PMC

A, Structure of JCP174-BODIPY TMR (JCP174-BT) composed of chloroisocoumarin small-molecule JCP174 (red) and BODIPY TMR fluorophore (yellow). B, C, In-gel ABP competition labeling with JCP174-BT. (B) Purified recombinant HsAPT1 or HsAPT2 was pre-treated with different concentrations of JCP174, palmostatin B, or DMSO (0) before labeling with JCP174-BT. Samples were resolved by SDS-PAGE and fluorescent probe signal visualized using a flatbed scanner (upper panel), with loading assessed via silver stain (lower panel). (C) Purified recombinant TgPPT1 WT and active site mutant (S128A) were treated with JCP174, palmostatin B or DMSO (0) and labeled with JCP174-BT. Samples were analyzed as in (B).

A, Intact U-2 OS mammalian cells were labeled with different concentrations of JCP174-BT, washed to remove unbound probe, and lysed. Lysates were resolved by SDS-PAGE with fluorescent probe labeling visualized using a flatbed scanner. Carets indicate doublet of interest ~25 kDa previously identified as HsAPT1 and HsAPT2. B, Intact U-2 OS mammalian cells were labeled with JCP174-BT, washed to remove unbound probe, and lysed. HsAPT1 was immunoprecipitated from the lysate, with input, supernatant, and elution samples resolved by SDS-PAGE. Fluorescent probe signal was visualized via flatbed scanner (upper panel). HsAPT1 identity was confirmed by western blot (lower panel). The red caret indicates HsAPT1 species on the fluorescent scan. C, Intact T. gondii tachyzoites were labeled with different concentrations of JCP174-BT, washed to remove unbound probe and lysed. Lysates were resolved by SDS-PAGE with fluorescent probe labeling visualized using a flatbed scanner. Carets indicate bands of interest ~31 kDa. D, Intact wild-type T. gondii or ΔTgPPT1 tachyzoites were labeled with JCP174-BT as in (C). Carets indicate species corresponding to TgPPT1 in labeling of wild-type tachyzoites.

A, 4-NPO biochemical esterase activity assay schematic. 4-nitrophenyl octanoate is converted to 4-nitrophenolate (yellow) via esterase-mediated hydrolysis and measured at 401 nm by UV-Vis spectroscopy. B, Histogram of the results of the 4-NPO assay screen with a substituted chloroisocoumarin library. Residual esterase activity of HsAPT1 and HsAPT2 is shown for each compound. Structures of potent compounds are highlighted. * indicates dichloroisocoumarin (DCI). C, D, Dose-response curves for the chloroisocoumarin hits against HsAPT1 (C) and HsAPT2 (D) plotting residual activity versus molar concantration (M). E, ABP competition labeling with the pan-serine reactive ABP FP-rho. U-2OS cells were pre-treated with DMSO control, 50 μM ML348, or 50 μM ML349 before labeling with 0.5 μM FP-rho for 5 minutes. Samples were resolved by SDS-PAGE and visualized for fluorescent probe signal with a flatbed scanner. Carets indicate HsAPT1 and HsAPT2. F, ABP competition labeling with the pan-serine reactive ABP FP-rho. Intact cells were pre-treated with different concentrations of JCP174 or palmostatin B (Palmo B) before labeling with FP-rho. Samples were prepared as in (E). Carets indicate doublet of interest at ~25 kDa identified as HsAPT1 and HsAPT2.

A, Intact wild-type T. gondii or ΔTgPPT1 tachyzoites were labeled with JCP174-BT or FP-rho, washed to remove unbound probe and lysed. T. gondii tachyzoite lysate was labeled with FP-rho. Lysates were resolved by SDS-PAGE with fluorescent probe labeling visualized using a flatbed scanner. Carets indicate species corresponding to TgPPT1 in labeling of wild-type tachyzoites. Asterisk (*) indicates m-Cherry expressed in ΔTgPPT1 parasites. B, Intact U-2 OS cells were labeled with JCP174-BT or FP-rho, washed to remove unbound probe, and lysed. U-2 OS lysate was labeled with FP-rho. Lysates were resolved via SDS-PAGE. Fluorescent signal was visualized with a flatbed scanner (fluorescence scan), with the coomassie stain of the same gel shown to indicate loading. Carets indicate the species corresponding to HsAPT1 and HsAPT2. C, Intact mammalian cells were labeled with JCP174-BT and processed as in (A). Pairs of oncogenic cell lines from derived from three tissue types (carcinomas of the breast, ovary and prostate) were chosen to contrast low metastatic/aggressive potential (MCF7, OVCAR-3, and LNCaP) versus high metastatic potential (MDA-MB-231, SKOV-3, and PC-3) []. For each oncogenic pair, wedges indicate low to high metastatic potential. Fluorescent signal was visualized with a flatbed scanner (top panel). Total HsAPT1 protein level was visualized by western blot (middle panel). GAPDH was used as a loading control (lower panel).