A potent and selective reaction hijacking inhibitor of Plasmodium falciparum tyrosine tRNA synthetase exhibits single dose oral efficacy in vivo
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Abstract
The Plasmodium falciparum cytoplasmic tyrosine tRNA synthetase (PfTyrRS) is an attractive drug target that is susceptible to reaction-hijacking by AMP-mimicking nucleoside sulfamates. We previously identified an exemplar pyrazolopyrimidine ribose sulfamate, ML901, as a potent reaction hijacking inhibitor of PfTyrRS. Here we examined the stage specificity of action of ML901, showing very good activity against the schizont stage, but lower trophozoite stage activity. We explored a series of ML901 analogues and identified ML471, which exhibits improved potency against trophozoites and enhanced selectivity against a human cell line. Additionally, it has no inhibitory activity against human ubiquitin-activating enzyme (UAE) in vitro. ML471 exhibits low nanomolar activity against asexual blood stage P. falciparum and potent activity against liver stage parasites, gametocytes and transmissible gametes. It is fast-acting and exhibits a long in vivo half-life. ML471 is well-tolerated and shows single dose oral efficacy in the SCID mouse model of P. falciparum malaria. We confirm that ML471 is a reaction hijacking inhibitor that is converted into a tight binding Tyr-ML471 conjugate by the PfTyrRS enzyme. A crystal structure of the PfTyrRS/ Tyr-ML471 complex offers insights into improved potency, while molecular docking into UAE provides a rationale for improved selectivity.
Description
DATA AVILABILITY STATEMENT : The following structures have been deposited in the PDB: PfTyrRS/Tyr-ML471 - PDB ID 9CLL (https://doi.org/10.2210/pdb9CLL/pdb). Additional data and source data are available in Supplementary Information.
SUPPORTING INFORMATION : FIGURE S1. Exposure-time dependent responses of trophozoite and schizont stage parasites to short pulses of ML901. (A,B) A tightly synchronized culture of CAM 3.II Rev parasites (>70% of parasites within a 5-h time window) was subjected to pulses of ML901 for 3 h, 6 h, 9 h, 24 h, or continued exposure for 48 h (A) or 30 h (B), initiated at (A) trophozoite (25–30 h.p.i.) and (B) schizont (43–48 h.p.i.) stages. Flow cytometric analysis of Syto-61-labelled parasites in the cycle after the initiation of treatment assessed cell viability. Data are representative of three (trophozoite) and two (schizont) independent experiments. LD50 values are shown in Table S1. FIGURE S2. Activity of ML471, artesunate and chloroquine against ex vivo field isolates. Compounds were assayed on P falciparum (A) isolates and P. vivax (B) Brazilian isolates collected from mono-infected patients. Data represent mean ± SEM. Median EC50 (nM) values, the range of values, and the numbers of isolates are shown in Table S2. FIGURE S3. Activity of ML471, ML901, MMV390048 and Methylene blue (MB) against immature (A) and mature (B) stage gametocytes. Gametocytocidal activity of the compounds was assessed against immature (>90% stage II/III) and mature (>95% stage V) stage gametocytes. MMV390048 and MB were used as controls. Data represent mean ± SEM from three independent experiments. Mean IC50 ± SEM values from the three independent experiments are presented in S3 Table. FIGURE S4. Activity of ML471 and ML901 against P. falciparum NF175 (A) and NF135 (B) liver stage schizonts. Human primary hepatocytes were infected with P. falciparum NF175 or NF135 sporozoites and cultured for four days. Anti-HSP70 was used to detect parasites in fixed cells using high content imaging. Atovaquone and MMV390048 were used as control compounds against NF175 and NF135 schizonts, respectively. Data values represent mean ± SEM from three independent experiments. IC50 values are shown in S4 Table. FIGURE S5. Activity of ML471 and Cabamiquine (DDD498) against transmissible gametes. Inhibition of male (A) and female (B) gamete formation was assessed in the P. falciparum Dual Gamete Formation Assay. Cabamiquine (DDD498) and DMSO were used as positive and vehicle controls, respectively. Data represent mean ± SEM from 4–5 independent experiments. IC50 values are shown in S5 Table. FIGURE S6. Pharmacokinetics profile and efficacy of ML471 in treating P. falciparum infected mice. (A) Pharmacokinetics profile (in blood) for SCID mice engrafted with human RBCs infected with P. falciparum, over the first day following treatment with ML471 at 50 mg/kg p. o. See S8 Table for pharmacokinetics values. (B) Therapeutic efficacy of ML471 in the SCID mouse P. falciparum model, dosed with ML471 for 4 days at 50 mg/kg p.o. per day (arrows), initiated on Day 3 post-infection. The chloroquine data are from [16]. FIGURE S7. Identification of Tyr-ML471 adduct made by P. falciparum culture and PfTyrRS enzyme. (A) MS/MS analysis of the Tyr-ML471 adduct made by P. falciparum following treatment with ML471 (1 μM) for 2 h (upper panel); and the synthetic conjugate at 0.2 μM(lower panel). (B,C) PfTyrRS (1 μM) was incubated with ML471 (10 μM), ATP (10 μM), tyrosine (20 μM) and 4 μMPftRNATyr for 1 h at 37˚C. Following protein denaturation and precipitation, the supernatant was subjected to LCMS analysis. (B) The extracted ion chromatograms of Tyr-ML471 adduct made by PfTyrRS (upper panel); and the synthetic conjugate at 1 μM PLOS PATHOGENS Reaction hijacking inhibitor of P. falciparum tyrosine tRNA synthetase (lower panel). The inset shows the MS analysis of the enzyme-generated Tyr-ML471. (C) MS/MS analysis of the enzyme-generated Tyr-ML471 (upper panel) and the synthetic conjugate at 1 μM(lower panel). FIGURE S8. Kinase GLO assays for ML901 and derivatives. Effects of increasing concentrations of ML723, ML111, ML864, ML470 (A) and ML107, ML676, ML681 (B) on ATP consumption by PfTyrRS. The reaction conditions are: PfTyrRS (25 nM), ATP (10 μM), tyrosine (200 μM), cognate tRNATyr (4.8 μM) and pyrophosphatase (1 unit/mL). Incubations were at 37˚C for 1 h. Data are mean ± SEM from three independent experiments. FIGURE S9. Comparison of the active site architecture of PfTyrRS in complex with Tyr-ML471 or Tyr-ML901 (7ROS). (A) Inhibitor/active site interactions for the A-chain of PfTyrRS with bound Tyr-ML471. (B) LigPlots of interacting residues for the A- and B-chains of PfTyrRSwith bound Tyr-ML471. (C) A-chain of Tyr-ML471-bound PfTyrRS showing the poses adopted by the ML471 isopropyl (aqua arrow) and His70, which are incompatible with a structured KMSKS loop. (D) A-chain of Tyr-ML901-bound PfTyrRS (7ROS) illustrating the ML901 difluoromethoxy group (red arrow) and the His70 conformation. The KMSKS loop is not resolved. (E) Overlay of the A-chains of Tyr-ML471- and Tyr-ML901-bound PfTyrRS. TABLE S1. The median lethal dose (LD50) values of different asexual blood stages after exposure of ML901. TABLE S2. P. vivax and P. falciparum ex vivo drug susceptibility. TABLE S3. Activity against immature (>90% stage II/III) and mature (>95% stage V) stage gametocytes (Pf3D7-pfs16-CBG99) for ML901, ML471 and antimalarial controls. TABLE S4. Activity against liver stage P. falciparum schizonts and against primary hepatocytes. TABLE S5. Activity against transmissible gametes. TABLE S6. Parasite Reduction Ratio. TABLE S7. Pharmacological and ADME characterization of ML471. TABLE S8. Pharmacological parameters for ML471 in the SCID mouse model. TABLE S9. Summary of resistance selection. ABLE S10. Copy Number Variants (CNV) in ML901-selected sample. TABLE S11. Copy Number Variants (CNV) in ML471-selected sample. TABLE S12. Tm values of TyrRSs determined by differential scanning fluorimetry (DSF). TABLE S13. X-ray diffraction data collection and refinement statistics. TEXT S1. Chemistry materials and methods.
SUPPORTING INFORMATION : FIGURE S1. Exposure-time dependent responses of trophozoite and schizont stage parasites to short pulses of ML901. (A,B) A tightly synchronized culture of CAM 3.II Rev parasites (>70% of parasites within a 5-h time window) was subjected to pulses of ML901 for 3 h, 6 h, 9 h, 24 h, or continued exposure for 48 h (A) or 30 h (B), initiated at (A) trophozoite (25–30 h.p.i.) and (B) schizont (43–48 h.p.i.) stages. Flow cytometric analysis of Syto-61-labelled parasites in the cycle after the initiation of treatment assessed cell viability. Data are representative of three (trophozoite) and two (schizont) independent experiments. LD50 values are shown in Table S1. FIGURE S2. Activity of ML471, artesunate and chloroquine against ex vivo field isolates. Compounds were assayed on P falciparum (A) isolates and P. vivax (B) Brazilian isolates collected from mono-infected patients. Data represent mean ± SEM. Median EC50 (nM) values, the range of values, and the numbers of isolates are shown in Table S2. FIGURE S3. Activity of ML471, ML901, MMV390048 and Methylene blue (MB) against immature (A) and mature (B) stage gametocytes. Gametocytocidal activity of the compounds was assessed against immature (>90% stage II/III) and mature (>95% stage V) stage gametocytes. MMV390048 and MB were used as controls. Data represent mean ± SEM from three independent experiments. Mean IC50 ± SEM values from the three independent experiments are presented in S3 Table. FIGURE S4. Activity of ML471 and ML901 against P. falciparum NF175 (A) and NF135 (B) liver stage schizonts. Human primary hepatocytes were infected with P. falciparum NF175 or NF135 sporozoites and cultured for four days. Anti-HSP70 was used to detect parasites in fixed cells using high content imaging. Atovaquone and MMV390048 were used as control compounds against NF175 and NF135 schizonts, respectively. Data values represent mean ± SEM from three independent experiments. IC50 values are shown in S4 Table. FIGURE S5. Activity of ML471 and Cabamiquine (DDD498) against transmissible gametes. Inhibition of male (A) and female (B) gamete formation was assessed in the P. falciparum Dual Gamete Formation Assay. Cabamiquine (DDD498) and DMSO were used as positive and vehicle controls, respectively. Data represent mean ± SEM from 4–5 independent experiments. IC50 values are shown in S5 Table. FIGURE S6. Pharmacokinetics profile and efficacy of ML471 in treating P. falciparum infected mice. (A) Pharmacokinetics profile (in blood) for SCID mice engrafted with human RBCs infected with P. falciparum, over the first day following treatment with ML471 at 50 mg/kg p. o. See S8 Table for pharmacokinetics values. (B) Therapeutic efficacy of ML471 in the SCID mouse P. falciparum model, dosed with ML471 for 4 days at 50 mg/kg p.o. per day (arrows), initiated on Day 3 post-infection. The chloroquine data are from [16]. FIGURE S7. Identification of Tyr-ML471 adduct made by P. falciparum culture and PfTyrRS enzyme. (A) MS/MS analysis of the Tyr-ML471 adduct made by P. falciparum following treatment with ML471 (1 μM) for 2 h (upper panel); and the synthetic conjugate at 0.2 μM(lower panel). (B,C) PfTyrRS (1 μM) was incubated with ML471 (10 μM), ATP (10 μM), tyrosine (20 μM) and 4 μMPftRNATyr for 1 h at 37˚C. Following protein denaturation and precipitation, the supernatant was subjected to LCMS analysis. (B) The extracted ion chromatograms of Tyr-ML471 adduct made by PfTyrRS (upper panel); and the synthetic conjugate at 1 μM PLOS PATHOGENS Reaction hijacking inhibitor of P. falciparum tyrosine tRNA synthetase (lower panel). The inset shows the MS analysis of the enzyme-generated Tyr-ML471. (C) MS/MS analysis of the enzyme-generated Tyr-ML471 (upper panel) and the synthetic conjugate at 1 μM(lower panel). FIGURE S8. Kinase GLO assays for ML901 and derivatives. Effects of increasing concentrations of ML723, ML111, ML864, ML470 (A) and ML107, ML676, ML681 (B) on ATP consumption by PfTyrRS. The reaction conditions are: PfTyrRS (25 nM), ATP (10 μM), tyrosine (200 μM), cognate tRNATyr (4.8 μM) and pyrophosphatase (1 unit/mL). Incubations were at 37˚C for 1 h. Data are mean ± SEM from three independent experiments. FIGURE S9. Comparison of the active site architecture of PfTyrRS in complex with Tyr-ML471 or Tyr-ML901 (7ROS). (A) Inhibitor/active site interactions for the A-chain of PfTyrRS with bound Tyr-ML471. (B) LigPlots of interacting residues for the A- and B-chains of PfTyrRSwith bound Tyr-ML471. (C) A-chain of Tyr-ML471-bound PfTyrRS showing the poses adopted by the ML471 isopropyl (aqua arrow) and His70, which are incompatible with a structured KMSKS loop. (D) A-chain of Tyr-ML901-bound PfTyrRS (7ROS) illustrating the ML901 difluoromethoxy group (red arrow) and the His70 conformation. The KMSKS loop is not resolved. (E) Overlay of the A-chains of Tyr-ML471- and Tyr-ML901-bound PfTyrRS. TABLE S1. The median lethal dose (LD50) values of different asexual blood stages after exposure of ML901. TABLE S2. P. vivax and P. falciparum ex vivo drug susceptibility. TABLE S3. Activity against immature (>90% stage II/III) and mature (>95% stage V) stage gametocytes (Pf3D7-pfs16-CBG99) for ML901, ML471 and antimalarial controls. TABLE S4. Activity against liver stage P. falciparum schizonts and against primary hepatocytes. TABLE S5. Activity against transmissible gametes. TABLE S6. Parasite Reduction Ratio. TABLE S7. Pharmacological and ADME characterization of ML471. TABLE S8. Pharmacological parameters for ML471 in the SCID mouse model. TABLE S9. Summary of resistance selection. ABLE S10. Copy Number Variants (CNV) in ML901-selected sample. TABLE S11. Copy Number Variants (CNV) in ML471-selected sample. TABLE S12. Tm values of TyrRSs determined by differential scanning fluorimetry (DSF). TABLE S13. X-ray diffraction data collection and refinement statistics. TEXT S1. Chemistry materials and methods.
Keywords
Plasmodium falciparum, Pyrazolopyrimidine ribose sulfamate, ML901, Potency
Sustainable Development Goals
SDG-02: Zero Hunger
SDG-03: Good health and well-being
SDG-03: Good health and well-being
Citation
Xie, S.C., Tai, C.-W., Morton, C.J., Ma, L., Huang, S.-C., Wittlin, S., et al. (2024) A potent and selective reaction hijacking inhibitor of Plasmodium falciparum tyrosine tRNA synthetase exhibits single dose oral efficacy in vivo. PLoS Pathogens 20(12): e1012429. https://doi.org/10.1371/journal. pat.1012429.