2. Academic Validation
  3. MLKL activity requires a splicing-regulated, druggable intramolecular interaction

MLKL activity requires a splicing-regulated, druggable intramolecular interaction

  • Mol Cell. 2025 Apr 17;85(8):1589-1605.e12. doi: 10.1016/j.molcel.2025.03.015.
Uris Ros 1,  Veronica Martinez-Osorio 2,  Pedro A Valiente 3,  Yasmin Abdelwahab 2,  Milos Gojkovic 2,  Raed Shalaby 2,  Silvia Zanna 2,  Julia Saggau 4,  Laurens Wachsmuth 2,  Harshal N Nemade 5,  Jonathan Zoeller 6,  Hannah Lottermoser 5,  Yu-Guang Chen 7,  Mohamed Ibrahim 8,  Konstantinos Kelepouras 4,  Lazaros Vasilikos 9,  Paula Bedoya 2,  Rafael A Espiritu 10,  Stefan Müller 2,  Veronika Altmannova 11,  D Peter Tieleman 12,  John Weir 11,  Julian Langer 6,  Matti Adam 5,  Henning Walczak 13,  W Wei-Lynn Wong 9,  Gianmaria Liccardi 4,  Martin Mollenhauer 5,  Manolis Pasparakis 2,  Nieves Peltzer 14,  Ana J García-Sáez 15
Affiliations

Affiliations

  • 1 Institute of Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne 50931, Germany; Max Planck Institute of Biophysics, Frankfurt am Main 60439, Germany. Electronic address: uris.ros@biophys.mpg.de.
  • 2 Institute of Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne 50931, Germany.
  • 3 Center for Protein Studies, Faculty of Biology, Havana University, Havana 10400, Cuba.
  • 4 Institute of Biochemistry I, Centre for Biochemistry, Faculty of Medicine, University of Cologne, Cologne 50931, Germany; Center for Molecular Medicine Cologne, Cologne 50931, Germany.
  • 5 Department for Experimental Cardiology, Faculty of Medicine, University of Cologne, and Clinic III for Internal Medicine, University Hospital Cologne, Cologne 50931, Germany.
  • 6 Max Planck Institute of Biophysics, Frankfurt am Main 60439, Germany.
  • 7 Centre for Cell Death, Cancer, and Inflammation (CCCI), UCL Cancer Institute, University College London, London WC1E 6DD, UK; Division of Hematology/Oncology, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei 11490, Taiwan.
  • 8 Institute of Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne 50931, Germany; Center for Molecular Medicine Cologne, Cologne 50931, Germany.
  • 9 Institute of Experimental Immunology, University of Zürich, Zürich 8057, Switzerland.
  • 10 Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen 72076, Germany.
  • 11 Friedrich Miescher Laboratory and Max Planck Institute, Tübingen 72076, Germany.
  • 12 Centre for Molecular Simulation, Department of Biological Sciences, University of Calgary, Calgary T2N 1N4, Canada.
  • 13 Institute of Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne 50931, Germany; Institute of Biochemistry I, Centre for Biochemistry, Faculty of Medicine, University of Cologne, Cologne 50931, Germany; Centre for Cell Death, Cancer, and Inflammation (CCCI), UCL Cancer Institute, University College London, London WC1E 6DD, UK.
  • 14 Institute of Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne 50931, Germany; Center for Molecular Medicine Cologne, Cologne 50931, Germany; Department of Genome Editing, Institute of Biomedical Genetics (IBMG), University of Stuttgart, Stuttgart 70569, Germany.
  • 15 Institute of Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne 50931, Germany; Max Planck Institute of Biophysics, Frankfurt am Main 60439, Germany. Electronic address: ana.garcia@biophys.mpg.de.
PMID: 40209701 DOI: 10.1016/j.molcel.2025.03.015
Abstract

Necroptosis is an inflammatory form of regulated cell death implicated in a range of human pathologies, whose execution depends on the poorly understood pseudokinase Mixed Lineage Kinase domain-like (MLKL). Here, we report that splicing-dependent insertion of a short amino acid sequence in the C-terminal α-helix (Hc) of MLKL abolishes cell killing activity and creates an anti-necroptotic isoform that counteracts cell death induced by the necroptosis-proficient protein in mice and humans. We show that interaction of Hc with a previously unrecognized hydrophobic groove is essential for Necroptosis, which we exploited in a strategy to identify small molecules that inhibit MLKL and substantially ameliorate disease in murine models of necroptosis-driven dermatitis and abdominal aortic aneurysm. Thus, alternative splicing of microexons controls the ability of MLKL to undergo an intramolecular rearrangement essential for Necroptosis with potential to guide the development of allosteric MLKL inhibitors for the treatment of human disease.

Keywords

MLKL; abdominal aortic aneurysm; drug discovery; inflammatory diseases; membrane permeabilization; microexons; necroptosis; skin inflammation; small molecule inhibitors; splicing variants.

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