The trypanocidal benzoxaborole AN7973 inhibits trypanosome mRNA processing

Abstract

Kinetoplastid parasites-trypanosomes and leishmanias-infect millions of humans and cause economically devastating diseases of livestock, and the few existing drugs have serious deficiencies. Benzoxaborole-based compounds are very promising potential novel anti-trypanosomal therapies, with candidates already in human and animal clinical trials. We investigated the mechanism of action of several benzoxaboroles, including AN7973, an early candidate for veterinary trypanosomosis. In all kinetoplastids, transcription is polycistronic. Individual mRNA 5'-ends are created by trans splicing of a short leader sequence, with coupled polyadenylation of the preceding mRNA. Treatment of Trypanosoma brucei with AN7973 inhibited trans splicing within 1h, as judged by loss of the Y-structure splicing intermediate, reduced levels of mRNA, and accumulation of peri-nuclear granules. Methylation of the spliced leader precursor RNA was not affected, but more prolonged AN7973 treatment caused an increase in S-adenosyl methionine and methylated lysine. Together, the results indicate that mRNA processing is a primary target of AN7973. Polyadenylation is required for kinetoplastid trans splicing, and the EC50 for AN7973 in T. brucei was increased three-fold by over-expression of the T. brucei cleavage and polyadenylation factor CPSF3, identifying CPSF3 as a potential molecular target. Molecular modeling results suggested that inhibition of CPSF3 by AN7973 is feasible. Our results thus chemically validate mRNA processing as a viable drug target in trypanosomes. Several other benzoxaboroles showed metabolomic and splicing effects that were similar to those of AN7973, identifying splicing inhibition as a common mode of action and suggesting that it might be linked to subsequent changes in methylated metabolites. Granule formation, splicing inhibition and resistance after CPSF3 expression did not, however, always correlate and prolonged selection of trypanosomes in AN7973 resulted in only 1.5-fold resistance. It is therefore possible that the modes of action of oxaboroles that target trypanosome mRNA processing might extend beyond CPSF3 inhibition.

Fig 1. Structures of benzoxaboroles studied in this paper.

The benzoxaborole scaffold is in cyan.

Fig 2. Effects of AN7973 on the cell cycle and protein synthesis.

A) Cumulative growth curve for bloodstream-form trypanosomes incubated with and without AN7973. Results are arithmetic mean ± standard deviation for three replicates. B) Cell cycle analysis by microscopy. The percentages of cells with different numbers of nuclei and kinetoplasts are shown. C) Effect on protein synthesis. 5x10⁶ Cells were incubated with AN7973 (10x EC₅₀) for various times, then (after washing and 15-min pre-incubation) with [³⁵S]-methionine for 30 min. Proteins were separated by SDS-PAGE and radioactive incorporation was assessed by autoradiography. The Coomassie-stained gel is shown as a loading control. Two prominent protein bands showing different effects of AN7973 are indicated as "a" and "b". All lanes are from the same gel and exposure; the gaps between the lanes are present because identical results for two partially resistant lines (also treated at 10x EC₅₀) have been deleted.

Fig 3. AN7973 affects mRNA processing.

A) Trypanosomes were incubated with AN7973 for different times (0–9 hours). RNA was subjected to denaturing agarose gel electrophoresis, blotted, and detected using radioactive probes. This panel shows Methylene blue-stained gel showing equal loading of total RNA. The three prominent bands are rRNA. B) As (A), blot probed with a [³²P]-end-labelled probe complementary to the spliced leader. The signals representing the ~140nt SLRNA and mature mRNAs are indicated. The cluster of bands around 2 kb probably represent the abundant mRNAs encoding the Variant Surface Glycoprotein, alpha and beta tubulin (both ~1.9 kb including 100nt poly(A)), and EF-1 alpha (1.8 kb) [91]. C) The blot from (B) was stripped then incubated with a beta-tubulin probe. Shadows from the SL probe remain. The positions of monomer, dimer and multimers from the tubulin repeats are indicated. D) The effect of AN7973 is not due to inhibition of SLRNA synthesis. Trypanosomes were treated either with AN7973 for 1h, or with Actinomycin D for 30 min, or with AN7973 followed by Actinomycin D, as indicated above the lanes. The lower panel is the rRNA loading control.

Fig 4. Effect of AN7973 on trans-splicing.

A) Schematic representation of the splicing reaction. SLRNA and Y structure during primer extension experiments. SLRNA is represented with various colours; red is the cap4 region, green is the spliced leader (SL), blue is the SL intron. The pre-mRNA is shown with a grey intergenic region and a magenta portion that represents the 5' end of a mature mRNA. The 5' and 3' ends of each RNA are indicated. During trans splicing, the SL intron forms a branched "Y" structure with the 2' hydroxyl of an adenosine located 5' to the polypyrimidine tract that is recognised by the splicing machinery. Meanwhile the SL is trans spliced to the 5'-end of the mRNA. B) Schematic representation of the primer extension assay. The 5'-end labelled oligonucleotide primer hybridises towards the 3' end of the SLRNA. Reverse transcription on the intact SLRNA extends (dashed line) towards the 5' end but terminates upon encountering the methylated residues of cap4. In the Y structure, the primer extends (dashed line) until the branch point, giving an 87nt product. C) Typical result from a primer extension experiment illustrated in (B). A primer complementary to the U3 snRNA was used as a control. Lane 1 (0’) shows the result from trypanosomes that were treated with DMSO only. Lanes 2 and 3 show the results from trypanosomes treated with AN7973 for 30 min and 60 min, and lane 4 is the result for 30 min Sinefungin. The arrowhead shows the primer extension product from unmethylated SLRNA. D) Quantification from several independent tests of AN7973 treatment; the ratio between Y structure and the U3 signal is shown. # indicates the number of data points used. Sinefungin results were taken only from experiments that included AN7973 on the same gel. The central line is the median, and boxes extend from the 25th to the 75th quartile. The whiskers extend to the most extreme data point that is no more than 1.5 times the inter-quartile range. Other points are outliers. Details for our initial experiments are in S1A and S1B Fig. After only 30 min incubation with AN7973, the difference between treated and untreated cells was significant using a one-way ANOVA. E-H) Trypanosomes were incubated with 10x EC50 of various known anti-trypanosomal drugs for 1h or 2h, then splicing was assayed. The % Y structure (relative to U3) is shown (individual points, plus mean and standard deviation). The 95% confidence intervals are shown in S1C–S1F Fig.

Fig 5. Effects of benzoxaboroles on the metabolome.

Fold increases are shown for selected metabolites, relative to untreated cells (mean and standard deviation). The numerical data are in S6 Table. SAM: S-adenosylmethionine; MTA: methylthioadenosine; ML: methyllysine; 2ML: dimethyllysine; 3ML: trimethyllysine; AL: acetyllysine.

Fig 6. EC₅₀s of selected benzoxaboroles for salivarian trypanosomes.

The graph shows EC₅₀s against T. brucei (in vitro, 48h assay for bloodstream forms, 72h for procyclic forms) and for selected compounds, also against T. congolense (in vitro) and T. vivax (ex vivo). Details for all tested compounds are in S1 Table.

Fig 7. Relationship between trans splicing inhibition and methylation changes.

A) Trypanosomes were incubated with 10x EC₅₀ of various compounds for 2h, then splicing was assayed as in Fig 4. The % Y structure (relative to the U3 control) is shown. Results are shown for one experiment (experiment 1) with a full compound set and for a second experiment with selected compounds. A time course for the second experiment is shown in S4A Fig. B) The mean increase in SAM and MTA after treatment with each compound is plotted on the y-axis; the x-axis shows mean and standard deviation of the % Y-structure remaining (data for experiment 1 in (A)). The AN numbers are colour-coded to indicate the time of incubation prior to metabolome analysis. C. Metabolite changes after AN5568 or Act D treatment (both 5h). Each point represents a metabolite; data are in S7 Table. Orange: significantly affected by Actinomycin D; cyan: significantly affected by AN5568; purple: significantly affected by both treatments. Labelled metabolites are: 1- Glutathione disulfide; 2- Lys-Val-Pro; 3- N6,N6,N6-Trimethyl-L-lysine; 4- N6-Acetyl-L-lysine.

Fig 8. In vitro splicing assay.

Permeablised procyclic-form trypanosomes (2.5 x 10⁸ / reaction) were incubated for 2 min in the presence of DMSO or AN7973, and then for 10 min in the presence of alpha-32P-UTP. Newly-made RNA was analysed by denaturing gel electrophoresis and autoradiography. The full-length SLRNA (~140nt) and the de-branched SLRNA intron (~100 nt) are indicated.

Fig 9. Effects of benzoxaborole treatment on formation of YFP-DHH1 granules.

A-C) Bloodstream-form trypanosomes with one DHH1 gene tagged in situ were washed with PBS, fixed with formaldehyde, then allowed to adhere to glass slides before imaging to detect YFP-tagged DHH1 (magenta) and DNA (cyan). Cells were (A) untreated, (B) incubated with Sinefungin (1μg/mL 30 min), or (C) treated with AN7973 (230 nM, 2h). D) Results for all tested oxaboroles. The average percentages of cells with clear peri-nuclear granules are plotted on the y-axis and the % Y-structure is on the x-axis. Times to show an effect on growth are as in Fig 6B. Results for the three replicate experiments are in S5 Fig and typical fields are shown in S6 and S7 Figs.

Fig 10. Ectopic expression of myc-tagged CPSF3 causes mild resistance to some benzoxaboroles.

A) Expression of myc-CPSF3 in three independent cloned cell lines that were used for the assays. Expression was induced by tetracycline. Tryparedoxin peroxidase (TxNPx) served as a loading control. B) Correlation between the effect of myc-CPSF3 induction and the extent of Y-structure inhibition in the splicing assay. All results are arithmetic mean and standard deviation. Before plotting of the regression line and calculation of the correlation coefficient, the data for AN3661 were excluded because it was used at an extremely high concentration.