Identification of drug-like molecules targeting the ATPase activity of dynamin-like EHD4
Saif Mohd, Andreas Oder, Edgar Specker, Martin Neuenschwander, Jens Peter Von Kries, Oliver Daumke
Abstract
Eps15 (epidermal growth factor receptor pathway substrate 15) homology domain-containing proteins (EHDs) comprise a family of eukaryotic dynamin-related ATPases that participate in various endocytic membrane trafficking pathways. Dysregulation of EHDs function has been implicated in various diseases, including cancer. The lack of small molecule inhibitors which acutely target individual EHD members has hampered progress in dissecting their detailed cellular membrane trafficking pathways and their function during disease.
Introduction
Dynamin superfamily proteins are involved in various cellular membrane remodeling events [1]. They harness energy from nucleotide hydrolysis to perform mechano-chemical work. The proteins oligomerize on the surface of membranes, leading to stimulation of their low basal nucleotide hydrolysis activity [2]. While most dynamin superfamily proteins are GTP-binding and hydrolyzing enzymes, Eps15-homolgy domain-containing proteins (EHDs) comprise a highly conserved dynamin-related ATPase family, with four members in eukaryotes (EHD1-4) [3].
Materials and method
EHD4 expression and purification
An N-terminal truncated construct of mouse EHD4 (Uniprot Q1MWP8, residues 22–541, EHD4ΔN) was expressed as N-terminal His6-fusion followed by a PreScission cleavage site in E. coli BL21 DE3 Rosetta (Novagen) from a modified pET28 vector, as described in [7]. Bacterial cultures were grown in Terrific Broth medium (TB) to an OD600 of 0.8 at 37°C. Protein expression was induced at 18°C by adding 100 μM isopropyl-ß-D-1-thiogalactopyranoside (IPTG) and cultures were grown for another 20 h at 18°C. Cells were sedimented, resuspended in 50 mM HEPES pH 7.5, 500 mM NaCl, 25 mM imidazole, 2.5 mM β-mercaptoethanol, 2 mM MgCl2 and stored at -20°C.
Results
Developing an EHD4 ATPase assay suitable for drug screening
We aimed for developing a prototypic small molecule inhibitor for the EHD family. From the four mammalian EHDs, only EHD2 and EHD4 can be bacterially expressed and purified in quantities compatible with HTS (S2A Fig) [4, 7]. EHD2 has a very low basal ATPase activity (kcat ~1 h-1) which can only be moderately stimulated in the presence of lipids (kcat~ 0.1 min-1) [4]. This low activity is not suited for reliable HTS.
Discussion
There are no chemical inhibitors for EHD proteins described to date. This study reports the identification of an EHD4 inhibitor by establishing a robust, reproducible EHD4 ATPase assay in the presence of liposomes, which is compatible with drug screening. Several problems had to be overcome during optimization of the MLG-based assay. The basal ATPase activity of EHD4ΔN is hardly above the background in the MLG-based assay and could therefore not be used for inhibitor screening.
Acknowledgments
We thank Dr. Marc Nazaré, Dr. Peter Lindermann and Jerome Paul for usage of and support with their Rotavac and Regina Piske and Helmut Kettenmann for granting continuous access to their plate reader. We thank the online scientific community for intense discussions, especially Dr. Adam Shapiro (Entasis Therapeutics), and Marius Weismehl for critical reading of the manuscript.
Citation: Mohd S, Oder A, Specker E, Neuenschwander M, Von Kries JP, Daumke O (2024) Identification of drug-like molecules targeting the ATPase activity of dynamin-like EHD4. PLoS ONE 19(7): e0302704. https://doi.org/10.1371/journal.pone.0302704
Editor: Ludger Johannes, Institut Curie, FRANCE
Received: April 9, 2024; Accepted: July 13, 2024; Published: July 29, 2024
Copyright: © 2024 Mohd et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: “We thank Deutsche Forschungsgemeinschaft (SFB958, project A12, to O.D.) and MDC for their funding and support. "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
