Clinical and veterinary trypanocidal benzoxaboroles target CPSF3

Abstract

African trypanosomes cause lethal and neglected tropical diseases, known as sleeping sickness in humans and nagana in animals. Current therapies are limited, but fortunately, promising therapies are in advanced clinical and veterinary development, including acoziborole (AN5568 or SCYX-7158) and AN11736, respectively. These benzoxaboroles will likely be key to the World Health Organization's target of disease control by 2030. Their mode of action was previously unknown. We have developed a high-coverage overexpression library and use it here to explore drug mode of action in Trypanosoma brucei Initially, an inhibitor with a known target was used to select for drug resistance and to test massive parallel library screening and genome-wide mapping; this effectively identified the known target and validated the approach. Subsequently, the overexpression screening approach was used to identify the target of the benzoxaboroles, Cleavage and Polyadenylation Specificity Factor 3 (CPSF3, Tb927.4.1340). We validated the CPSF3 endonuclease as the target, using independent overexpression strains. Knockdown provided genetic validation of CPSF3 as essential, and GFP tagging confirmed the expected nuclear localization. Molecular docking and CRISPR-Cas9-based editing demonstrated how acoziborole can specifically block the active site and mRNA processing by parasite, but not host CPSF3. Thus, our findings provide both genetic and chemical validation for CPSF3 as an important drug target in trypanosomes and reveal inhibition of mRNA maturation as the mode of action of the trypanocidal benzoxaboroles. Understanding the mechanism of action of benzoxaborole-based therapies can assist development of improved therapies, as well as the prediction and monitoring of resistance, if or when it arises.

Keywords: CPSF73; N-myristoyltransferase; Ysh1; drug discovery; genetic screening.

Fig. 1.

Assembly and validation of the T. b. brucei overexpression library. (A) Schematic illustrating the pRPa^OEX construct and library assembly. BLA, blasticidin resistance gene; EP and RRNA arrows, procyclin and RRNA promotors; HYG and RRNA, hygromycin and ribosomal RNA targeting sequences; TetO, Tet operator. Partially Sau3AI-digested gDNA and BbsI-digested pRPa^OEX were semifilled (red G’s and T’s). Other relevant restriction sites are shown. (B) The schematic illustrates T. b. brucei library construction, screening, sequencing, and mapping. See SI Appendix, SI Materials and Methods and text for further details. Blue/pink or black/green barcodes indicate fragments overexpressed in each direction. (C) A representative 35-kbp genomic region indicates coverage and fragment junctions corresponding to Sau3AI sites (red). Barcoded reads as in B. Black bars, CDSs, and two convergent polycistrons. (D) Growth curves for the induced library with (closed circles) or without (open circles) DDD85646. For the former, arrows indicate the addition of Tet (blue) or Tet + drug (black). (E) Genome-scale map of hits in the DDD85646 screen (structure indicated). The arrowhead and inset box indicate the primary hit, encoding N-myristoyltransferase (NMT, green). Black bars and black/green peaks as in B and C; gray, all mapped reads.

Fig. 2.

Screens with benzoxaboroles identify CPSF3 as the putative target. (A) Genome-scale map of hits in the acoziborole screen (structure indicated). The arrowhead indicates the primary hit on chromosome 4. (Inset) Primary hit, Tb927.4.1340 (green bar) encoding CPSF3. Other details as in Fig. 1E. (B) Genome-scale map of hits in the SCYX-6759 screen. Other details as in A.

Fig. 3.

Validation of T. b. brucei CPSF3 as the benzoxaborole target. (A) Inducible overexpression (24 h +tetracycline, Tet) of CPSF3^GFP was demonstrated by protein blotting; two independent clones. Dose–response curves with (open circles) and without (closed circles) overexpression of CPSF3^GFP for (B) acoziborole: EC₅₀ no Tet, 0.38 ± 0.01 µM; +Tet, 2.19 ± 0.21 µM; 5.7-fold shift; (C) SCYX-6759: EC₅₀ no Tet, 0.17 ± 0.01 µM; +Tet, 0.71 ± 0.04 µM; 4.2-fold shift; (D) AN11736: EC₅₀ no Tet, 0.63 ± 0.03 nM; +Tet, 2.29 ± 0.06 nM; 3.6-fold shift; (E) DDD85646: EC₅₀ no Tet, 2.7 ± 0.05 nM; +Tet, 2.73 ± 0.17 nM. Error bars, ±SD; n = 3. (F) Inducible CPSF3 knockdown (24 h +Tet) was demonstrated by protein blotting; two independent clones. (G) Cumulative cell growth was monitored during CPSF3 knockdown. Wild-type (open squares), no Tet (open circles), +Tet (closed circles); two of the three latter populations display recovery after 3 d, as often seen in similar experiments, resulting from disruption of the RNA interference cassette in some cells. Error bars, ±SD; n = 3 independent knockdown strains. Fluorescence microscopy reveals the subcellular localization of CPSF3^GFP in cells expressing (H) a native tagged allele or (I) an overexpressed copy. (Scale bars, 10 µM.)

Fig. 4.

Molecular docking and editing of the T. b. brucei CPSF3 active site. (A) The phylogenetic tree shows the relationship among CPSF3 proteins from humans and protozoal parasites, all discussed in the text. (Right) Domain structure for each protein. Domains: gray, metallo-β-lactamase (residues 48–250 in T. brucei); green, β-CASP (residues 271–393 in T. brucei); blue, Zn-dependent metallo-hydrolase (residues 408–463 in T. brucei); rust, C-terminal (residues 502–746 in T. brucei). (B) Docking model for acoziborole (yellow structure) bound to T. brucei CPSF3. Gray spheres, zinc atoms; dotted lines, protein–ligand interactions. (C) Modified docking model illustrating a steric clash between acoziborole (red patch) and a Tyr residue (beige) in human CPSF3, in place of Asn²³² in T. brucei CSPF3. The acoziborole molecular surface is otherwise represented in gray. Other binding site residues that differ between the human and T. brucei enzymes are indicated. (D) Dose–response curves with wild-type T. b. brucei (closed circles) or T. b. brucei expressing Asn²³²His substituted CPSF3 (open squares and triangles, two independent clones). Acoziborole EC₅₀: wild-type, 0.31 ± 0.01 µM; Asn²³²His, 1.59/1.44 ± 0.04/0.05 µM; 5.1/4.6-fold shift. Error bars, ±SD. (E) Modified docking model illustrating a steric clash between acoziborole (red patch) and the His residue (beige) in Asn²³²His substituted CPSF3.