Small Molecule Conjugation of Hsp70 Inhibits Necroptosis

Necroptosis is a subtype of regulated necrosis in which signaling through receptor interacting protein kinase 3 (RIPK3) triggers caspase independent, immunogenic cell death morphologically identical to necrosis. Necroptosis is physiologically relevant in viral disease, ischemia reperfusion injury, necrotizing pancreatitis, and systemic inflammatory response syndrome. Although many necroptotic stimuli exist, our research focuses on TNF mediated necroptosis. Following TNF-TNFR1 binding and concurrent inhibition of caspase 8, Receptor Interacting Protein Kinase 1 and 3 (RIPK1/3) bind through their Receptor Homotypic Interacting Motif (RHIM) domains, activate via phosphorylation, and form an amyloid-like structure termed the necrosome. RIPK3 recruits and phosphorylates Mixed Lineage Kinase-Like (MLKL) protein. This induces MLKL polymerization, which putatively causes direct cell membrane disruption resulting in cell death. The steps downstream of the MLKL phosphorylation remain poorly defined. We designed and carried out a forward small molecule screen, which identified an inhibitor of necroptosis (Necroptosis Blocking Compound 1, NBC1) downstream of MLKL phosphorylation. Positive, negative, and biotinylated analogs of this compound led to identification of the drug target (Hsp70) by LC-MS. Biochemical data suggests that NBC1 conjugates Hsp70 and blocks necroptosis by inhibiting MLKL polymerization.