Neuroprotective effects of a novel demeclocycline derivative lacking antibiotic activity: from a hit to a promising lead compound

RODRIGO TOMAS-GRAU; FLORENCIA GONZALEZ LIZARRAGA; DIEGO PLOPER; CESAR LUIS AVILA; SERGIO B. SOCÍAS; PIERRE BESNAULT; AURORE TOURVILLE; ROSA MELLA; PATRICIA VILLACÉ LOZANO; CLARISA SALADO; JEAN MICHEL BRUNEL; LAURENT FERRIÉ; RITA RAISMAN-VOZARI; PATRICK MICHEL; BRUNO FIGADÉRE; ROSANA CHEHÍN

Paris

Congreso; Federation of European Neuroscience Societies (FENS); 2022

The antibiotic tetracycline demeclocycline (DMC) was recently reported to rescue α-Synuclein (α- Syn) fibril-induced pathology (Braun et al., NPJ Parkinsons Dis, 2021). However, their antibiotic activity precludes its potential use for long-term neuroprotective treatments. Here, our aim was to synthesize a DMC derivative named DDMC with low antibiotic activity and improved neuroprotective and anti-aggregant properties.MethodsDDMC synthesis: DDMC was obtained by removal the dimethylamino substituent at position 4 and reduction of the hydroxyl group at position 12 on ring A of DMC.α-SynPFF production: Recombinant wild-type human α-Syn was expressed and purified according to Torres-Bugeau et al., J Biol Chem (2012). α-Syn preformed fibrils (α-SynPFF) were obtained as previously described (Gonzalez-Lizárraga et al., Sci Rep, 2017).Antimicrobial activity: Minimum inhibitory concentration (MIC) was determined using three bacterial strains: GRAM (-) Pseudomonas aeruginosa PA01, Escherichia coli ATCC 25922 and GRAM (+) Staphylococcus aureus ATCC 25923.SH-SY5Y viability: Toxicity was determined by MTT (Mosmann, J Immunol Methods, 1983). The number of lysosomes and subcellular location was studied with LysoTrackerTM Deep Red and Lysosomal-Associated Membrane Protein 1 (LAMP-1).α-Syn aggregation: Thioflavin T (ThT) (LeVine, Protein Sci, 1993) and Congo Red (Klunk et al., Methods Enzymol, 1989) assays were used to estimate the amount of fibrils formed in each condition studied. Transmission Electron Microscopy (TEM) was used to visualize α-Syn fibrils according to Gonzalez-Lizárraga et al., Sci Rep, 2017.α-Syn seeding: Ex-vitro, α-Syn kinetics were determined with the ThT assay. The lag phase was calculated according to Lau et al., Nat Neurosci, 2020. In-vitro, using SH-SY5Y cells overexpressing the fusion protein α-Syn-tRFP (Innoprot #P30707-02).Neuroimmflamation activity: Primary mouse microglial cells were used to estimate TNFα and glutamate (Glut) release induced by α-SynPFF according to Acuña et al., Cells, 2019.