Pyroptosis inhibiting nanobodies block Gasdermin D pore formation

Abstract

Gasdermin D (GSDMD) is a key mediator of pyroptosis, a pro-inflammatory form of cell death occurring downstream of inflammasome activation as part of the innate immune defence. Upon cleavage by inflammatory caspases, the N-terminal domain of GSDMD forms pores in the plasma membrane resulting in cytokine release and eventually cell death. Targeting GSDMD is an attractive way to dampen inflammation. In this study, six GSDMD targeting nanobodies were characterized in terms of their binding affinity, stability, and effect on GSDMD pore formation. Three of the nanobodies inhibited GSDMD pore formation in a liposome leakage assay, although caspase cleavage was not perturbed. We determined the crystal structure of human GSDMD in complex with two nanobodies at 1.9 Ang resolution, providing detailed insights into the GSDMD-nanobody interactions and epitope binding. The pore formation is sterically blocked by one of the nanobodies that binds to the oligomerization interface of the N-terminal domain in the multi-subunit assembly. Our biochemical and structural findings provide new tools for studying inflammasome biology and build a framework for the design of novel GSDMD targeting drugs.

Antagonistic nanobodies reveal mechanism of GSDMD pore formation and unexpected therapeutic potential

“…As the inhibitory nanobodies stabilize GSDMD into a monomeric intermediate, they can function as valuable tools to further elucidate molecular details of membrane insertion and pore formation in relevant cell types using live cell microscopy. In a parallel study, Kopp et al solved the crystal structure of VHHGSDMD-2 and VHHGSDMD-6 in complex with full length GSDMD (Kopp et al, submitted) 49 . VHHGSDMD-2 binds to an epitope of full length GSDMD that forms the oligomerization interface in GSDMD pores, confirming the biochemically determined interference with oligomerization.…”