Indirubin

The aryl hydrocarbon receptor (AhR) was shown to be an important regulator of inflammatory processes at epithelial barriers, and is thus considered a therapeutic target for several chronic inflammatory diseases, such as inflammatory bowel disease. We aimed to identify and validate new AhR agonists that sustainably attenuate intestinal inflammation. Using a high-throughput luciferase reporter gene assay, 90 AhR ligands were identified out of 7448 approved and investigational drugs. Out of these, 15 AhR ligands were selected based on substance class, half maximal effective concentration, known toxicity and pharmacokinetic/pharmacodynamic profiles, and preclinical/clinical evaluation status for other indications. While Febuxostat, Nitazoxanide, Rabeprazole, 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester, 3-Indolepropionic acid, and Indirubin, were already known as AhR agonists, Nabumetone, Teriflunomide, Timapiprant/OC000459, and Caffeic acid phenylethyl ester have not yet been directly described in this context. Six compounds (Daidzein/Equol, as well as compounds no. 19, 22, 49, and 64, not yet disclosed due to pending patent applications) were newly described as AhR agonists. Hit compounds were studied in silico for their molecular interactions with AhR and in vitro for potential immunotoxicity and their ability to induce interleukin (IL)-10 and/or to suppress IL-1β in murine macrophages without significant cytochrome P450 1A1 induction in Caco-2 cells. Five compounds that met these criteria were functionally tested using organoid-based Transwell®-like models derived from gut biopsies. Five candidates restored the epithelial barrier, as evidenced by increased transepithelial electrical resistance and induction of the tight junction proteins claudin-1/-2 and occludin, while exhibiting anti-inflammatory effects, i.e., decreased expression of toll-like receptor 4. Out of these, one compound was selected for future in vivo preclinical studies.

Indirubin, a naturally occurring bisindole alkaloid, has demonstrated clinical efficacy in the management of chronic myeloid leukemia while exhibiting multiple pharmacological activities including antitumor, anti-inflammatory, and broad-spectrum antibacterial effects. To enhance indirubin biosynthesis in the engineered Escherichia coli BL21 (DE3) host, an integrated metabolic engineering strategy was systematically executed in a sequential manner. Firstly, the Methylophaga aminisulfidivorans flavin-containing monooxygenase (MaFMO) was heterologously overexpressed under the regulation of the strong P_tac promoter. Then, fermentation conditions were systematically optimized, including adjusting the pH to 9.5 and supplementing with 15 mM cysteine to facilitate the conversion of indole to indirubin. Under the conditions of 15 mM cysteine and pH 9.5, the indirubin yield reached 162.16 mg/L. Guided by hotspot prediction and literature precedents, rational mutagenesis was performed on MaFMO. Through mutation of key residues, engineered strain A125C achieved a yield of 187.36 mg/L. Subsequent structural analysis revealed that substitution with cysteine at residue 125 introduced enhanced side chain polarity and steric volume. Additionally, a NADPH regeneration plasmid was introduced to facilitate the recycling of cofactors. Finally, exogenous tryptophan (4 g/L) was added as the precursor, engineered strain A125C-mdh leaded to 203.64 mg/L indirubin.