The LRRK2 kinase has emerged as a priority therapeutic target due to its well-documented role in Parkinson-s diseases (PD). One of LRRK2’s key partners is the phosphatase PP1, which dephosphorylates LRRK2. Therefore, modulating this protein/protein interaction represents a promising therapeutic strategy for PD. We have developed a bi-functional peptide, PEP 3, capable of crossing the blood-brain barrier (BBB) and disrupting the LRRK2/PP1 interaction. In vitro competition assays confirmed that PEP 3 specifically targets this interaction. The in vivo imaging demonstrated that the peptide remains detectable in the mouse brain up to 6 hours or retro-orbital vein post-injection. The PEP 3 peptide does not show chronic toxicity in CD1 mice and is resistant to degradation by mouse, human, dog and monkey serum proteases. In mouse models overexpressing alpha-synuclein, repeated intraperitoneal injections of PEP 3 for 15 and 30 days were well tolerated. Immunohistochemistry revealed a significant reduction of dopaminergic cell death in the substantia nigra, and quantification of phosphorylated pathological alpha-synuclein showed decreased levels in the PEP 3-treated group compared to controls. These findings support the potential of PEP 3 as a therapeutic agent for Parkinson’s disease.
References
[1]
Menozzi, E. and Schapira, A.H.V. (2025) Prospects for Disease Slowing in Parkinson Disease. Annual Review of Pharmacology and Toxicology, 65, 237-258. https://doi.org/10.1146/annurev-pharmtox-022124-033653
[2]
Surmeier, D.J., Obeso, J.A. and Halliday, G.M. (2017) Selective Neuronal Vulnerability in Parkinson Disease. Nature Reviews Neuroscience, 18, 101-113. https://doi.org/10.1038/nrn.2016.178
[3]
Lees, A.J., Hardy, J. and Revesz, T. (2009) Parkinson’s Disease. The Lancet, 373, 2055-2066. https://doi.org/10.1016/s0140-6736(09)60492-x
[4]
Spillantini, M.G., Schmidt, M.L., Lee, V.M., Trojanowski, J.Q., Jakes, R. and Goedert, M. (1997) Α-Synuclein in Lewy Bodies. Nature, 388, 839-840. https://doi.org/10.1038/42166
[5]
Daneman, R. and Prat, A. (2015) The Blood-Brain Barrier. Cold Spring Harbor Perspectives in Biology, 7, a020412. https://doi.org/10.1101/cshperspect.a020412
[6]
Oller-Salvia, B., Sánchez-Navarro, M., Giralt, E. and Teixidó, M. (2016) Blood-Brain Barrier Shuttle Peptides: An Emerging Paradigm for Brain Delivery. Chemical Society Reviews, 45, 4690-4707. https://doi.org/10.1039/c6cs00076b
[7]
Andrieu, J., Re, F., Russo, L. and Nicotra, F. (2019) Phage-Displayed Peptides Targeting Specific Tissues and Organs. Journal of Drug Targeting, 27, 555-565. https://doi.org/10.1080/1061186x.2018.1531419
[8]
Zhang, X., Brossas, J.Y., Parizot, C., Zini, J.M. and Rebollo, A. (2018) Identification and Characterization of Novel Enhanced Cell Penetrating Peptides for Anti-Cancer Cargo Delivery. Oncotarget, 9, 5944-5957. https://doi.org/10.18632/oncotarget.23179
[9]
Arrouss, I., Nemati, F., Roncal, F., Wislez, M., Dorgham, K., Vallerand, D., et al. (2013) Specific Targeting of Caspase-9/PP2A Interaction as Potential New Anti-Cancer Therapy. PLOS ONE, 8, e60816. https://doi.org/10.1371/journal.pone.0060816
[10]
Dominguez-Berrocal, L., Cirri, E., Zhang, X., Andrini, L., Marin, G.H., Lebel-Binay, S., et al. (2019) New Therapeutic Approach for Targeting Hippo Signalling Pathway. Scientific Reports, 9, Article No. 4771. https://doi.org/10.1038/s41598-019-41404-w
[11]
Bruzzoni-Giovanelli, H., Alezra, V., Wolff, N., Dong, C., Tuffery, P. and Rebollo, A. (2018) Interfering Peptides Targeting Protein-Protein Interactions: The Next Generation of Drugs? Drug Discovery Today, 23, 272-285. https://doi.org/10.1016/j.drudis.2017.10.016
[12]
Kolupaeva, V. and Janssens, V. (2012) PP1 and PP2A Phosphatases-Cooperating Partners in Modulating Retinoblastoma Protein Activation. The FEBS Journal, 280, 627-643. https://doi.org/10.1111/j.1742-4658.2012.08511.x
[13]
Takai, A., Eto, M., Hirano, K., Takeya, K., Wakimoto, T. and Watanabe, M. (2018) Protein Phosphatases 1 and 2A and Their Naturally Occurring Inhibitors: Current Topics in Smooth Muscle Physiology and Chemical Biology. The Journal of Physiological Sciences, 68, 1-17. https://doi.org/10.1007/s12576-017-0556-6
[14]
Heroes, E., Lesage, B., G？rnemann, J., Beullens, M., Van Meervelt, L. and Bollen, M. (2013) The PP1 Binding Code: A Molecular‐Lego Strategy That Governs Specificity. The FEBS Journal, 280, 584-595. https://doi.org/10.1111/j.1742-4658.2012.08547.x
[15]
Sheppeck, J.E., Gauss, C. and Chamberlin, A.R. (1997) Inhibition of the Ser-Thr Phosphatases PP1 and PP2A by Naturally Occurring Toxins. Bioorganic & Medicinal Chemistry, 5, 1739-1750. https://doi.org/10.1016/s0968-0896(97)00146-6
[16]
Lobbestael, E., Zhao, J., Rudenko, I.N., Beylina, A., Gao, F., Wetter, J., et al. (2013) Identification of Protein Phosphatase 1 as a Regulator of the LRRK2 Phosphorylation Cycle. Biochemical Journal, 456, 119-128. https://doi.org/10.1042/bj20121772
[17]
Taymans, J. and Baekelandt, V. (2014) Phosphatases of Alpha-Synuclein, LRRK2, and Tau: Important Players in the Phosphorylation-Dependent Pathology of Parkinsonism. Frontiers in Genetics, 5, Article ID: 382. https://doi.org/10.3389/fgene.2014.00382
[18]
Lobbestael, E., Baekelandt, V. and Taymans, J. (2012) Phosphorylation of LRRK2: From Kinase to Substrate. Biochemical Society Transactions, 40, 1102-1110. https://doi.org/10.1042/bst20120128
[19]
Taymans, J. (2017) Regulation of LRRK2 by Phosphatases. In: Rideout, H., Ed., Advances in Neurobiology, Springer International Publishing, 145-160. https://doi.org/10.1007/978-3-319-49969-7_8
[20]
Nichols, R.J. (2017) LRRK2 Phosphorylation. In: Rideout, H., Ed., Advances in Neurobiology, Springer International Publishing, 51-70. https://doi.org/10.1007/978-3-319-49969-7_3
[21]
Dzamko, N., Deak, M., Hentati, F., Reith, A.D., Prescott, A.R., Alessi, D.R., et al. (2010) Inhibition of LRRK2 Kinase Activity Leads to Dephosphorylation of Ser910/Ser935, Disruption of 14-3-3 Binding and Altered Cytoplasmic Localization. Biochemical Journal, 430, 405-413. https://doi.org/10.1042/bj20100784
[22]
Doggett, E.A., Zhao, J., Mork, C.N., Hu, D. and Nichols, R.J. (2011) Phosphorylation of LRRK2 Serines 955 and 973 Is Disrupted by Parkinson’s Disease Mutations and LRRK2 Pharmacological Inhibition. Journal of Neurochemistry, 120, 37-45. https://doi.org/10.1111/j.1471-4159.2011.07537.x
[23]
Dong, C.Z., Bruzzoni-Giovanelli, H., Yu, Y., Dorgham, K., Parizot, C., Zini, J.M., et al. (2020) Identification of Peptides Interfering with the LRRK2/PP1 Interaction. PLOS ONE, 15, e0237110. https://doi.org/10.1371/journal.pone.0237110
[24]
Boudjelloul, R., Rebollo, A., Loisel, S., Charlotte, F., Franetich, J.F. and Savier, E. (2023) Differential Expression of PP2A and Set in Hepatocellular Carcinoma: Antitumoral Effect of an Interfering Peptide Blocking the Interaction. Clinics in Oncology, 8, Article 2028.
[25]
Makky, A., Bousset, L., Polesel-Maris, J. and Melki, R. (2016) Nanomechanical Properties of Distinct Fibrillar Polymorphs of the Protein Α-Synuclein. Scientific Reports, 6, Article No. 37970. https://doi.org/10.1038/srep37970
[26]
Stefanis, L. (2011) Α-Synuclein in Parkinson’s Disease. Cold Spring Harbor Perspectives in Medicine, 2, a009399. https://doi.org/10.1101/cshperspect.a009399
[27]
Burré, J., Sharma, M., Tsetsenis, T., Buchman, V., Etherton, M.R. and Südhof, T.C. (2010) Α-Synuclein Promotes Snare-Complex Assembly in Vivo and in Vitro. Science, 329, 1663-1667. https://doi.org/10.1126/science.1195227
[28]
Cresto, N., Gaillard, M., Gardier, C., Gubinelli, F., Diguet, E., Bellet, D., et al. (2020) The C-Terminal Domain of LRRK2 with the G2019S Mutation Is Sufficient to Produce Neurodegeneration of Dopaminergic Neurons in Vivo. Neurobiology of Disease, 134, Article 104614. https://doi.org/10.1016/j.nbd.2019.104614
[29]
Cresto, N., Gardier, C., Gaillard, M., Gubinelli, F., Roost, P., Molina, D., et al. (2021) The C-Terminal Domain of LRRK2 with the G2019S Substitution Increases Mutant A53T Α-Synuclein Toxicity in Dopaminergic Neurons in Vivo. International Journal of Molecular Sciences, 22, Article 6760. https://doi.org/10.3390/ijms22136760
[30]
Garretti, F., Monahan, C., Sette, A., Agalliu, D. and Sulzer, D. (2022) T Cells, Α-Synuclein and Parkinson Disease. Handbook of Clinical Neurology, 184, 439-455.
[31]
Li, X., Wang, Q.J., Pan, N., Lee, S., Zhao, Y., Chait, B.T., et al. (2011) Phosphorylation-Dependent 14-3-3 Binding to LRRK2 Is Impaired by Common Mutations of Familial Parkinson’s Disease. PLOS ONE, 6, e17153. https://doi.org/10.1371/journal.pone.0017153
[32]
Nichols, R.J., Dzamko, N., Morrice, N.A., Campbell, D.G., Deak, M., Ordureau, A., et al. (2010) 14-3-3 Binding to LRRK2 Is Disrupted by Multiple Parkinson’s Disease-Associated Mutations and Regulates Cytoplasmic Localization. Biochemical Journal, 430, 393-404. https://doi.org/10.1042/bj20100483
[33]
Cresto, N., Gardier, C., Gubinelli, F., Gaillard, M., Liot, G., West, A.B., et al. (2019) The Unlikely Partnership between LRRK2 and Α‐Synuclein in Parkinson’s Disease. European Journal of Neuroscience, 49, 339-363. https://doi.org/10.1111/ejn.14182
[34]
Fu, B.M. (2021) Quantification of in Vitro Blood-Brain Barrier Permeability. In: Zhao, F. and Leong, K.W., Eds., Methods in Molecular Biology, Springer, 217-228. https://doi.org/10.1007/978-1-0716-1708-3_18
