High mobility group box 1 (HMGB1) is a damage-associated molecular pattern (DAMP) protein that mediates inflammatory responses after infection or injury through toll-like receptor 4 (TLR4). Recently, our group has (1) shown that TLR4/myeloid differentiation factor 2 (MD-2)/HMGB1 complex formation is required for HMGB1 to induce cytokine production, and (2) identified a peptide inhibitor of HMGB1 (P5779) that acts by directly interrupting HMGB1/MD-2 binding. To understand the mechanism of HMGB1-triggered TLR4 activation, we explored the protein-protein interactions by modeling and simulating HMGB1:TLR4:MD-2 complex formation. In addition, using fingerprint similarity searches and docking and molecular dynamic (MD) simulation studies, we identified several folic acid derived-drugs as P5779 mimetopes. In surface plasmon resonance (SPR) binding studies, these drugs showed direct interaction with TLR4/MD-2 but not HMGB1. They also inhibited HMGB1 and MD-2 binding and suppressed HMGB1-induced TNF release in human macrophages in the nanomolar range. We assert from our findings that the anti-inflammatory effects of these folic acid analogue drugs are mediated at least partially through inhibition of TLR4-dependent signaling.

Folic acid derived-P5779 mimetics regulate DAMP-mediated inflammation through disruption of HMGB1:TLR4:MD-2 axis

256th National Meeting (2018)

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