Carboxylic Acid Derivatives of Amlexanox Display Enhanced Potency toward TBK1 and IKKepsilonand Reveal Mechanisms for Selective Inhibition.

Chronic, low-grade inflammation is a hallmark of obesity, which is a risk factor for the development of type 2 diabetes. The drug amlexanox inhibits inhibitor of NF-κB kinase ϵ (IKKϵ) and TANK binding kinase 1 (TBK1) to promote energy expenditure and improve insulin sensitivity. Recent clinical studies have demonstrated efficacy in a subset of diabetic patients with underlying adipose tissue inflammation, albeit with moderate potency, necessitating the need for improved analogs. Herein we report crystal structures of TBK1 in complex with amlexanox and a series of analogs that modify its carboxylic acid moiety. Removal of the carboxylic acid or mutation of the adjacent Thr156 residue significantly reduces potency toward TBK1, whereas conversion to a short amide or ester nearly abolishes inhibitory effects. IKKϵ is less affected by these modifications, possibly due to variation in its hinge residues allowing for increased conformational plasticity. Installation of a tetrazole carboxylic acid bioisostere improved potency to 200 and 400 nM towards IKKe and TBK1, respectively. Despite improvements in in vitro potency, no analog produced a greater response in adipocytes than amlexanox, perhaps because of altered absorption and distribution. The structure-activity relationships (SAR) and co-crystal structures described herein will aid in future structure-guided inhibitor development utilizing the amlexanox pharmacophore for the treatment of obesity and type 2 diabetes.