Neuroprotective Effects of a Novel Demeclocycline Derivative Lacking Antibiotic Activity: From a Hit to a Promising Lead Compound

Abstract

The antibiotic tetracycline demeclocycline (DMC) was recently reported to rescue α-synuclein (α-Syn) fibril-induced pathology. However, the antimicrobial activity of DMC precludes its potential use in long-term neuroprotective treatments. Here, we synthesized a doubly reduced DMC (DDMC) derivative with residual antibiotic activity and improved neuroprotective effects. The molecule was obtained by removal the dimethylamino substituent at position 4 and the reduction of the hydroxyl group at position 12a on ring A of DMC. The modifications strongly diminished its antibiotic activity against Gram-positive and Gram-negative bacteria. Moreover, this compound preserved the low toxicity of DMC in dopaminergic cell lines while improving its ability to interfere with α-Syn amyloid-like aggregation, showing the highest effectiveness of all tetracyclines tested. Likewise, DDMC demonstrated the ability to reduce seeding induced by the exogenous addition of α-Syn preformed fibrils (α-Syn_PFF) in biophysical assays and in a SH-SY5Y-α-Syn-tRFP cell model. In addition, DDMC rendered α-Syn_PFF less inflammogenic. Our results suggest that DDMC may be a promising drug candidate for hit-to-lead development and preclinical studies in Parkinson's disease and other synucleinopathies.

Keywords: Parkinson’s disease; neuroprotection; novel tetracycline.

Figure 1

The novel TC DDMC is innocuous in bacteria and human cells. (a) Synthesis of DDMC via the treatment of DMC with zinc in acetic acid and water for 2 h at room temperature. The hydrogen atoms incorporated due to the reduction step are shown in pink. The dashed-line box points to the structural signature related to binding with amyloid-like aggregates. (b) Antibiotic activities of DMC and DDMC against P. aeruginosa (PAO1), E. coli (ATCC 25922), and S. aureus (ATCC 25923). MICs are shown in µM. (c) MTT cell viability assay in SH-SY5Y cells after 24 h treatment with DMC or DDMC (10 or 50 µM final concentrations) and hydrogen peroxide (H₂O₂) as a positive control (50 µM). All experiments were performed in sextuplicate, and relative cell viability was expressed as a percentage relative to the untreated control cells. Statistical analysis was performed as described in Materials and Methods, and significant differences are indicated in the figures as follows: **** p < 0.001 vs. control; ns (not significant)—p > 0.05 vs. control. (d) The effect of 10 µM DDMC on lysosome numbers was assessed using LysoTracker^TM Deep Red staining in SH-SY5Y cells and confocal microscopy. (e) The number of LAMP1-positive vesicles was estimated using immunostaining and captured with a confocal microscope. Lysosome quantification for (c) and (d) was performed with the granularity module in CellProfiler 4.2.1 [21].

Figure 2

Comparative effect of DMC and DDMC on the inhibition of α-Syn aggregation. (a) Effect of DMC and DDMC on α-Syn aggregation as measured by the fluorescence emission intensity of 25 μM ThT in a solution containing 70 μM α-Syn incubated in the presence of 10 or 50 μM DMC or DDMC, after 120 h of incubation. Statistical significance is indicated as: * p = 0.0148; **** p < 0.0001 vs. Control (-); # p = 0.0152 DDMC 10 μM vs. DDMC 50 μM, ## p = 0.0025 DMC 10 μM vs. DDMC 10 μM, #### p < 0.0001 DMC 10 μM vs. DMC 50 μM. (b) Absorbance of 20 µM Congo red in a solution containing 70 μM α-Syn incubated in the presence or absence of 10 µM DMC or DDMC. Statistical significance is indicated as: **** p < 0.0001 vs. Control (-); # p = 0.025 DMC 10 μM vs. DDMC 10 μM. Values represent the mean ± SEM (n = 5), and statistical analyses were performed as described in Materials and Methods. (c) Transmission electron microscopy (TEM) of α-Syn samples incubated for 120 h in the presence of 10 μM DMC or DDMC.

Figure 3

Effect of DDMC on α-Syn seeding induced by externally added α-Syn_PFF. (a) The fluorescence emission intensity of 25 μM ThT as a function of time (0-120 hr) in a solution containing 70 μM monomeric α-Syn (α-Syn_m) alone (black circles) or seeded with α-Syn_PFF (α-Syn_m+α-Syn_PFF) in the absence (green triangles) or presence of DDMC (α-Syn_m+α-Syn_PFF+DDMC) (orange squares) after 120 h of incubation. For each time point, data values represent the mean + SEM. (b) Lag phase quantification based on the kinetics of aggregation of the seeding assay as described in Materials and Methods. Statistical significance is indicated as: **** p < 0.01; ** p < 0.01 vs. α-Syn_m.

Figure 4

Effect of DDMC on α-Syn_PFF-induced seeding of cytoplasmic α-Syn. (a–c) SH-SY5Y transgenic cell line (Innoprot #P30707-02) expressing α-Syn-tRFP treated with α-Syn_PFF or α-Syn_PFF+DDMC (10 µM). The lower panel corresponds to an enlargement of the region indicated with the white arrows together with DAPI counterstaining. (d) Granularity was quantified using CellProfiler 4.2.1. Statistical significance is indicated as: *** p < 0.001 vs. Control; ### p < 0.01 vs. α-Syn_PFF; ns not significant vs. Control.

Figure 5

DDMC mitigates the inflammogenic properties of α-Syn amyloid-like fibrils. (a) TNFα and (b) Glut release from microglial cells undergoing (i) no treatment (control) or an exposure to (ii) α-Syn_PFF; (iii) α-Syn_PFF together with 0.7 µM DDMC (α-Syn_PFF+DDMC) or to iv) α-SynPFF formed in the presence of 10 µM DDMC (α-SynPFF:DDMC) with subsequent dialysis. Bars represent the mean ± S.E.M (n = 8). Statistical significance is indicated as: *** p < 0.001; **** p < 0.001 vs. α-Syn_PFF; #### p < 0.0001 vs. control; ns—p > 0.05. Note that 0.7 µM DDMC corresponds to the residual concentration of DDMC theoretically remaining in α-Syn_PFF+DDMC samples before dialysis.