De novo Cyclic Peptides Allow Visualisation of the Monomeric and Functional Amyloid Conformations of the Kinase RIPK3

Receptor Interacting Protein Kinase 3 (RIPK3) is a central regulator of necroptosis and a key mediator of inflammatory signaling. Its function is orchestrated through RIP Homotypic Interaction Motif (RHIM)-dependent interactions with other RHIM-containing adapter proteins, forming amyloid-structured necrosomes that trigger kinase activation and subsequently lead to immunogenic cell lysis. Necroptosis eliminates infected or damaged cells and provokes a strong inflammatory response. Dysregulated necroptosis is implicated in chronic inflammatory diseases and associated with ischaemic injury. Despite separate structural elucidation of the isolated RIPK3 kinase domain and the amyloid fibrils formed by the RIPK3 RHIM-containing region, visualizing full-length human RIPK3 in live cells remains challenging due to a lack of specific tools. To address this, we employed Random Non-standard Peptide Integrated Discovery (RaPID) mRNA display to identify cyclic peptides that bind the RHIM-containing region of RIPK3 during its assembly into amyloid fibrils. Three peptides were selected for characterisation and demonstrate utility in visualizing RIPK3 in human cells. These peptides represent promising tools for probing RIPK3 localisation and modulating its function.