In vitro pharmacological profiling of ITE amide bioisosteric aryl hydrocarbon receptor agonist

Abstract

Previously, we reported ITE-derived bioisostere 2-(1 H -indole-3-carbonyl)-N-methyl thiazole-4-carboxamide (ITE-CONHCH 3 ) as a high-affinity ligand and potent agonist of the aryl hydrocarbon receptor (AhR) with anti-colitis properties. In the current study, we present its initial in vitro pharmacological profiling. Physicochemical analysis revealed low aqueous solubility (22 µM), excellent chemical stability in human and mouse plasma, and strong plasma protein binding (>98%). Off-target screening against steroid and nuclear receptors confirmed high AhR selectivity. In Caco-2 cells, ITE-CONHCH 3 exhibited high permeability with no active efflux. Interestingly, the compound partitioned substantially into mouse red blood cells (47%) but only weakly into human RBCs (16%). In reporter gene assays, ITE-CONHCH 3 displayed ∼40-fold higher agonist potency in human cells compared to mouse cells. Its ability to activate AhR was further validated by a cell-free AhR-ARNT heterodimerization assay and by the induction of canonical ( CYP1A1 ) and non-canonical ( PAI-1 ) genes expression in Caco-2 cells. In conclusion, ITE-CONHCH 3 possesses a favorable pharmacological profile supporting its potential preclinical development. However, the observed differences in RBC partitioning and AhR potency between species must be considered when interpreting mouse-derived data.