(M1330-03-21) Discovery of Pan-EphB Tyrosine Kinase Inhibitors to Reverse Metabolic Syndrome in Obese Mice

Purpose: Metabolic syndrome (MS) is associated with cluster of cardiometabolic pathological events including obesity, insulin resistance, type-2 diabetes mellitus (T2DM), cardiovascular diseases, dyslipidaemia, and non-alcoholic steatohepatitis (NASH)^1-3. The global prevalence of metabolic syndrome (MS) including insulin resistance and obesity had created one of the most pressing public health dilemmas and significant financial burden to the healthcare system. Despite the surge of glucagon-like peptide-1 (GLP-1) agonists in treatment of metabolic disorders, recent studies showed that 40% of body-weight loss resulted from the lean mass loss^4-7. Additional side effects resulting from use of GLP-1 agonist include induction of chronic intestinal pseudo-obstruction^8,9, increased risk of gastrointestinal disorders, arthritic disorders, interstitial nephritis, and drug-induced pancreatitis¹⁰. Therefore, there is an urgent need to develop novel therapeutic strategies focusing on molecular targets with minimal adverse effects. The large family of Eph (erythropoietin-producing hepatocellular carcinoma) receptor tyrosine kinases is validated in the progression of multiple disorders, including Alzheimer’s disease, neuropathic pain, opioid use disorders, malignancies, fibrotic diseases, and viral infections^11-14. EphB1/ephrin-B2 signaling is involved in the progression and development of obesity-associated colorectal tumors¹⁵. EphB2 receptor tyrosine kinase showed promotion for hepatic fibrosis¹⁶. Also, knockdown of Ephb2 in hepatocytes ameliorated inflammation and fibrosis in NASH mouse model¹⁷. Additionally, genetic and pharmacological modulation of EphB4 improved insulin resistance and glucose intolerance in obese mice¹⁸. Hepatic overexpression of EphB4 decreased the expression levels of insulin receptor (InsR) and increased hepatic and systemic insulin resistance in vivo, while EphB4 liver-specific knockout mice (EphB4 LKO) improved insulin resistance and glucose intolerance with no effect on the whole-body weight¹⁸. This validates that EphB4 is critical for insulin-induced liver InsR degradation and the role of the EphB4 tyrosine kinase signaling pathway in insulin resistance and hyperglycemia¹⁸. Collectively, this can suggest that targeting EphB signaling can offer plausible therapeutic strategy to alleviate metabolic syndrome including insulin resistance and liver fibrosis. Therefore, our objectives were tailored to introduce pan-EphB tyrosine kinase inhibitors that can mitigate the progression of complications associated with metabolic syndrome.

Methods: We leveraged our resolved X-ray crystal structure of the hEphB1 kinase domain bound to CTC (PDB ID: 6UMW)¹⁹, where CTC showed potential occupancy for hinge binding domain (S2) and DFG gatekeeper motif (S3) sub-pockets oriented towards E668 and F763 supported with hydrogen bonds with T697 and M700. We pursued molecular simulations and a rational based drug design approach against the EphB1 catalytic binding domain using the Open Eye molecular modeling package. We probed different heteroaryl groups, not related to tetracycline, applying ligand based drug design to deduce structural similarity based on Tanimoto’s co-efficient to be called as STA analogs²⁰. We did the IC50 profiling of all synthesized STA analogs against EphB1, EphB2, EphB3, and EphB4 tyrosine kinase activity using ADP-Glo assay. Based on the assay, STA-013 was selected as a candidate for further in vitro and in vivo studies. STA-013 (10 mM in 100% DMSO) was sent to MRC PPU International Center for premier kinase profiling at the University of Dundee, UK, to test compound selectivity against 140 kinase enzymes at a concentration of 5 µM. The premier kinome profiling assay showed its potential to inhibit EphB1, EphB2, and EphB4 tyrosine kinases without affecting other tyrosine kinases and tyrosine-like kinases. In vitro phenotypic screening of STA-013 was done in the embryonic mouse 3T3-L1 fibroblasts. STA-013 inhibited the lipid accumulation after differentiation to adipocytes in the embryonic mouse 3T3-L1 fibroblast using Oil Red O staining (Sigma-Aldrich, 01391) in a dose-dependent manner, compared to vehicle-treated cells. Systemic injection of STA-013 was carried out on diet induced obese (DIO) mice for 6 weeks while the total body-weight, body-mass composition, and the metabolic profiles were monitored. In vivo molecular target was validated using Western blotting analysis.

Results: STA-013 inhibited EphB1, EphB2, and EphB4 TKs in vitro. Systemic injections resulted in weight loss with significant reduction of fat mass while preserving lean mass and a significant improvement in glucose homeostasis in DIO mice. STA-013 showed elevated signal for insulin receptor-β (InsR-β) as evident from the Western blot analysis of harvested liver tissue and brown adipose tissue samples validating its potential to reverse the effect of insulin on InsR degradation, allowing restoration of glucose homeostasis and mitigation of insulin resistance.

Conclusion: We demonstrate that STA-013 induced weight loss while preserving the lean mass, improved glucose homeostasis, and mitigated insulin resistance. These results highlight the translational potential of reversing the effect of insulin on InsR degradation via pharmacological inhibition of EphB1, EphB2, and EphB4 tyrosine kinases. These findings can contribute to the validation of pan-EphB inhibition in metabolic syndrome to deliver future candidates with better potency and selectivity profiles.

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Acknowledgements: Mahmoud Salama Ahmed is supported by start-up funds offered by the School of Pharmacy at TTUHSC and the Office of Innovation and Research at TTUHSC as well as NINDS/NIH by 1R01NS136485-01A1. We thank the CPRIT imaging core at TTUHSC, Amarillo funded by the Core Facility Support Award (grant number RP200572) from the Cancer Prevention and Research Institute of Texas (CPRIT). We would like to acknowledge OpenEye molecular modeling software for supporting a free academic license to pursue our in silico molecular simulations. Funding support from the American Heart Association (Grant # 953881), Diabetes Action (Grant# 523) and Texas Tech University School of Veterinary Medicine to Prasanth K. Chelikani is acknowledged.


Structure-based drug design of the second generation of thienopyridine-based analogs (STA-001 to STA- 022). a, Design strategy for STA compounds. Starting with the CTC skeleton to adopt a ring-opening strategy at 1C distance, followed by structure extension. c-d, Surface representation showing the potential of STA-001 to fill in S2 and S3 of hEphB1 kinase domain supported with 3D visualization showing dotted green hydrogen bonds (referred by yellow arrows) with E668 and T697. e-f, Surface representation showing the potential of STA-021 to fill in S1, Hinge region, and DFG pockets of hEphB1 kinase domain supported with 3D visualization showing dotted green hydrogen bonds (referred by yellow arrows) with E668.


IC50 profiling the second generation of thienopyridine-based analogs (STA-001 to STA-022). against EphB1, EphB2, EphB3, and EphB4 tyrosine kinase activity using ADP-Glo assay. Residual activity was calculated using two independent biological replicates, represented by mean ± SEM.


STA-013 promoted whole-body weight loss, restored glucose homeostasis, and mitigated insulin resistance in a high-fat diet (HFD)-induced obesity mouse model. a, Schematic for STA-013 administration to HFDinduced obese mice at 25 mg/kg, i.p. for 6 weeks. b-c, STA-013 significantly reduced whole-body weight for HFD-obese mice compared to vehicle-treated HFD-obese mice. d-e, STA-013 restored glucose homeostasis for HFD-obese mice significantly compared to vehicle-treated HFD-obese mice using the glucose tolerance test (GTT). f-g, STA-013 significantly mitigated insulin resistance for HFD-obese mice compared to vehicle-treated HFD-obese mice using an insulin tolerance test (ITT). h-i, STA-013 reduced fat-mass while preserving the lean muscle mass for HFD-obese mice compared to vehicle treated HFD-obese mice using NMR (Minispec LF110®, Bruker). j-k, STA-013 decreased total cholesterol and free fatty acids in the plasma for HFD-obese mice significantly compared to vehicle-treated HFD-obese mice. l-m, STA-013 decreased liver fibrosis for isolated livers from HFD-obese mice significantly, compared to vehicle-treated HFD-obese mice using oil red staining. Data are mean ± s.e.m.; unpaired two-sided t-test. *P < 0.05, **P < 0.01, ***P < 0.001.