Fig. 3.

Unique mode of action of AWZ1066S was indicated by its fast kill rate and was investigated through a proteomic target identification approach using two photoreactive chemical probes. (A) The Wolbachia depletion rates of AWZ1066S measured for three different treatment lengths (1, 2, and 6 d) in the B. malayi microfilaria time-kill assay. Doxycycline, minocycline, moxifloxacin, and rifampicin were used as positive controls (only doxycycline for day 1). Data are expressed as mean ± SD of five replicates for each time point. (B) Two photoreactive chemical probes used for target identification of AWZ1066S in Wolbachia. Structures (bifunctional, photoreactive, and clickable side chains are highlighted in red) and their anti-Wolbachia activity in the cell assay (n = 3). (C) Fifty-three putative protein targets of photoreactive probes 1 and 2 assigned (as shown in the Venn diagram) based on statistical testing [right-sided t test, permutation-based false discovery rate = 0.001, Artificial within groups variance (S0) = 3] of label-free quantification (LFQ) intensities measured in samples derived from probe-treated cells and control, DMSO-treated cells (n = 3). Protein targets presented in the left-hand block are from W. pipientis and in the right-hand block are from A. albopictus. Numbers within the heatmap legend represent Log₂ LFQ t test differences. Protein ID codes above the heatmap given in red indicate 22 proteins identified as common targets of both probes.