Delivery of caspase inhibitors through GSDMD pores to inhibit pyroptosis

Caspase-1, -4, -5, and -11 activate Gasdermin D (GSDMD) to form pores in the plasma membrane. In addition to releasing interleukin (IL)-1β and IL-18, GSDMD pores cause a lytic, proinflammatory form of cell death called pyroptosis. Blocking this pathway holds therapeutic promise for the treatment of inflammatory disorders, but clinical trials of cell permeable caspase inhibitors have been unsuccessful. Here, we describe covalent inhibitors of proinflammatory caspases that are impermeable to healthy cells but effectively block caspase-1 driven pyroptosis and IL-1β secretion. Their failure to inhibit apoptosis implies inhibitor entry through GSDMD pores. Propidium iodide entered rescued cells, confirming transient membrane permeabilization via caspase-1 and GSDMD. Caspase-1 inhibition prevented rather than delayed cell death, likely due to the activation of membrane repair mechanisms neutralizing the initial GSDMD pores. Notably, inhibiting caspase-1 and -11 suppressed IL-1β and IL-18 production in a mouse model of endotoxic shock. These findings underscore the therapeutic potential of exploiting GSDMD pores for the delivery of caspase inhibitors, offering a novel strategy for treating inflammatory diseases.