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Disruption of endocytosis through chemical inhibition of clathrin heavy chain function


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.

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All accession codes supporting the findings of this study are available within the paper and its Supplementary Information. Structures are accessible under the PDB code 6E4L. There is no restriction on data availability.


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We thank S. Vanneste for fruitful discussions, R. Kumar for providing the pDONR211-AP2S plasmid, D. Martinez Molina for help with the CETSA protocol and M. De Cock for help in preparing the manuscript. This work was supported by the Research Foundation-Flanders (project No. G022516N to E.R., project No. G009415N to D.V.D and project No. G0E5718N to E.R. and J.F.); the European Research Council (ERC Co T-Rex, grant No. 682436 to D.V.D); the Deutsche Forschungsgemeinschaft (No. TRR186/A08 to V.H.); the Agency for Innovation by Science and Technology for postdoctoral (K.M.) and predoctoral (W.D. and S.D.M) fellowships; the China Science Council for a predoctoral fellowship (Q.L.); the joint research projects (Nos. VS.025.13N and VS.095.16N) within the framework of cooperation between the Research Foundation-Flanders and the Bulgarian Academy of Sciences (K.M.); and the Belgian Science Policy Office for a postdoctoral fellowship to non-EU researchers (I.S.).

Author information

W.D. and E.R. initiated the work. W.D., I.S. and E.R. designed the experiments. W.D., I.S., B.D., A.M. and J.W. performed SAR. W.D., K.M., A.S. and K.G. performed affinity purification and MS analysis. H.B. and V.H. performed the X-ray crystallography. I.S., S.D.M. and S.N.S. performed the in vitro binding assay. W.N. did the molecular docking. W.D., I.S. and Q.L. performed CETSA. W.D., I.S. and Q.L. performed DARTS. W.D., I.S., E.M., D.V.S., and D.V.D. carried out the imaging and data analysis. I.S. performed the cloning and generated transgenic Arabidopsis cell cultures. W.D., I.S., A.D. and D.A. performed ATP measurements. M.V. and J.F. contributed to the HeLa cell assays. K.Y. generated the TPLATE antibody. Q.L. and R.D.R performed TEM. W.D., I.S. and E.R. wrote the manuscript. All authors commented on the results and the manuscript.

Correspondence to Eugenia Russinova.

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Supplementary Figures 1–12, Supplementary Tables 1–3

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Further reading

Fig. 1: ES9 binds CHC.
Fig. 2: ES9 binds the terminal domain of CHC.
Fig. 3: ES9-17 is not a protonophore.
Fig. 4: ES9-17 is a CME inhibitor.
Fig. 5: ES9-17 targets CHC.