Trimer-to-Monomer Disruption Mechanism for a Potent, Protease-Resistant Antagonist of Tumor Necrosis Factor-α Signaling

Aberrant tumor necrosis factor-alpha (TNF alpha) signaling is associated with many inflammatory diseases. The homotrimeric quaternary structure of TNF alpha is essential for receptor recognition and signal transduction. Previously, we described an engineered alpha/beta-peptide inhibitor that potently suppresses TNF alpha activity and resists proteolysis. Here, we present structural evidence that both the alpha/beta-peptide inhibitor and an all-alpha analogue bind to a monomeric form of TNF alpha. Calorimetry data support a 1:1 inhibitor/TNF alpha stoichiometry in solution. In contrast, previous cocrystal structures involving peptide or small-molecule inhibitors have shown the antagonists engaging a TNF alpha dimer. The structural data reveal why our inhibitors favor monomeric TNF alpha. Previous efforts to block TNF alpha-induced cell death with peptide inhibitors revealed that surfactant additives to the assay conditions cause a more rapid manifestation of inhibitory activity than is observed in the absence of additives. We attributed this effect to a loose surfactant TNF alpha association that lowers the barrier to trimer dissociation. Here, we used the new structural data to design peptide inhibitors bearing a surfactant-inspired appendage intended to facilitate TNF alpha trimer dissociation. The appendage modified the time course of protection from cell death.