

T. brucei (10⁵cells/mL) were treated with 1 (200 nM), ethidium bromide (200 nM), H₂O, or DMSO (0.1% vol/vol) for 24 h as part of a 30 h time-course in HMI-9 medium (Supplementary Figs S7 and S8). Cells were fixed with PBS containing 70% methanol, treated with RNase A (500 μg/mL) and DNA was stained with propidium iodide (7.5 μM). (a,c) Histograms of DNA content per cell. 10,000 trypanosomes were analyzed per sample. Chromosomal content (e.g. “2C”) is indicated for each peak. (b,d) Proportion of cells with DNA content from 2C–8C. Mean percentage of cells +/− standard deviation were determined from three independent experiments. *P < 0.05, determined by Student’s t-test comparing DNA content of vehicle (DMSO or H₂O) to drug-treated trypanosomes.

Figure 8. Delayed killing effects of Compound 1 on T. brucei. T. brucei (5 × 10⁵cells/mL) were treated with 1 (1 μM) or DMSO (0.1% vol/vol) for 6 h.

Cells were washed twice with HMI-9 medium, resuspended at 1 × 10⁵cells/mL, and cultured for 48 h. Trypanosome densities were determined with a hemocytometer. (a) Flow chart summarizing trypanosome treatment and recovery protocol prior to analysis. (b) Cell density before (“Start”) and after 6 h treatment with DMSO or 1 (1 μM). (c) Cell density after wash and resuspension at 1 × 10⁵cell/mL (“Start”) and after 48 h incubation in drug-free medium. Mean cell density (values above each bar) ± standard deviation is presented from two independent experiments, four separate biological replicates. “” indicates no cells observed.