Neuroprotective activity of dimeric dipeptide mimetics of neurotrophin-3 loop 4 in a cell model of Parkinson's disease

Introduction. Parkinson's disease (PD) is a widespread neurodegenerative disorder characterized by the progressive loss of dopaminergic (DA) neurons in the nigrostriatal system of the brain. Since current PD therapy is predominantly symptomatic, there is an urgent need to develop novel disease-modifying therapeutic strategies. Neurotrophins represent promising candidates for this role. Specifically, neurotrophin-3 (NT-3) improves the survival and functional state of DA neurons during neurodegenerative processes. However, the clinical application of neurotrophins is limited by their low bioavailability. At the Federal research center for innovator and emerging biomedical and pharmaceutical technologies, low-molecular-weight dimeric dipeptide mimetics of NT-3 were synthesized based on the central β-turn fragment of its loop 4. These include GTS-301LL (bis-(N-monosuccinyl-L-asparaginyl-L-asparagine) hexamethylenediamine), GTS-304LL (bis-(N-monoglutaryl-L-asparaginyl-L-asparagine) hexamethylenediamine) and its LD-stereoisomer.
Objective. To evaluate the neuroprotective activity of the obtained mimetics on human neuroblastoma SH-SY5Y cells under conditions of 6-OHDAinduced toxicity (a cell model of PD).
Methods. The dipeptides were added to the culture medium at concentrations ranging from 10−5 to 10−8 M, either 24 hours before or immediately after 6-OHDA exposure. NT-3 (100 ng/mL) was used as a positive control.
Results. NT-3 improved cell viability when added both 24 hours before and immediately after the neurotoxin. Among the NT-3 mimetics, GTS-301LL and GTS-304LL exhibited the most pronounced neuroprotective activity, increasing cell viability at a concentration of 10−5 M when added 24 hours before 6-OHDA, and at concentrations of 10−5 to 10−7 M when added after 6-OHDA. GTS-304LD showed neuroprotective activity only when added 24 hours prior to 6-OHDA at the concentrations range 10−5 – 10−6 M.
Conclusion. These findings indicate that further investigation of the antiparkinsonian activity of dipeptide mimetics of NT-3 loop 4 is highly promising.

1. Luo Y, Qiao L, Li M, et al. Global, regional, national epidemiology and trends of Parkinson's disease from 1990 to 2021: findings from the Global Burden of Disease Study 2021. Front Aging Neurosci. 2025 Jan 10;16:1498756. doi: 10.3389/fnagi.2024.1498756.

2. Su D, Cui Y, He C, et al. Projections for prevalence of Parkinson's disease and its driving factors in 195 countries and territories to 2050: modelling study of Global Burden of Disease Study 2021. BMJ. 2025 Mar 5;388:e080952. doi: 10.1136/bmj-2024-080952.

3. Váradi C. Clinical Features of Parkinson's Disease: The Evolution of Critical Symptoms. Biology (Basel). 2020 May 19;9(5):103. doi: 10.3390/biology9050103.

4. Han JW, Ahn YD, Kim WS, et al. Psychiatric Manifestation in Patients with Parkinson's Disease. J Korean Med Sci. 2018 Nov 1;33(47):e300. doi: 10.3346/jkms.2018.33.e300.

5. Pardo-Moreno T, García-Morales V, Suleiman-Martos S, et al. Current Treatments and New, Tentative Therapies for Parkinson's Disease. Pharmaceutics. 2023 Feb 25;15(3):770. doi: 10.3390/pharmaceutics15030770.

6. Kim TY, Lee BD. Current therapeutic strategies in Parkinson's disease: Future perspectives. Mol Cells. 2025 Nov;48(11):100274. doi: 10.1016/j.mocell.2025.100274.

7. Skaper SD. Neurotrophic Factors: An Overview. Methods Mol Biol. 2018;1727:1-17. doi: 10.1007/978-1-4939-7571-6_1.

8. Povarnina PYu, Gudasheva TA, Dorofeev VL. Involvement of neurotrophins in the pathogenesis of Parkinson's disease. Farmakokinetika i farmakodinamika = Pharmacokinetics and pharmacodynamics. 2025;(4):9-16. (In Russ.). doi: 10.37489/2587-7836-2025-4-9-16. EDN: WHRWJQ.

9. Stefani A, Pierantozzi M, Cardarelli S, et al. Neurotrophins as Therapeutic Agents for Parkinson's Disease; New Chances From Focused Ultrasound? Front Neurosci. 2022 Mar 25;16:846681. doi: 10.3389/fnins.2022.846681.

10. Mansoor S, Jindal A, Badu NYA, et al. Role of Neurotrophins in the Development and Treatment of Neurodegenerative Diseases: A Systematic Review. Cureus. 2024 Nov 19;16(11):e74048. doi: 10.7759/cureus.74048.

11. Fedotcheva TA, Shimanovsky NL. Current State of the NeurotrophinBased Pharmaceutics in the Treatment of Neurodegenerative Diseases and Neuroinflammation. Med Sci (Basel). 2025 Dec 29;14(1):15. doi: 10.3390/medsci14010015.

12. Bahlakeh G, Rahbarghazi R, Mohammadnejad D, et al. Current knowledge and challenges associated with targeted delivery of neurotrophic factors into the central nervous system: focus on available approaches. Cell Biosci. 2021 Oct 12;11(1):181. doi: 10.1186/s13578-021-00694-2.

13. Patent RUS № RU2800369C9/ 04.10.23. Byul. №28. Seredenin SB, Gudasheva TA, Vakhitova YuV, et al. Dimeric dipeptide mimetics of neurotrofin-3. (In Russ.). Доступно по: https://patents.google.com/patent/RU2800369C9/ru. Ссылка активна на 14.09.2026.

14. Gudasheva TA, Sazonova NM, Tarasyuk AV, et al. The first dipeptide mimetic of neurotrofin-3: design and pharmacological properties. Doklady Biological Sciences. 2022;505(1):303-309. (In Russ.). doi: 10.31857/S2686738922040059.

15. Shirayama Y, Chen AC, Nakagawa S, et al. Brain-derived neurotrophic factor produces antidepressant effects in behavioral models of depression. J Neurosci. 2002 Apr 15;22(8):3251-61. doi: 10.1523/JNEUROSCI.22-08-03251.2002.

16. Riveles K, Huang LZ, Quik M. Cigarette smoke, nicotine and cotinine protect against 6-hydroxydopamine-induced toxicity in SH-SY5Y cells. Neurotoxicology. 2008 May;29(3):421-7. doi: 10.1016/j.neuro.2008.02.001.

17. Cohen RI, Marmur R, Norton WT, et al. Nerve growth factor and neurotrophin-3 differentially regulate the proliferation and survival of developing rat brain oligodendrocytes. J Neurosci. 1996 Oct 15;16(20):6433-42. doi: 10.1523/JNEUROSCI.16-20-06433.1996.

18. Ueda Y, Walsh E, Nakanishi H, Yoshida K. A colorimetric assay method for the evaluation of neurotrophic activity in vitro. Neurosci Lett. 1994 Jan 3;165(1-2):203-7. doi: 10.1016/0304-3940(94)90745-5.

19. Numan S, Seroogy KB. Expression of trkB and trkC mRNAs by adult midbrain dopamine neurons: a double-label in situ hybridization study. J Comp Neurol. 1999 Jan 18;403(3):295-308. doi: 10.1002/(sici)1096-9861(19990118)403:3<295::aid-cne2>3.0.co;2-l.

20. Hyman C, Juhasz M, Jackson C, et al. Overlapping and distinct actions of the neurotrophins BDNF, NT-3, and NT-4/5 on cultured dopaminergic and GABAergic neurons of the ventral mesencephalon. J Neurosci. 1994 Jan;14(1):335-47. doi: 10.1523/JNEUROSCI.14-01-00335.1994.

21. Gu S, Huang H, Bi J, et al. Combined treatment of neurotrophin-3 gene and neural stem cells is ameliorative to behavior recovery of Parkinson's disease rat model. Brain Res. 2009 Feb 27;1257:1-9. doi: 10.1016/j.brainres.2008.12.016.

22. Altar CA, Boylan CB, Fritsche M, et al. Efficacy of brain-derived neurotrophic factor and neurotrophin-3 on neurochemical and behavioral deficits associated with partial nigrostriatal dopamine lesions. J Neurochem. 1994 Sep;63(3):1021-32. doi: 10.1046/j.1471-4159.1994.63031021.x.

23. Omar NA, Kumar J, Teoh SL. Neuroprotective effects of Neurotrophin-3 in MPTP-induced zebrafish Parkinson's disease model. Front Pharmacol. 2023 Nov 28;14:1307447. doi: 10.3389/fphar.2023.1307447.

24. Xicoy H, Wieringa B, Martens GJ. The SH-SY5Y cell line in Parkinson's disease research: a systematic review. Mol Neurodegener. 2017 Jan 24;12(1):10. doi: 10.1186/s13024-017-0149-0.

25. Ioghen OC, Ceafalan LC, Popescu BO. SH-SY5Y Cell Line In Vitro Models for Parkinson Disease Research-Old Practice for New Trends. J Integr Neurosci. 2023 Jan 16;22(1):20. doi: 10.31083/j.jin2201020.

26. Wen S, Aki T, Unuma K, Uemura K. Chemically Induced Models of Parkinson's Disease: History and Perspectives for the Involvement of Ferroptosis. Front Cell Neurosci. 2020 Dec 23;14:581191. doi: 10.3389/fncel.2020.581191.

27. Nguyen TL, Kim CK, Cho JH, et al. Neuroprotection signaling pathway of nerve growth factor and brain-derived neurotrophic factor against staurosporine induced apoptosis in hippocampal H19-7/IGF-IR [corrected]. Exp Mol Med. 2010 Aug 31;42(8):583-95. doi: 10.3858/emm.2010.42.8.060.

28. Brunet A, Datta SR, Greenberg ME. Transcription-dependent and -independent control of neuronal survival by the PI3K-Akt signaling pathway. Curr Opin Neurobiol. 2001 Jun;11(3):297-305. doi: 10.1016/s0959-4388(00)00211-7.

29. Reichardt LF. Neurotrophin-regulated signalling pathways. Philos Trans R Soc Lond B Biol Sci. 2006 Sep 29;361(1473):1545-64. doi: 10.1098/rstb.2006.1894.

30. Almeida RD, Manadas BJ, Melo CV, et al. Neuroprotection by BDNF against glutamate-induced apoptotic cell death is mediated by ERK and PI3-kinase pathways. Cell Death Differ. 2005 Oct;12(10):1329-43. doi: 10.1038/sj.cdd.4401662.

31. Salinas M, Diaz R, Abraham NG, et al. Nerve growth factor protects against 6-hydroxydopamine-induced oxidative stress by increasing expression of heme oxygenase-1 in a phosphatidylinositol 3-kinase-dependent manner. J Biol Chem. 2003 Apr 18;278(16):13898-904. doi: 10.1074/jbc.M209164200.

32. Tan YX, Wang DF, Li XM, Liu XM. [Effects of nerve growth factor pretreatment on apoptosis of neurons and expression of Bcl-2, Bax protein in brain tissue following cerebral ischemia/reperfusion injury in gerbils]. Zhongguo Wei Zhong Bing Ji Jiu Yi Xue. 2007 Jun;19(6):358-60. Chinese.