Abstract Body (Do not enter title and authors here): Introduction/Background. The global prevalence of obesity has fueled a recent surge in anti-obesity medications. However, the undesirable complications of current medications necessitate the development of a newer generation of drugs. Despite the known role of EphB tyrosine kinases in insulin signaling, the druggability of these kinases remains largely unexplored. We resolved the crystal structure EphB1 tyrosine kinase along with chlortetracycline (PDB ID: 6UMW) and ADP (PDB ID: 7KPM) within the ATP-binding catalytic domain. This prompted us to explore the druggability potential of these kinases selectively in obesity preclinical settings.
Hypothesis. Our novel small molecules, not related to the tetracycline scaffold, will inhibit EphB tyrosine kinase forward signaling to introduce novel tools to investigate the mechanisms that reverse the progression of obesity and associated metabolic disorders.
Methods / Approach. We leveraged the resolved X-ray crystal structure to apply computational molecular modeling for designing and synthesis of novel thienopyridine-based analogs, not related to the tetracycline scaffold, named as STA analogs. This was followed by plethora of biochemical validation and preclinical testing in diet-induced obese mice models.
Results. This results in introduction of a series of 22 thienopyridine-based analogs (termed STA analogs) that showed potential pan-inhibitory profiles for EphB1, EphB2, and EphB4 tyrosine kinase signaling. Of these analogs, STA-013 potently inhibited EphB1 (IC₅₀ = 883 nM), EphB2 (IC₅₀ = 3.52 µM), and EphB4 (IC₅₀ = 1.02 µM) tyrosine kinases. This was supported with a full kinome profiling assay against 140 tyrosine and tyrosine-like kinases to show the selectivity of STA-013. Systemic injections of STA-013 resulted in weight loss and improved glucose clearance homeostasis significantly in high-fat diet-induced obese mice models. This was associated with evidence of an increase in metabolism during the light cycle. The molecular mechanism associated with STA-013 administration was fully investigated to show the inhibition of the EphB phosphorylated signal associated with increased p-AKT/AKT signaling. Also, STA-013 showed an elevated signal for InsR-β to suggest the reversal effect of insulin on InsR degradation to allow the restoration of glucose homeostasis.
Conclusions. Herein, we report the first-in-class pan-EphB tyrosine kinase inhibitors targeting obesity and associated metabolic disorders.