(T1430-03-15) Identification of EphB1/2 Tyrosine Kinase Inhibitors for Chemotherapy-Induced Peripheral Neuropathic Pain

Purpose: Chemotherapy induced peripheral neuropathy (CIPN) is defined by persistent pain among cancer survivors after the administration of chemotherapeutic agents such as paclitaxel or cisplatin. CIPN affects more than two thirds of cancer survivors within one month of treatment. CIPN has been particularly difficult to treat due to its multitude of pathological iterations, such as neuronal hyperexcitability, axonal degradation, mitochondrial disruption, and others. Opioid-based medications are the first therapeutic regimens for treating CIPN that can contribute to worsening of the opioid crisis and addiction. The large family of Eph (erythropoietin-producing hepatocellular carcinoma) receptor tyrosine kinases are involved in different physiological processes, including axon guidance, topographic mapping, neuronal migration, and synapse formation. Pathologically, EphB tyrosine kinases have been identified in different EphB-associated diseases, including Alzheimer’s disease, anxiety, glioblastoma, non-alcoholic steatohepatitis, and peripheral neuropathic pain. Our research group applied structure-based drug repurposing strategy to introduce the synergism of three tetracyclines (Minocycline, Chlortetracycline, and Demeclocycline) family members that showed the competitive inhibitory profile along with ATP-binding catalytic domain of EphB1 tyrosine kinase to reverse thermal hyperalgesia and mechanical allodynia in inflammatory pain models. However, the IC50 for this approach is in the low micromolar range, requiring a near-maximal dose of all three antibiotics in combination. Additionally, we resolved the crystal structure of EphB1 catalytic binding domain of tyrosine kinase bound with chlortetracycline (PDB ID: 6UMW)7. Therefore, the role of EphB1/2 tyrosine kinase signaling in the progression of peripheral neuropathy is well-validated. However, there are neither FDA-approved drugs, nor preclinical new chemical entities that can target EphB1/2 tyrosine kinase signaling within the settings of CIPN. Our overarching hypothesis is that our novel small molecules, not related to the tetracycline scaffold, will inhibit EphB1/2 tyrosine kinase signaling that either block or reverse mechanical allodynia in CIPN. We leveraged our resolved X-ray crystal structure of the hEphB1 kinase domain bound to CTC, 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.

Methods: We pursued molecular simulations and a rational-based drug design approach against the EphB1 catalytic binding domain using the Open Eye molecular modeling package to adopt the ring opening strategy for tetracycline ring while recruiting thienopyridine ring inspired by 3-[4-amino-3-(3-chloro-4-fluorophenyl) thieno [3,2-c] pyridin-7-yl]-benzene sulfonamide-based analogs (previously reported EphB inhibitor). Where the genesis of our structural design was to overcome the ring strain and offer ring flexibility within the catalytic binding domain. We probed different heteroaryl groups, not related to tetracycline, to be called as AHM analogs. To prove efficacy, we tested our analogues using ADP-Glo kinase chemiluminescent assay for EphB1/2. Additionally, we tested our compounds in vivo to demonstrate analgesic effects against mechanical allodynia via a Von Frey assay. All animal testing was done in accordance to the IACUC procedures approved by TTUHSC.

Results: The first generation of 10 AHM analogs showed better occupancy for the catalytic domain than CTC; via hydrophobic-hydrophobic interactions and three hydrogen bonds with E668, T697, and M700. Our structural analysis deciphered the potential targeting of the hEphB1 catalytic binding domain via three sub-pockets (S1, S2, and S3), where CTC and AHM-009 showed potential filling for S2 and S3 sub-pockets oriented towards E668 and F763. Based on the ADP-Glo kinase chemiluminescent assay, the ring opening strategy was not enough to increase the potency, where all of the synthesized analogs showed modest inhibitory activity against EphB1/2 tyrosine kinase at IC50 > 100 μM. Next, we assembled the second generation of 23 AHM analogs to fill in S1, hinge binding domain (S2), and DFG gatekeeper motif (S3) of the hEphB1 catalytic binding domain with established CNS-MPO scores to improve the inhibitory profiles against EphB1/2 tyrosine kinase at IC50s ranging from 120-600 nM. The kinome profiling assays done at the University of Dundee showed the ability of AHM-020 (5μM, around 10-fold of the IC50) to inhibit EphB1/2 selectively at 66% and 60%, respectively without affecting 138 tyrosine and tyrosine-like kinases. Our in vivo validation initially involved the increase of the expression for p-EphB1/2 in the isolated spinal cords and dorsal root ganglion for mice treated with paclitaxel, compared to vehicle treated cohorts. Our in vivo behavioral studies suggested the potential of AHM-020 to reverse the mechanical allodynia in paclitaxel-induced peripheral neuropathic pain in a dose-dependent manner. These findings can contribute to the EphB1/2 tyrosine kinase inhibition in CIPN to deliver future candidates with better potency and selectivity profiles.

Conclusion: The synthesized compounds our lab has produced have shown the ability to inhibit the activity of EphB1 with an IC₅₀ in the nanomolar range, as well as specificity to EphB1/2. The lead is also able to alleviate the symptoms of chemotherapy induced peripheral neuropathy in animal models.

References: 1 Lai, K. O. & Ip, N. Y. Synapse development and plasticity: roles of ephrin/Eph receptor signaling. Curr Opin Neurobiol 19, 275-283 (2009).
2 Soskis, M. J. et al. A chemical genetic approach reveals distinct EphB signaling mechanisms during brain development. Nature Neuroscience 15, 1645-1654 (2012).
3 Cibert-Goton, V. et al. Involvement of EphB1 receptors signalling in models of inflammatory and neuropathic pain. PLoS One 8, e53673 (2013).
4 Pasquale, E. B. Eph receptors and ephrins in cancer: bidirectional signalling and beyond. Nature reviews. Cancer 10, 165-180 (2010).
5 Han, Y., Song, X. S., Liu, W. T., Henkemeyer, M. & Song, X. J. Targeted mutation of EphB1 receptor prevents development of neuropathic hyperalgesia and physical dependence on morphine in mice. Mol Pain 4, 60 (2008).
6 O'Neal, W. T. et al. Ephrin-Eph signaling as a potential therapeutic target for the treatment of myocardial infarction. Med Hypotheses 80, 738-744 (2013).
7 Ahmed, M. S. et al. Identification of tetracycline combinations as EphB1 tyrosine kinase inhibitors for treatment of neuropathic pain. Proc Natl Acad Sci U S A 118 (2021).

Acknowledgements: OpenEye Scientific for free academic software license. Jerry H. Hodge School of Pharmacy and Office of Health Sciences Research and Innovation. NIH Research Project (R01) grant 1R01NS136485-01A1


Depiction of first series of scaffold analogs, detailing IC50 values against EphB1/2, and CNS-MPO values.


Depiction of second series of scaffold analogs, detailing IC50 values against EphB1/2, and CNS-MPO values.


a. Kinome map, EphB1/2 have been highlighted.
b. In vivo testing of HBA-020 against mechanical.allodynia via a Von Frey assay.