Pegasus: A novel dopamine agonist with neuroprotective effect

Home Pegasus: A novel dopamine agonist with neuroprotective effect

SkyBio participated at FENS (Federation of European Neuroscience Societies) 2022, introducing to the international scientific comunity its molecule Pegasus: A novel dopamine agonist with neuroprotective effect.

Aims: Continuous and safe delivery of dopamine (DOPA) across the blood brain barrier (BBB) is a main obstacle for successful substitution therapy in Parkinson’s disease. Our aim is to take advantage of the well-known capacity of doxycycline (DOXY) to cross the BBB1, together with its neuroprotective activity2, in order to obtain a DOXY-DOPA hybrid molecule, which we call Pegasus, with potential application in Parkinson’s disease.

Methods: Pegasus was synthetized by removing the dimethylamino at C4 and introducing an amino group at C9, which was covalently coupled to dopamine through a linker. Confirmation of intact dopaminergic receptor activation was performed with cAMPNOMAD cells (INNOPROT). Cytotoxicity was studied with the MTT assay in SH-SY5Y and Bv2 cells, and with HEK293T Cytochrome C-tGFP apoptosis reporter cell line (INNOPROT). Neuroprotective properties were studied as follows: i) inhibition of α-Synuclein (α-Syn) aggregation by ThT assay; ii) antioxidant capability by using CellRoxTM reagent; iii) inhibition of proinflammatory cytokine IL-1β release by microglial cells; iv) influence on lysosomal number in SH-SY5Y cells with LysoTrakerTM.

Pegasus chemical synthesis

From doxyxciline:

  1. Synthesis of doxycycline methyl iodide
  2. Sybthesis of 4-dedimethylaminodoxycyline
  3. Synthesis of 4-dedimethylamino-9nitro-doxycycline (4)

From dopamine:

  1. Synthesis of di-O-benzyl-N-Boc-dopamine
  2. Synthesis of di-O-benzyl-dopamine (6)
  3. Addition of 6 to succinic anhydride to afford 7
  4. Synthesis of the conjugate doxycycline-dopamine [N1 –(4- dedimethylaminodoxycycline-9-yl)-N4-(3,4-dihydroxyphenethyl)succinimide (8)
  5. Hydrogenolysis of 8: synthesis of Pegasus

Pegasus has anti-aggregant and antinflammatory activity

The ability of Pegasus to interfere α-Syn amyloid aggregation was studied using thioflavin T (ThT) fluorescence emission according to Le Vine3. a) 25 μM ThT in a solution containing 70 μM α-Syn alone (α-Syn) or with the addition of Pegasus at 10 and 50 μM after 120 h at 37 °C . A solution containing 70 μM α-Syn and doxycycline (Doxy) was included as an internal control. b) Proinflammatory cytokine IL-1b assay. The effect of Pegasus on the release of IL-1b (Invitrigen BMS 6002) was studied in LPS-stimulated microglial Bv2 cells.

Pegasus has the antioxidant properties of its precursor

Effect of Pegasus on the formation of reactive oxygen species in cell culture. (a) SH-SY5Y cells (INNOPROT#P020303) were treated for 24 h with either PBS, α-Syn fibrils (α-SynPFF), α-Syn fibrils  in the presence of Pegasus (α-SynPFF+Pegasus), Pegasus. As  internal control, we also studied in the same conditions α-SynPFF in the presence of Doxy (α-SynPFF+Doxy) or Doxy. The formation of ROS was assayed using CellRox®. (b) Quantification of ROS response on figures was performed with CellProfiler4.

Pegasus does not induce apoptosis

Apoptosis assay in HEK293T Cytochrome C-tGFP cell line. Confocal microscopy images of Cytochrome C-tGFP after incubation with 200 µM of Pegasus for 24 h in the HEK293/Cytochrome C-tGFP cell line (INNOPROT#P30801). a) Magnification 40 X b) Inset with digital magnification.

Pegasus does not affect lysosomes

Effect of Pegasus on lysosomal activity in SH-SY5Y cells. Confocal microscopy images showing the localization and number of lysosomes after treatment with PBS (Control) or 200 µM of Pegasus for 24 h. Cells were stained with DAPI and LysoTracker®. a) SH-SY5Y t-RFP cells (INNOPROT). b) Cells were stained with DAPI. (c) Granules/field quantification.

Pegasus stimulateD1 and D2 receptors

Figure 6. Activation of D1 dopamine receptors by Pegasus. a) Dose-response curves in the HEK_cAMPNmd_DRD1 cell line (INNPPROT®). b) Dose-response curves in the U2OS_cAMPNmd_DRD2 cell line (INNPPROT®). Cells were treated for 24 hs. Data points represent the mean ± SD for each condition for a single experiment performed in triplicate.


With the aim of creating a novel dopamine agonist for Parkinson´s disease (PD) that possesses the known anti-inflammogenic, anti-oxidant anti-aggregant, and BBB-crossing properties of DOXY, we have synthesized Pegasus.

Preliminary data demonstrates that Pegasus retains the anti-inflammatory and anti-oxidant properties of DOXY, and the potential to cross the BBB (-XLogP= -0,03). Furthermore, the anti-aggregant effect on α-Syn was also conserved. In addition, it resulted not toxic in cell cultures and does not affect lysosomes, vital organelles affected in PD. Importantly, Pegasus was able to activate D1 and D2 dopamine receptors.

We conclude that Pegasus has potential to be further developed as a novel multitarget dopamine agonist for PD.

  • Ability to activate D1 and D2 receptors increasing the intracytoplasmic cAMP level
  • – Non-antibiotic against Gram (+) and Gram (-)
  • – Non-toxic until 20 μM in SH-SY5Y and Bv2
  • cell lines.
  • – Improves the antiaggregant properties of doxycycline.
  • – Conserves the antioxidant and anti-inflammatory properties of doxycycline in cell models.


1-Domercq, M. & Matute, doi: 10.1016/ (2004).

2- González-Lizárraga et. al,  doi: 10.1038/srep41755 (2017)

3- Le Vine, doi: 10.1016/s0076-6879(99)09020 (1999)

4- Stirling et. al., doi:10.1186/s12859-021-04344-9 (2021)


Chehín R1, Ploper D1, Pernicone A2, Manzano V2, Socías SB1, Avila CL1, Vera Pingitore E1, Chaves S1, Kolender A2, Tomas-Grau RH1, González‐Lizárraga F1, Luong M2,  Varela O2.

1Instituto de Investigación en Medicina Molecular y Celular Aplicada (IMMCA) (CONICET-UNT-SIPROSA), Pasaje Dorrego 1080, 4000 Tucumán, Argentina. 2 Universidad de Buenos Aires, Facultad de Ciencias Exactas y Naturales, Departamento de Química Orgánica, Ciudad Universitaria, Pabellón 2, C1428EHA, Buenos Aires, Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)-UBA, Centro de Investigación en Hidratos de Carbono (CIHIDECAR), Argentina.