N-myristoyltransferase inhibitors as new leads to treat sleeping sickness

Abstract

African sleeping sickness or human African trypanosomiasis, caused by Trypanosoma brucei spp., is responsible for approximately 30,000 deaths each year. Available treatments for this disease are poor, with unacceptable efficacy and safety profiles, particularly in the late stage of the disease when the parasite has infected the central nervous system. Here we report the validation of a molecular target and the discovery of associated lead compounds with the potential to address this lack of suitable treatments. Inhibition of this target-T. brucei N-myristoyltransferase-leads to rapid killing of trypanosomes both in vitro and in vivo and cures trypanosomiasis in mice. These high-affinity inhibitors bind into the peptide substrate pocket of the enzyme and inhibit protein N-myristoylation in trypanosomes. The compounds identified have promising pharmaceutical properties and represent an opportunity to develop oral drugs to treat this devastating disease. Our studies validate T. brucei N-myristoyltransferase as a promising therapeutic target for human African trypanosomiasis.

Figure 1. Identification of NMT lead series inhibitors

a. Chemical evolution of DDD85646 from the initial high-throughput screening hit DDD64588. Combining the structure-activity-relationships from the two strategies led to the development of the potent compound DDD85646. Potencies were determined for all compounds synthesised against recombinant TbNMT and huNMT, as well as against BSF T. brucei and MRC5 proliferation in vitro. b. Correlation between the inhibitions of recombinant TbNMT and BSF T. brucei proliferation for 175 members of the pyrazole sulfonamide series. Data shown are replicates of between 2 and 22 independent potency determinations using 10-point curves. Robustness of TbNMT and trypanosome proliferation assays are exemplified through routinely reported parameters of Z’ (0.703 ± 0.050, n=169 and 0.695 ± 0.095, n>1000 for TbNMT and trypanosome assays respectively) and reproducible potencies of standards (DDD73498 (TbNMT assay) pIC₅₀ = 6.52 ± 0.14, n=276 and pentamidine (trypanosome assay) pEC₅₀ = 8.37 ± 0.41, n=497).

Figure 2. TbNMT inhibitor cures acute trypanosomiasis in vivo

a. Mean total and free blood concentration time profiles following single oral administration of DDD85646 at 10 and 50 mg kg⁻¹ free base to female NMRI mice (n=3 per dose group). EC₉₉ is calculated from the average EC₅₀ of 2.46 ± 1.8 nM and Hill slope of 4.84 ± 0.6 (n=5). Solid lines are total plasma concentrations and dashed lines are the predicted free plasma concentrations (fraction unbound in plasma = 0.063). b. Kaplan Meier survival plot for female NMRI mice (n=5 per dose group) following infection with T. b. brucei strain 427 (variant 221) (inoculum 1 × 10⁴ parasites). Oral treatment with DDD85646 commenced 3 days after infection at the indicated doses (all b.i.d for 4 days).

Figure 3. TbNMT inhibitors have rapid trypanocidal effects in vitro and in vivo

a. Parasitaemia in mice (n=3 per group) with (red) or without (black) DDD85646 treatment (50 mg kg⁻¹, oral, b.i.d); for method see Figure 2b. Arrows represent dose administration times. Data: mean ± s.d. b. T. b. brucei proliferation in culture determined by counting motile parasites in presence (red) or absence (black) of 50 nM DDD85646. Data: mean ± s.d. for 3 determinations. c. Blood smears of infected mice and culture samples were stained by Giemsa and observed by light microscopy. Treated cells showed typical BigEye phenotype. d. Scanning electron micrograph of T. b. brucei treated with 10 nM DDD85646 for 24 h. Inset shows an untreated control cell. e. Transmission electron micrograph of sagittal section of flagellar pocket of T. b. brucei treated with 5 nM DDD85646 for 72 h. Inset shows a section of flagellar pocket of an untreated control cell. Asterisks mark flagellar pockets. Dashed lines: cell detection limits. Scale bars: 500 nm.

Figure 4. Pyrazole sulfonamide series acts ‘on-target’ in the trypanosome

a. Fluorographs of SDS-PAGE gels loaded with lysates of BSF T. b. brucei cells labelled with either [³H]-myristic acid (lanes 1-4) or [³⁵S]-methionine (lanes 5 and 6) after pre-incubation with (+) or without (−) 0.5 μM DDD85646 for 6 h. Gels were incubated with or without 0.2 M NaOH in methanol, as indicated, prior to fluorography. b. Wild-type (“single marker”, SM) parasites and T. b. brucei over-expressing myc-tagged NMT were incubated with 0-100 nM DDD85646 for 64 h; motile cells were counted using a haemocytometer. Closed circles, T. brucei over-expressing NMT (n=3); open circles, wild-type cells (n=3). Levels of myc-tagged NMT expression were confirmed via western blotting. c. T. b. brucei expressing ARF1Q71L (GTP-locked form of ARF1) under tetracycline control were treated with 10 nM DDD85646 for 6 h. Cells were then subjected to live/dead FACS analysis. Data shown represent mean ± s.d. from 2 independent experiments. Levels of myc-tagged ARF1 mutant expression were analysed via western blotting.

Figure 5. Characterisation of pyrazole sulfonamide interactions with NMT

a, DDD64558 potency against TbNMT (IC₅₀) determined in the presence of 0.5 (closed circles) and 16 μM (open circles) CAP5.5 peptide substrate. Each data point represents mean ± s.d. (n=4). b, Kinetics of binding of DDD85646 to TbNMT and huNMT1 determined by SPR. c, X-ray crystal structure of DDD85646 bound to LmNMT. Left panel shows the LmNMT binding site with protein backbone (pink ribbon), solvent accessible surface (grey), DDD85646 (stick representation, carbon atoms gold, nitrogen blue, chlorine green, oxygen red and sulphur yellow), myristoyl CoA (C atoms cyan) and an omit map (F_o-F_c, 3.0 sigma) around DDD85646 (blue). Right panel shows in stick representation DDD85646 and residues forming the active site (C atoms grey). Key residues are highlighted (C atoms yellow) as are water molecules (red spheres) and H-bonds (dashed lines).