New, Potent, Small Molecule Agonists of Tyrosine Kinase Receptors Improve Dry Eye Disease

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin3 (NT-3) bind to tyrosine kinase (Trk) receptors, TrkA, TrkB, and TrkC, respectively. This study investigated the efficacy of novel molecule agonists of Trk receptors in an in vivo model of dry eye disease. Compared to vehicle, mice subjected to desiccating stress and treated with agonists pan and C1 showed improved corneal barrier function, while C1 also increased GC densities. NanoString analyses revealed upregulation of specific mRNA transcripts (Ptger4, Tnfaip3, Il1a and Ptger4, Tlr3, Osal1) in pan and C1 treated corneas compared to vehicle-treated corneas. Western blots showed pan and C1 decreased vehicle-induced NFkB nuclear translocation after DS for one day and increased PTGER4 and TNFAIP3 protein levels after 5 days of DS in corneal epithelium lysates. These results were confirmed by immunostaining using antibodies for TNFAIP3 and PTGER4 in wholemount corneas. We conclude that small-molecule agonists of Trk receptors improve dry eye disease by decreasing NFkB activation and increasing protein expression of anti-inflammatory molecules TNFAIP3 and PTGER4. Surprisingly, the most efficacious small molecule agonists were not TrkA selective, but TrkC and panTrk, suggesting that wider exploration of TrkB and C and pan Trk agonists are warranted in efforts to treat dry eye disease. For this, small molecule TrkC agonist C1) and a pan-Trk agonist (pan) were synthesized. C57BL/6J mice were subjected to desiccating stress (DS) and received bilateral eye drops of C1, pan or vehicle (2x/day). Dry eye signs, inflammation and expression of Corneal barrier function, and conjunctival goblet cell (GC) densities were measured as part of the dry eye phenotype. Corneal epithelial lysates were collected for either western blot or RNA extraction. Extracted total RNAs were used for NanoString analyses. Immunofluorescent staining was performed on wholemount corneas using anti-TNFAIP3 and anti-PTGER4 antibodies.