Various genetic alterations of the fibroblast growth factor receptor (FGFR) family have been detected across a wide range of cancers. However, inhibition of FGFR signaling by kinase inhibitors demonstrated limited clinical effectiveness. Herein, we evaluated the transforming activity and sensitivity of 160 nonsynonymous FGFR mutations and ten fusion genes to seven FGFR tyrosine kinase inhibitors (TKI) using the mixed-all-nominated-in-one (MANO) method, a high-throughput functional assay. The oncogenicity of 71 mutants was newly discovered in this study. The FGFR TKIs showed anti-proliferative activities against the wild-type FGFRs and their fusions, while several hotspot mutants were relatively resistant to those TKIs. The drug sensitivities assessed with the MANO method were well concordant with those evaluated using in vitro and in vivo assays. Comprehensive analysis of published FGFR structures revealed a possible mechanism through which oncogenic FGFR mutations reduce sensitivity to TKIs. It was further revealed that recurrent compound mutations within FGFRs affect the transforming potential and TKI-sensitivity of corresponding kinases. In conclusion, our study suggests the importance of selecting suitable inhibitors against individual FGFR variants. Moreover, it reveals the necessity to develop next-generation FGFR inhibitors, which are effective against all oncogenic FGFR variants.

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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.