Pyroptosis inhibiting nanobodies block Gasdermin D pore formation

TLDR

Gasdermin D is a key mediator of pyroptosis, forming membrane pores after caspase cleavage that release cytokines and trigger cell death, making it an attractive target to reduce inflammation. The study characterizes six GSDMD‑targeting nanobodies for binding affinity, stability, and impact on pore formation. Structural analysis revealed that two nanobodies bind distinct epitopes on GSDMD, with one sterically blocking the oligomerization interface to prevent pore assembly. Three nanobodies effectively inhibit GSDMD pore formation without affecting caspase cleavage, offering a basis for inflammasome‑targeted therapeutics.

Abstract

Human 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 in the cytosol, 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 are characterized in terms of their binding affinity, stability, and effect on GSDMD pore formation. Three of the nanobodies inhibit GSDMD pore formation in a liposome leakage assay, although caspase cleavage was not perturbed. We determine the crystal structure of human GSDMD in complex with two nanobodies at 1.9 Å 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 pore assembly. Our biochemical and structural findings provide tools for studying inflammasome biology and build a framework for the design of GSDMD targeting drugs.