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

Abstract

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 IB kinase (IKK) and TANK binding kinase 1 (TBK1) to promote energy expenditure and improve insulin sensitivity. 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 the inhibitory effects. IKK is less affected by these modifications, possibly due to variation in its hinge that allows for increased conformational plasticity. Installation of a tetrazole carboxylic acid bioisostere improved potency to 200 and 400 nM toward IKK and TBK1, respectively. Despite improvements in the in vitro potency, no analog produced a greater response in adipocytes than amlexanox, perhaps because of altered absorption and distribution. The structure-activity relationships and cocrystal structures described herein will aid in future structure-guided inhibitor development using the amlexanox pharmacophore for the treatment of obesity and type 2 diabetes.

Essential Roles for the Non-Canonical IκB Kinases in Linking Inflammation to Cancer, Obesity, and Diabetes

“…In a clinical trial of amlexanox for 42 patients with obesity and T2D or nonalcoholic fatty liver disease (NCT01975935), a subset of patients responded with a reduction in blood glucose [116]. While designing and validating analogues of amlexanox with more potent TBK1/IKKε inhibition activities and minimal toxicity are in progress [161,162], PIAA exhibited higher potency than amlexanox in β-cell regeneration in diabetic zebrafish with minimal deleterious effects [48]. Thus, further creation of new molecular structures with potent TBK1 and/or IKKε inhibition activities and minimal toxicity using the PIAA as a scaffold will allow us to develop legitimate strategies for maintaining energy and glucose homeostasis, and impeding subsequent tumor progression.…”

Synthesis of deuterium‐labelled amlexanox and its metabolic stability against mouse, rat, and human microsomes

“…Each is an average of three separate determinations. Since the isopropyl group on amlexanox is solvent exposed, incorporation of deuterium is not expected to affect inhibitor potency. Indeed, the in vitro potencies of amlexanox ( 1 ) and d 2 ‐amlexanox ( 5 ) against TBK1 are 0.6 ± 0.1μM and 1.1 ± 0.3μM, respectively (Figure and Supporting Information).…”

“…The structures of metabolites 2 and 3 have been confirmed by synthesis. 13 As part of a program toward making analogues of amlexanox, 14,15 we decided to synthesize a derivative 5 ( Figure 1) in which deuterium is introduced into two sites of metabolism on the C-7 isopropyl function. The deuterium kinetic isotope effect associated with placing deuterium at the site of metabolic derivatization slows metabolic activation and thus is expected to increase the lifetime of the active drug in vivo.…”

S100 proteins as therapeutic targets

“…While these anti-allergic compounds exhibit promising effects on S100-mediated pathologies they are not selective S100 inhibitors. For example, amlexanox also inhibits IκB kinase ε and TANK-binding kinase 1, proteins that promote a proinflammatory response associated with the development of obesity (Beyett et al 2018;Reilly et al 2013). These observations suggest that the antiinflammatory responses observed with amlexanox and other anti-allergics are likely due to the modulation of multiple cellular pathways.…”