Organisms use multiple forms of programmed cell death to protect against invading pathogens, including apoptosis and necroptosis. Necroptosis is highly immunogenic and causes lytic cell death. Dysregulation of necroptosis has been implicated in multiple disease states, including inflammatory bowel disease and ischaemic injury after stroke. Central to necroptosis is a functional amyloid complex referred to as the necrosome, which is comprised largely of the protein RIPK3. The necrosome forms when the ~18‑residue receptor homotypic interaction motif (RHIM) within RIPK3 adopts an amyloid cross-β structure and recruits additional monomers. Oligomerisation of RIPK3 results in autophosphorylation of this kinase and subsequent phosphorylation of downstream effector proteins, causing lytic cell death.
There are currently no small molecules available that selectively bind to the amyloidogenic RHIM region of RIPK3. Here, we have aimed to identify cyclic peptides that bind with high specificity to the RIPK3 RHIM region. The RIPK3 RHIM region was used as the target for screening against a cyclic peptide library in a selection known as random non-standard peptide integrated discovery (RaPID). From this screen, several novel cyclic peptide binders were identified. These peptides therefore have the potential to bind specifically to the RIPK3 RHIM amyloid fibril interface and may have use as diagnostic tools to detect RIPK3 functional amyloid formation. Further characterisation has revealed that these peptides independently form assemblies that display amyloid-like properties. Investigation of peptide:RIPK3 RHIM binding is underway using fluorescent colocalization studies, transmission electron microscopy and label-free assays utilising RIPK3 autofluorescence.