CMT-3 targets different α-synuclein aggregates mitigating their toxic and inflammogenic effects

Abstract

Parkinson's disease (PD) is a neurodegenerative disorder for which only symptomatic treatments are available. Repurposing drugs that target α-synuclein aggregation, considered one of the main drivers of PD progression, could accelerate the development of disease-modifying therapies. In this work, we focused on chemically modified tetracycline 3 (CMT-3), a derivative with reduced antibiotic activity that crosses the blood–brain barrier and is pharmacologically safe. We found that CMT-3 inhibited α-synuclein amyloid aggregation and led to the formation of non-toxic molecular species, unlike minocycline. Furthermore, CMT-3 disassembled preformed α-synuclein amyloid fibrils into smaller fragments that were unable to seed in subsequent aggregation reactions. Most interestingly, disaggregated species were non-toxic and less inflammogenic on brain microglial cells. Finally, we modelled the interactions between CMT-3 and α-synuclein aggregates by molecular simulations. In this way, we propose a mechanism for fibril disassembly. Our results place CMT-3 as a potential disease modifier for PD and possibly other synucleinopathies.

The Chemically-Modified Tetracycline COL-3 and Its Parent Compound Doxycycline Prevent Microglial Inflammatory Responses by Reducing Glucose-Mediated Oxidative Stress

“…The expression and purification of recombinant human αS were performed as previously described [12,29]. The purity of recombinant αS was evaluated by SDS-PAGE, and contaminating endotoxins were removed from protein samples using a Pierce Spin column (#88275; Thermo Fisher Scientific, Courtaboeuf, France).…”

“…The anti-inflammatory properties of DOX may also explain why this tetracycline provided relief against L-DOPA-induced dyskinesia in a PD rat model [11]. Interestingly, DOX was also found capable of preventing amyloid aggregation of α Synuclein (αS) and tau, two seeding-prone proteins involved in PD [12,13] and Alzheimer's disease [14] pathologies, respectively, suggesting that this tetracycline has the potential of a multimodal neuroprotective drug.…”

The non-antibiotic tetracycline COL-3 prevents microglial inflammatory responses by reducing glucose-mediated oxidative stress.

“…That might be explained by the structural differences of these two molecules: the presence of methyl groups that play a role in the antibiotic activity of DOX might not influence or even reduce the anti-inflammatory/antioxidant response shaped by this tetracycline. Gonzalez-Lizarraga et al [17] described that COL-3 is also more effective than DOX as an antiaggregant molecule due to these structural distinctions. In this way, COL-3 not only efficiently prevented the formation of toxic α-synuclein amyloid aggregates in vitro , but also disaggregated α-synuclein amyloid fibrils, while DOX was only efficient in the former analysis.…”

“…To date, more than eight CMTs are available [15]. Because CMTs might represent a safer treatment compared to antimicrobial treatment with tetracyclines, since their administration in laboratory animals does not produce tetracycline resistant microorganisms in the oral and intestinal flora [16], some of them, as CMT-1, CMT3 and CMT-8 have been tested in pre-clinical and clinical applications, including cancer trials and neurological disease studies [10,17,18].…”

“…COL-3 molecule is highly lipophilic, so it can also cross the blood-brain barrier and thus act within the brain [14,26]. COL-3 mechanism of action in the central nervous system includes anti-apoptotic effects, anti-protein aggregation, removal of ROS, inhibition of matrix metalloproteinase, and protection against mitochondrial dysfunctions, among others [2,17]. COL-3 has been also proposed as an anti-inflammatory molecule, as it can inhibit lipopolysaccharide (LPS)-induced microglial activation and cytokine expression in the brain [26]; however, the mechanism through which this molecule reduces neuroinflammation is yet to be described.…”