Fig. 3: Kell protease activity and its inhibition by thiorphan: effects on erythrocytes and malaria parasite growth.

A Bar graph depicting Kell protease activity on the erythrocyte surface. Erythrocytes (2 × 10⁶) were incubated with or without the substrate Glut-FAAF-AMC (10 µM), followed by the measurement of Kell’s proteolytic activity at excitation/emission wavelengths (λex/λem: 340/440 nm). Results are based on three independent biological replicates (n = 3). B Bar graph illustrating the inhibition of Kell protease activity on the erythrocyte surface in the presence of varying concentrations of Thiorphan. The data represent three independent biological replicates (n = 3). C Schematic representation (created by Biorender.com) of the zinc-dependent affinity purification process for isolating Kell from the erythrocyte surface, emphasizing the metalloprotease’s reliance on zinc for activity. D Bar graph demonstrating Thiorphan’s inhibitory effect on Kell’s enzymatic activity. The assay was performed using 500 ng of purified Kell in the presence of increasing concentrations of Thiorphan. Data represents three independent biological replicates (n = 3). E Bar graph illustrates the percentage of protease activity between wild-type (WT) and Kell_null derived reticulocytes under identical experimental conditions. Error bars represent standard deviations from the mean of three biological replicates (n = 3). Statistical significance was assessed using a two-tailed Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). F Schematic representation (created by Biorender.com) of the in vitro antimalarial activity of thiorphan in two experimental conditions in the first condition, uninfected red blood cells (RBCs) were pretreated with Thiorphan at various concentrations for 2 h at 37 °C, followed by extensive washing to remove the inhibitor. Purified schizonts were then added to the pretreated RBCs. In the second condition, schizonts were added to untreated RBCs in the presence of different concentrations of Thiorphan. After 10 h of incubation, the successful invasion was quantified by counting ring-stage parasites. G Dose-response curve showing the IC₅₀ values of thiorphan in two experimental conditions. Data points represent the average of three biological replicates (n = 3), with 95% confidence intervals (C.I.) calculated from nonlinear regression curves using GraphPad Prism 8.0 software. H Representative Giemsa-stained images comparing control and experimental conditions (at Thiorphan IC₅₀ concentration) for both experimental conditions. I, J Dose-response curves showing 72-h growth inhibition assays with Thiorphan on Plasmodium strains Pf3D7 and PfRKL-9 at different concentrations. Giemsa-stained images illustrate untreated parasites and those treated with Thiorphan at IC₅₀ concentrations. All experiments were conducted with at least three independent biological replicates (n = 3), and the data were presented as the mean ± standard deviation (SD). Statistical significance is denoted as *p < 0.05 and **p < 0.01 calculated through the student’s t-test.