Figure 4. Synthesis and activity of 3, a specific PfA-M17 inhibitor.

(A) Scheme 1. Synthesis of 3: (i) Boronic acid, Pd(PPh₃)₂Cl₂, Na₂CO₃, THF, 100 °C, 2 hr, (ii) NH₂OH.HCl, KOH, RT, 16 hr. Inhibition constant for 3 toward recombinant, purified PfA-M17 is shown. (B) Binding mode of 3 bound to PfA-M17. Solvent-accessible surface of PfA-M17 (grey) with active site ions shown in grey spheres. Stick representation (magenta) shows the binding positions of 3. Molecular interactions between 3 and PfA-M17 are indicated by dashed lines; water molecules are represented by red spheres. (C) Killing action of 3 over 72 hr as determined by SYBR Green I assay. The EC₅₀ value was calculated from four biological replicates performed in triplicate and data plotted as the mean ± standard error of the mean. (D) Parasite killing rate was determined by incubating Pf3D7 parasites in 10 x EC₅₀ as previously determined for either 24 or 48 hr before the drug was washed off and parasites allowed to grow for a further 48 hr. Survival was determined via Sybr Green I assay and compared to vehicle (DMSO)-treated controls. Shown is the mean ± standard deviation (n=4). Statistical significance was determined using a one-way ANOVA. (E) Synchronized parasites at 4 hr post-invasion (hpi) were treated over two cycles (C1, cycle 1; C2, cycle 2) with either 5 x or 10 x EC₅₀ or DMSO at the concentration present in the 10 x EC₅₀ treatment. Representative Giemsa-stained smears from two biological replicates show delay in parasite maturation to schizogony (5 x EC₅₀) or trophozoite stage (10 x EC₅₀).

Figure 4—figure supplement 1. Compound 3 is a potent and selective PfA-M17 inhibitor.

Dose response curves showing aminopeptidase activity (fluorescence units per sec, FU/s) in the presence of increasing concentration (shown as log nM) of 3 for PfA-M1 (A) and PfA-M17 (B). Three separate dose-response curves prepared from three separate protein purifications are shown, with the final K_i (Bwire et al., 2020) value is the mean ± SEM of the three independent values (n=3). (C) Binding of 3 (magenta sticks) to PfA-M17 (grey cartoon). Interactions between 3 and PfA-M17 are shown by black dash lines and distances (Å) of key interactions (excluding zinc coordination) are indicated above dashed lines. Residues involved in hydrogen bonding and hydrophobic packing interactions are shown in grey sticks and residues numbers are indicated.