Autoimmune diseases, such as psoriasis and arthritis, show a patchy distribution of inflammation despite systemic dysregulation of adaptive immunity. Thus, additional tissue-derived signals, such as danger-associated molecular patterns (DAMPs), are indispensable for manifestation of local inflammation. S100A8/S100A9 complexes are the most abundant DAMPs in many autoimmune diseases. However, regulatory mechanisms locally restricting DAMP activities are barely understood. We now unravel for the first time, to our knowledge, a mechanism of autoinhibition in mice and humans restricting S100-DAMP activity to local sites of inflammation. Combining protease degradation, pull-down assays, mass spectrometry, and targeted mutations, we identified specific peptide sequences within the second calcium-binding EF-hands triggering TLR4/MD2-dependent inflammation. These binding sites are free when S100A8/S100A9 heterodimers are released at sites of inflammation. Subsequently, S100A8/S100A9 activities are locally restricted by calcium-induced (S100A8/S100A9)2 tetramer formation hiding the TLR4/MD2-binding site within the tetramer interphase, thus preventing undesirable systemic effects. Loss of this autoinhibitory mechanism in vivo results in TNF-α-driven fatal inflammation, as shown by lack of tetramer formation in crossing S100A9-/- mice with 2 independent TNF-α-transgene mouse strains. Since S100A8/S100A9 is the most abundant DAMP in many inflammatory diseases, specifically blocking the TLR4-binding site of active S100 dimers may represent a promising approach for local suppression of inflammatory diseases, avoiding systemic side effects.

tTF-NGR consists of the extracellular domain of tissue factor and the peptide GNGRAHA, a ligand of the surface protein aminopeptidase N and of integrin αvβ3. Both surface proteins are upregulated on endothelial cells of tumor vessels. tTF-NGR shows antitumor activity in xenografts and inhibition of tumor blood flow in cancer patients. We performed random TMS(PEG)12 PEGylation of tTF-NGR to improve the antitumor profile of the molecule. PEGylation resulted in an approximately 2-log step decreased procoagulatory activity of the molecule. Pharmacokinetic studies in mice showed a more than 1-log step higher mean area under the curve. Comparison of the LD10 values for both compounds and their lowest effective antitumor dose against human tumor xenografts showed an improved therapeutic range (active/toxic dose in mg/kg body weight) of 1/5 mg/kg for tTF-NGR and 3/>160 mg/kg for TMS(PEG)12 tTF-NGR. Results demonstrate that PEGylation can significantly improve the therapeutic range of tTF-NGR.

Mechanistic promiscuity: The (2-alkyl)-3-hydroxy-4(1H)-quinolone-cleaving dioxygenase Hod has an α/β-hydrolase fold and a Ser/His/Asp triad in its active site. Isatoic anhydride, a suicide substrate of serine hydrolases, inactivates Hod by covalent modification of the active-site serine, thus indicating that the α/β-hydrolase fold can accommodate dioxygenase chemistry without completely abandoning hydrolase-like properties.

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