Fig. 5: The M-833 series demonstrates target engagement for PfSTART1 while the mutant form prevents compound binding.

A Isothermal titration calorimetry (ITC) analysis of M-833 series and recombinant PfSTART(WT) or PfSTART(N330K). Representative thermograms of 90 µM PfSTART1 titrated into 10 µM M-833, W-991, and negative control 4. The bottom panel comprises the data after integration of the peaks and a fitted offset applied. The binding curve shows the fit to a single-site binding model. DP = differential power. The table represents summary of the mean affinity, enthalpy, and entropy obtained for M-833, W-991 and 4 binding to PfSTART1 from n = 2 experiments. Error represents standard deviation. ND = not determined. The second replicate and values for individual parameters are provided in Fig. S4C. B Solvent proteome profiling assays. Parasite lysate was treated with DMSO or 10 µM W-991, challenged with an acetic acid/ethanol/formic acid mixture (AEF) and soluble fractions extracted and analysed via western blots probed with anti-PfSTART1. Western blot replicates are shown in Fig. S4D. C Volcano plots depict differential soluble protein abundance analysis (moderated t-test based on limma package) of parasite lysate treated with W-991 (100 µM) or the DMSO control (n = 3 biological replicates, mean +/− SD) after solvent-induced protein precipitation (7–25% AEF). Non-significant (ns) proteins are plotted in grey, and significant stabilised proteins are in red. Hit selection cut-offs of 0.73 log2 fold-change and p < 0.01 are indicated with dashed lines. Significant stabilisation hits are shown in the bar graphs representing the average of three biological replicates of relative soluble protein abundance in DMSO- or W-991-treated parasite lysate samples after solvent-induced protein precipitation, plotted for Gradient 1 ‘G1’ (7–15% AEF) and Gradient 2 ‘G2’ (17-25% AEF). Error bars represent standard deviation. Source data are provided as a Source Data file.