Discovery of a novel peptidomimetic for the neuroprotective treatment of glaucoma

Purpose: BDNF activates downstream cell signaling pathways through NTRK2 receptor, leading to neuronal survival and triggering key functions in Retinal Ganglion cells (RGCs). Glaucomatous blockage stops retrograde transport of this neuroprotective molecule and results in dysregulation of other cellular processes. However, there are several clinical concerns about its therapeutic properties for glaucoma disease. In the current study, we discovered BDNF-mimicking peptide as an alternative molecule to the clinical challenges.Methods: The peptide library was screened using NTRK2 protein and evaluated by ELISA. After sequencing, peptide-receptor interactions were investigated and a consensus construct was selected. The affinity binding and function of the construct was confirmed by testing against the native structure of NTRK2 in SH-SY5Y cells using flow-cytometry and MTT assays, respectively. The neuroprotective activities of RNYS on primary RGC culture were assessed in glaucomatous condition. Accordingly, a suitable pressure system was set up based on a Lab-on-a chip strategy using PMMA sheets. PMMA surface was chemically modified and a desirable cellular microenvironment was created. The purified RGCs were cultured in glaucoma condition and cell-to-cell communications were observed by ICC assay.Results: Polyclonal phage ELISA indicated that target populations were enriched round by round and the panning process was truly effective. The results of monoclonal phage-ELISA showed that all clones had principally bound to NTRK2 but fifteen best clones were sequenced, which eleven of them have only one sequence. The resulting peptides mimicked loop 2 of BDNF and strongly interacted with the surface residues of NTRK2 protein at the IgC2 domain. One sequence was synthesized as a mimetic construct (named as RNYS). RNYS was prevented neuronal degeneration of ATRA-treated SH-SY5Y at 3.4 nM (as an optimal concentration) with equal efficacy to or even better than BDNF at 2nM. Also in glaucomatous condition, RNYS increased neuronal survival approximately similar to BDNF after 6, 12, and 24 hours. However, it worked better than BDNF after longer elevated pressure.Conclusion: In this condition, RNYS can mimic BDNF activities in vitro due to its unique sequence. Small size of RNYS might be an advantage for drug design in future.