Shooting three inflammatory targets with a single bullet: Novel multi-targeting anti-inflammatory glitazones.

In search for effective multi-targeting drug ligands (MTDLs) to address low-grade inflammatory changes of metabolic disorders, we rationally designed some novel glitazones-like compounds. This was achieved by incorporating prominent pharmacophoric motifs from previously reported COX-2, 15-LOX and PPARγ ligands. Challenging our design with pre-synthetic docking experiments on PPARγ showed encouraging results. In vitro tests have identified 4 compounds as simultaneous partial PPARγ agonist, potent COX-2 antagonist (nanomolar IC50 values) and moderate 15-LOX inhibitor (micromolar IC50 values). We envisioned such outcome as a prototypical balanced modulation of the 3 inflammatory targets. In vitro glucose uptake assay defined six compounds as insulin-sensitive and the other two as insulin-independent glucose uptake enhancers. Also, they were able to induce PPARγ nuclear translocation in immunohistochemical analysis. Their anti-inflammatory potential has been translated to effective inhibition of monocyte to macrophage differentiation, suppression of LPS-induced inflammatory cytokine production in macrophages, as well as significant in vivo anti-inflammatory activity. Ligand co-crystallized PPARγ X-ray of one of MTDLs has identified new clues that could serve as structural basis for its partial agonism. Docking of the most active compounds into COX-2 and 15-LOX active sites, pinpointed favorable binding patterns, similar to those of the co-crystallized ligands. Finally, in silico assessment of pharmacokinetics, physicochemical properties, drug-likeness and ligand efficiency indices was performed. Hence, we anticipate that the prominent biological profile of such series will rationalize relevant anti-inflammatory drug development endeavors.

An in vitro test system for compounds that modulate human inflammatory macrophage polarization.

Macrophages undergo activation by pathophysiological stimuli to pro-inflammatory and bactericidal, or wound-healing and anti-inflammatory phenotypes, termed M1 or M2, respectively. Dysregulation of the M1-M2 balance is often associated with inflammatory diseases. Therefore, mechanisms of macrophage polarization may reveal new drug targets. We profiled six compounds with claimed modulatory effects on macrophage polarization using peripheral blood monocyte-derived macrophages. Based on the distinct mRNA or protein expression in macrophages stimulated either with M1 [lipopolysaccharide (LPS) + interferon-γ, IFNγ] or M2 interleukin-4 (IL-4) stimuli, we selected a combination of M1 (IL1ß, tumor necrosis factor-α,TNFα, CC chemokine receptor 7, CCR7 and CD80) and M2 (chemokine (C-C motif) ligand 22, CCL22, CD200R and mannose receptor C type 1, MRC1) markers to monitor drug effects on "M1 polarization" or cells "pre-polarized to M1". Azithromycin (25-50 µM), tofacitinib (2.5-5 µM), hydroxychloroquine (40 µg/ml) and pioglitazone (15-60 µM) exhibit an anti-inflammatory profile because they downregulated M1 markers and upregulated some M2 markers when given both before and after M1 polarization. Lovastatin given before M1 polarization downregulated M1 marker genes but enhanced the M1 phenotype in macrophages pre-polarized with LPS and IFNγ. Methotrexate (1.25-5 µM) did not modulate macrophage polarization. We have, thus, established a test system suitable to identify novel compounds or repurposed drugs that modulate inflammatory macrophage plasticity. Compounds with potential to reduce expression of molecules involved in inflammatory T cell activation (IL-1ß, TNFα, CD80), while enhancing production of a major chemokine involved in recruitment of Tregs (CCL22) may be of interest for treating chronic inflammatory diseases.