Disruption of endocytosis through chemical inhibition of clathrin heavy chain function

Abstract

Clathrin-mediated endocytosis (CME) is a highly conserved and essential cellular process in eukaryotic cells, but its dynamic and vital nature makes it challenging to study using classical genetics tools. In contrast, although small molecules can acutely and reversibly perturb CME, the few chemical CME inhibitors that have been applied to plants are either ineffective or show undesirable side effects. Here, we identify the previously described endosidin9 (ES9) as an inhibitor of clathrin heavy chain (CHC) function in both Arabidopsis and human cells through affinity-based target isolation, in vitro binding studies and X-ray crystallography. Moreover, we present a chemically improved ES9 analog, ES9-17, which lacks the undesirable side effects of ES9 while retaining the ability to target CHC. ES9 and ES9-17 have expanded the chemical toolbox used to probe CHC function, and present chemical scaffolds for further design of more specific and potent CHC inhibitors across different systems.

Figure 1. ES9 binds clathrin heavy chain.

(a) and (b) Protein extracts from Arabidopsis PSB-D cell cultures were treated with ES9 (250 μM) or DMSO (Ø) for 30 min and the thermal denaturation curves for endogenous clathrin heavy chain (CHC) (a) and tubulin (b) were recorded across 12 temperature points (30-65°C). The relative band intensity compared to the lowest temperature (30°C) sample was measured by Western blot with anti-CHC and anti-tubulin antibodies. Error bars in (a) and (b) indicate standard error of the mean (SEM) of three biological replicates. For the uncropped blots, see Supplementary Fig. 9. (c) Protein extracts from Arabidopsis PSB-D cell cultures were incubated with ES9 (250 μM) or DMSO (Ø) for 30 min and digested with different concentrations of pronase. An undigested sample was included as control. The relative band intensity compared to the DMSO control was measured by Western blot with anti-CHC and anti-ATP synthase subunitβ (ATPβ) antibodies. Error bars indicate SEM, individual data points are shown. *P<0.05, for a one-way analysis of variance (ANOVA) test with a Dunnett’s multiple comparisons test, and compared to the undigested control; (n=3); n, biological replicates. For the uncropped blots, see Supplementary Fig. 11. (d) Changes in the thermodynamic stability of the Arabidopsis CHC1 N-terminal domain in the presence of different concentrations of ES9 and the inactive analog ES9-6. Controls are respective to ES9 and ES9-6 treatments. The data shown are representative of three experiments. RFU, relative fluorescence units.

Figure 2. ES9 binds the terminal domain of clathrin heavy chain.

(a) The structure of the human clathrin heavy chain1 (CHC1) N-terminal domain (nTD) (green cartoon) is shown in complex with ES9 (salmon) (b). Omitted electron density (2Fo–Fc) of ES9, contoured at 1σ level, is observed inside the hydrophobic clathrin box, which is located between the first and second blades of the nTD. (c) The interaction distances between the nTD residues and ES9 are shown with yellow dashes. The zoom-in view of the benzonitro moiety of ES9 indicates that the oxygen of the nitro group interacts electrostatically with R64. (d) A 60-degree rotated view of ES9 focuses on the bromothiophene moiety. Whereas the sulfur atom of the thiophene group is shown in close contact to the carboxyl oxygen of S67, the bromide atom points toward the carboxyl oxygen of L82 by a σ hole.

Figure 3. ES9-17 is not a protonophore.

(a) Structure of ES9-17 (14). (b) Dose-response of ES9-17-mediated FM4-64 uptake inhibition. The data are plotted as the ratio of the intracellular over the plasma membrane (PM) FM4-64 fluorescence intensities. The EC₅₀ is 13 μM. At least 3 cells were measured per seedling, n=6 seedlings per treatment. (c) Strongly reduced FM4-64 uptake (2 μM, 30 min) in Arabidopsis root epidermal cells in the presence of ES9-17 (30 μM) when compared to the DMSO (Ø) control. The samples were pretreated with either ES9-17 (30 μM) or DMSO for 30 min. (d) Measurements of the ATP concentration in wild type Arabidopsis PSB-D cell cultures treated for 30 min with ES9 (10 μM), ES9-17 (30 μM) and ES9-6 (50 μM), showing ATP concentration relative to the DMSO control. Error bars indicate standard error of the mean of three biological replicates. (e) Confocal images of Arabidopsis epidermal root cells stained with Lyso Tracker Red DND 99 (30 min) and treated additionally for 30 min with DMSO (Ø), ES9 (10 μM) or ES9-17 (30 μM). Scale bars, 10 μm.

Figure 4. ES9-17 is a CME inhibitor.

(a) ES9-17 inhibited the recruitment of the plasma membrane-localized BRI1-GFP to the Brefeldin A (BFA) body. Samples were pretreated with 50 μM cycloheximide (CHX) for 1 h and treated with either DMSO (Ø) or ES9-17 (30 μM) for 30 min, followed by a combined application of FM4-64 (2 μM) and BFA (50 μM) for an additional 30 min in the presence of the inhibitor. Scale bar, 10 μm. (b) Confocal images of Arabidopsis root epidermal cells expressing PEPR1-GFP. Seedlings were treated with ES9-17 (30 μM) or DMSO (Ø) for 30 min, followed by elicitation with 100 nM Pep1. The internalization of PEPR1-GFP was followed until 90 min post elicitation. Scale bar, 20 μm. (c) Histograms representing the measured endocytic foci life time of clathrin light chain1 (CLC1)-GFP (CLC1::CLC1-GFP/Col-0) in the presence of ES9-17 (30 μM) or DMSO (Ø), n=1276 and 1069 measurements, from 10 and 4 seedlings respectively. Kymographs represent a line trace (horizontal axis) over a time period (vertical axis), taken from a time lapse (2 f/sec) of Arabidopsis root epidermal cells, that illustrate the life times of endocytic foci labeled by CHC1-GFP. Scale bar, 10 sec.

Figure 5. ES9-17 targets clathrin heavy chain.

(a) and (b) Protein extracts from Arabidopsis PSB-D cell cultures were treated with ES9-17 (250 μM) or DMSO (Ø) for 30 min. The thermal denaturation curves for endogenous clathrin heavy chain (CHC) (a) and tubulin (b) were observed across 12 temperature points (30-65°C). The relative band intensity compared to the lowest temperature (30°C) sample was measured by Western blot using anti-CHC and anti-tubulin antibodies. Error bars in (a) and (b) indicate standard error of the mean (SEM) of three biological replicates. For the uncropped blots, see Supplementary Fig. 9. (c) Protein extracts from Arabidopsis PSB-D cell cultures were incubated with ES9-17 (250 μM) or DMSO (Ø) for 30 min and digested with different concentrations of pronase. An undigested sample was included as a control. The relative band intensity compared to the DMSO control was measured by Western blot with anti-CHC and anti-ATP synthase subunitβ (ATPβ) antibodies. Error bars indicate SEM, individual data points are shown. **P<0.01 for a one-way analysis of variance (ANOVA) test with a Dunnett’s multiple comparisons test, and compared to the undigested control. n=4 biological replicates. For the uncropped blots, see Supplementary Fig. 12. (d) Boxplot representation of the root growth fold change over 48 h for wild type Arabidopsis Col-0 seedlings treated with ES9-17 (12 μM). Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by the R software, outliers are represented by dots. (n=80); n, number of seedlings in 3 independent experiments. *P<0.05, ***P<0.001 for a one-way analysis of variance (ANOVA) test with a Dunnett’s multiple comparisons test.