Fig 1.
Structures of ML901 and derivatives and adenosine 5’-sulfamate (AMS).
(A) ML901, (B) ML471, (C) ML676, (D) ML681, (E) ML723, (F) ML107, (G) ML470, (H) ML864, (I) ML111, (J) AMS, (K) Tyr-ML471.
Table 1.
Activities of pyrazolopyrimidine sulfamates as inhibitors of parasite growth.
P. falciparum (3D7, ring stage) cultures were exposed to different nucleoside sulfamates for 72 h and the 50% Inhibitory Concentration (IC50) for growth inhibition assessed using the lactate dehydrogenase (PfLDH) assay. Alternatively, synchronized Cam3.IIrev parasite cultures were subjected to 6-h pulses of nucleoside sulfamates, at the trophozoite (25–30 h.p.i.) stage. 50% growth inhibition (IC50) was determined in the cycle following treatment, using a SYBR Green I assay. HepG2 cell cultures were exposed to nucleoside sulfamates for 72 h and growth inhibition (IC50) assessed using CellTiter-Glo reagent. Data represent the mean of three independent experiments and error bars correspond to SEM. AMS = Adenosine 5’-sulfamate. n = Number of biological repeats. Medicines for Malaria Venture (MMV) designations for the compound names are in brackets.
Fig 2.
ML471 exhibits improved short-exposure activity against P. falciparum cultures, associated with rapid parasite killing.
(A) Synchronized Cam3.IIrev parasite cultures were subjected to 6-h pulses of ML901, ML471, ML107 and ML723, at the trophozoite (25–30 h.p.i.) stage. Growth inhibition was determined in the cycle following treatment. Data represent the mean of three independent experiments and error bars correspond to SEM. (B) 3D7 parasite cultures were treated for 0 to 120 h with ML471 or compounds with fast (artemisinin, chloroquine), moderate (pyrimethamine) or slow (atovaquone) killing profiles, at 10 times their respective IC50_48h values. Following removal of inhibitor, serial dilutions of cultures were established, and assessed after 18 days of culturing.
Table 2.
Inhibitory activity of selected pyrazolopyrimidine sulfamates in E1 enzyme assays.
Homogeneous Time-Resolved Fluorescence (HTRF) enzyme assays were employed to evaluate the 50% Inhibitory Concentrations (IC50) values for compounds against Atg7 (autophagy-related protein-7), NAE (NEDD8-activating enzyme), UAE (ubiquitin activating enzyme) and SAE (SUMO-activating enzyme) with appropriately tagged ubiquitin-like proteins and E2 conjugating enzymes as described in the Methods. GABARAP = GABAA receptor-associated protein. Data represent mean ± SEM. n = Number of independent experiments.
Fig 3.
Pharmacokinetics profiles and in vivo efficacy of ML471.
(A, B) Rat pharmacokinetics for ML471. Rats were dosed with ML471 at 1 mg/kg i.v. (blue) or 1, 10 or 25 mg/kg p.o. (green, red, orange), and blood (A) and plasma (B) samples were collected for analysis. See S7 Table for pharmacokinetics values. (C) Pharmacokinetics profile (in blood), for SCID mice engrafted with human RBCs infected with P. falciparum, over the first day following treatment with ML471 at 100 or 200 mg/kg p.o.. See S8 Table for pharmacokinetics values. (D) Therapeutic efficacy of ML471 in the SCID mouse P. falciparum model, dosed with ML471 at 100 or 200 mg/kg p.o. on Day 3 post-infection (arrowed). The chloroquine data are from [16].
Fig 4.
Identification of ML471 conjugates in P. falciparum and effects of nucleoside sulfamates on enzyme stability and activity.
P. falciparum-infected RBCs were treated with 1 μM ML471 for 2 h. Extracts were subjected to LCMS and the expected mass for amino acid-ML471 conjugates searched. (A) Upper panel shows the extracted ion chromatograms of the anticipated Tyr-ML471 adduct at m/z 552.1871 extracted from ML471 treated P. falciparum culture (black trace) and untreated control (red trace). Lower panel shows the synthetic Tyr-ML471 conjugate at 0.2 μM. The inset shows the MS analysis of the parasite-generated Tyr-ML471, and the structure of Tyr-ML471. Profiles are typical of data from 3 independent experiments. (B,C) First derivatives of melting curves for PfTyrRS (B) and HsTyrRS (C) (2.3 μM) in the apo form or after incubation at 37°C with ML901, ML471 or AMS, in the presence of 10 μM ATP and 20 μM tyrosine. For PfTyrRS, 50 μM nucleoside sulfamate and 4 μM PftRNATyr were incubated with substrates for 2 h. For HsTyrRS, 200 μM nucleoside sulfamate and 8 mg/mL yeast tRNA were incubated with substrates for 4 h. Data are representative of three independent experiments. (D) ATP consumption by PfTyrRS in the presence and absence of the cognate tRNATyr. ATP consumption in the absence of tRNATyr derives from turnover of Tyr-AMP generated in the initial phase of the TyrRS reaction. The reaction component concentrations are: PfTyrRS (25 nM), ATP (10 μM), tyrosine (200 μM), pyrophosphatase (1 unit/mL) and cognate tRNATyr (4.8 μM), if present; and incubations were at 37°C for 1 h. Data are the average of three independent experiments and error bars correspond to SEM. (E) Effects of increasing concentrations of ML471, ML901 and AMS on ATP consumption by PfTyrRS. Assay conditions are the same as in (D), with cognate tRNATyr. Data represent mean ± SEM from three or four independent experiments.
Table 3.
Activities of pyrazolopyrimidine sulfamates in selected biochemical assays.
ATP consumption by PfTyrRS (25 nM) in the presence of ATP (10 μM), tyrosine (200 μM), pyrophosphatase (1 unit/mL) and cognate tRNATyr (4.8 μM), was measured using the Kinase Glo assay after incubation at 37°C for 1 h. The Tm values for PfTyrRS and HsTyrRS (2.3 μM) were measured in the apo form or after incubation at 37°C for 2 h (PfTyrRS) or 4 h (HsTyrRS) with the nucleoside sulfamates (50 μM with PfTyrRS and 200 μM with HsTyrRS) in the presence of 10 μM ATP, 20 μM tyrosine, 4 μM cognate tRNATyr (PfTyrRS) or 8 mg/mL yeast tRNA (HsTyrRS). *KD values (apparent) are estimated from differential scanning fluorimetry (DSF) analysis using an irreversible protein thermal unfolding model that has been described previously [59]. Data values represent mean ± SEM. AMS = Adenosine 5’-sulfamate. n = Number of biological repeats.
Fig 5.
Docking of ML901 and ML471 into structures of PfTyrRS and UAE provides insights into selectivity.
(A) Active site of PfTyrRS/Tyr-ML901 (7ROS) B-chain (His70 depicted in green) with docked ML901 (aqua carbons). The model is overlayed with ML901 (depicted with yellow carbons) with the pose adopted upon docking into the A-chain. (B) Active site of PfTyrRS/Tyr-ML901 (7ROS) B-chain (His70 depicted in green) with docked ML471 (aqua carbons). The model is overlayed with ML471 (depicted with yellow backbone) with the pose adopted upon docking into the A-chain. The red arrow illustrates the different conformations adopted by the difluoromethoxy and isopropyl groups. (C, D) The structure of 7ROS B-chain with bound Tyr-ML901 is overlayed with B-chain-docked ML901 (C) and ML471 (D). The red arrows illustrate the different conformations adopted by the difluoromethoxy and isopropyl groups. The purple arrows illustrate the twisted ribose group in the Tyr-ML901 conjugate. By contrast, in the docked nucleoside sulfamates, the ring systems are co-planar. (E,F) ML901 (E) and ML471 (F) were docked into the ATP-binding site of human UAE (6DC6). A H-bond made by ML901 with residue Arg 551 is indicated with a red arrow. Asn577 and Arg551 (blue arrows) flank the hydrophobic isopropyl group in the ML471 dock.
Fig 6.
Comparison of the crystal structures of Tyr-ML471- and Tyr-ML901-bound PfTyrRS reveals differential mobility of the “KMSKS” loop.
(A) Crystal structure of the dimeric PfTyrRS/Tyr-ML471 complex showing chain A (green), chain B (blue), and bound Tyr-ML471 (red, stick representation). (B) Architecture of the B-chain of PfTyrRS with bound Tyr-ML471, showing direct interactions with active site residues. (C) B-chain of Tyr-ML471-bound PfTyrRS showing the poses adopted by the ML471 isopropyl group (blue arrow) and His70, which are incompatible with a structured KMSKS loop. (D) B-chain of Tyr-ML901-bound PfTyrRS (7ROS). The conformation of the ML901 difluoromethoxy group (red arrow) allows His70 to interact with Met248 of the KMSKS loop, leading to stabilisation. (E) Overlay of the B-chains of Tyr-ML471- and Tyr-ML901-bound PfTyrRS.