Design, Synthesis, and Evaluation of a Novel Conjugate Molecule with Dopaminergic and Neuroprotective Activities for Parkinson's Disease.

The escalating prevalence of Parkinson's disease (PD) underscores the need for innovative therapeutic interventions since current palliative measures, including the standard l-Dopa formulations, face challenges of tolerance and side effects while failing to address the underlying neurodegenerative processes. Here, we introduce DAD9, a novel conjugate molecule that aims to combine symptomatic relief with disease-modifying strategies for PD. Crafted through knowledge-guided chemistry, the molecule combines a nonantibiotic doxycycline derivative with dopamine, preserving neuroprotective attributes while maintaining dopaminergic agonism. This compound exhibited no off-target effects on PD-relevant cell functions and sustained antioxidant and anti-inflammatory properties of the tetracycline precursor. Furthermore, it effectively interfered with the formation and seeding of toxic α-synuclein aggregates without producing detrimental oxidative species. In addition, DAD9 was able to activate dopamine receptors, and docking simulations shed light onto the molecular details of this interaction. These findings position DAD9 as a potential neuroprotective dopaminergic agonist, promising advancements in PD therapeutics.

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

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-SynPFF) in biophysical assays and in a SH-SY5Y-α-Syn-tRFP cell model. In addition, DDMC rendered α-SynPFF 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.

Doxycycline Interferes With Tau Aggregation and Reduces Its Neuronal Toxicity.

Tauopathies are neurodegenerative disorders with increasing incidence and still without cure. The extensive time required for development and approval of novel therapeutics highlights the need for testing and repurposing known safe molecules. Since doxycycline impacts α-synuclein aggregation and toxicity, herein we tested its effect on tau. We found that doxycycline reduces amyloid aggregation of the 2N4R and K18 isoforms of tau protein in a dose-dependent manner. Furthermore, in a cell free system doxycycline also prevents tau seeding and in cell culture reduces toxicity of tau aggregates. Overall, our results expand the spectrum of action of doxycycline against aggregation-prone proteins, opening novel perspectives for its repurposing as a disease-modifying drug for tauopathies.

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

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.

Tetracycline repurposing in neurodegeneration: focus on Parkinson's disease.

The prevalence of Parkinson's disease, which affects millions of people worldwide, is increasing due to the aging population. In addition to the classic motor symptoms caused by the death of dopaminergic neurons, Parkinson's disease encompasses a wide range of nonmotor symptoms. Although novel disease-modifying medications that slow or stop Parkinson's disease progression are being developed, drug repurposing, which is the use of existing drugs that have passed numerous toxicity and clinical safety tests for new indications, can be used to identify treatment compounds. This strategy has revealed that tetracyclines are promising candidates for the treatment of Parkinson's disease. Tetracyclines, which are neuroprotective, inhibit proinflammatory molecule production, matrix metalloproteinase activity, mitochondrial dysfunction, protein misfolding/aggregation, and microglial activation. Two commonly used semisynthetic second-generation tetracycline derivatives, minocycline and doxycycline, exhibit effective neuroprotective activity in experimental models of neurodegenerative/ neuropsychiatric diseases and no substantial toxicity. Moreover, novel synthetic tetracyclines with different biological properties due to chemical tuning are now available. In this review, we discuss the multiple effects and clinical properties of tetracyclines and their potential use in Parkinson's disease treatment. In addition, we examine the hypothesis that the anti-inflammatory activities of tetracyclines regulate inflammasome signaling. Based on their excellent safety profiles in humans from their use for over 50 years as antibiotics, we propose the repurposing of tetracyclines, a multitarget antibiotic, to treat Parkinson's disease.

Exploiting the therapeutic potential of ready-to-use drugs: Repurposing antibiotics against amyloid aggregation in neurodegenerative diseases.

Neurodegenerative diseases are chronic and progressive disorders that affect specific regions of the brain, causing gradual disability and suffering that results in a complete inability of patients to perform daily functions. Amyloid aggregation of specific proteins is the most common biological event that is responsible for neuronal death and neurodegeneration in various neurodegenerative diseases. Therapeutic agents capable of interfering with the abnormal aggregation are required, but traditional drug discovery has fallen short. The exploration of new uses for approved drugs provides a useful alternative to fill the gap between the increasing incidence of neurodegenerative diseases and the long-term assessment of classical drug discovery technologies. Drug re-profiling is currently the quickest possible transition from bench to bedside. In this way, experimental evidence shows that some antibiotic compounds exert neuroprotective action through anti-aggregating activity on disease-associated proteins. The finding that many antibiotics can cross the blood-brain barrier and have been used for several decades without serious toxic effects makes them excellent candidates for therapeutic switching towards neurological disorders. The present review is, to our knowledge, the first extensive evaluation and analysis of the anti-amyloidogenic effect of different antibiotics on well-known disease-associated proteins. In addition, we propose a common structural signature derived from the antiaggregant antibiotic molecules that could be relevant to rational drug discovery.

Lessons learned from protein aggregation: toward technological and biomedical applications.

The close relationship between protein aggregation and neurodegenerative diseases has been the driving force behind the renewed interest in a field where biophysics, neurobiology and nanotechnology converge in the study of the aggregate state. On one hand, knowledge of the molecular principles that govern the processes of protein aggregation has a direct impact on the design of new drugs for high-incidence pathologies that currently can only be treated palliatively. On the other hand, exploiting the benefits of protein aggregation in the design of new nanomaterials could have a strong impact on biotechnology. Here we review the contributions of our research group on novel neuroprotective strategies developed using a purely biophysical approach. First, we examine how doxycycline, a well-known and innocuous antibiotic, can reshape α-synuclein oligomers into non-toxic high-molecular-weight species with decreased ability to destabilize biological membranes, affect cell viability and form additional toxic species. This mechanism can be exploited to diminish the toxicity of α-synuclein oligomers in Parkinson's disease. Second, we discuss a novel function in proteostasis for extracellular glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in combination with a specific glycosaminoglycan (GAG) present in the extracellular matrix. GAPDH, by changing its quaternary structure from a tetramer to protofibrillar assembly, can kidnap toxic species of α-synuclein, and thereby interfere with the spreading of the disease. Finally, we review a brighter side of protein aggregation, that of exploiting the physicochemical advantages of amyloid aggregates as nanomaterials. For this, we designed a new generation of insoluble biocatalysts based on the binding of photo-immobilized enzymes onto hybrid protein:GAG amyloid nanofibrils. These new nanomaterials can be easily functionalized by attaching different enzymes through dityrosine covalent bonds.

Repurposing doxycycline for synucleinopathies: remodelling of α-synuclein oligomers towards non-toxic parallel beta-sheet structured species.

Synucleinophaties are progressive neurodegenerative disorders with no cure to date. An attractive strategy to tackle this problem is repurposing already tested safe drugs against novel targets. In this way, doxycycline prevents neurodegeneration in Parkinson models by modulating neuroinflammation. However, anti-inflammatory therapy per se is insufficient to account for neuroprotection. Herein we characterise novel targets of doxycycline describing the structural background supporting its effectiveness as a neuroprotector at subantibiotic doses. Our results show that doxycycline reshapes α-synuclein oligomers into off-pathway, high-molecular-weight species that do not evolve into fibrils. Off-pathway species present less hydrophobic surface than on-pathway oligomers and display different ß-sheet structural arrangement. These structural changes affect the α-synuclein ability to destabilize biological membranes, cell viability, and formation of additional toxic species. Altogether, these mechanisms could act synergically giving novel targets for repurposing this drug.

Structural characterization of heparin-induced glyceraldehyde-3-phosphate dehydrogenase protofibrils preventing α-synuclein oligomeric species toxicity.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional enzyme that has been associated with neurodegenerative diseases. GAPDH colocalizes with α-synuclein in amyloid aggregates in post-mortem tissue of patients with sporadic Parkinson disease and promotes the formation of Lewy body-like inclusions in cell culture. In a previous work, we showed that glycosaminoglycan-induced GAPDH prefibrillar species accelerate the conversion of α-synuclein to fibrils. However, it remains to be determined whether the interplay among glycosaminoglycans, GAPDH, and α-synuclein has a role in pathological states. Here, we demonstrate that the toxic effect exerted by α-synuclein oligomers in dopaminergic cell culture is abolished in the presence of GAPDH prefibrillar species. Structural analysis of prefibrillar GAPDH performed by small angle x-ray scattering showed a particle compatible with a protofibril. This protofibril is shaped as a cylinder 22 nm long and a cross-section diameter of 12 nm. Using biocomputational techniques, we obtained the first all-atom model of the GAPDH protofibril, which was validated by cross-linking coupled to mass spectrometry experiments. Because GAPDH can be secreted outside the cell where glycosaminoglycans are present, it seems plausible that GAPDH protofibrils could be assembled in the extracellular space kidnapping α-synuclein toxic oligomers. Thus, the role of GAPDH protofibrils in neuronal proteostasis must be considered. The data reported here could open alternative ways in the development of therapeutic strategies against synucleinopathies like Parkinson disease.

The Ile13 residue of microcin J25 is essential for recognition by the receptor FhuA, but not by the inner membrane transporter SbmA.

Entry of the peptide antibiotic microcin J25 (MccJ25) into target cells is mediated by the outer membrane receptor FhuA and the inner membrane protein SbmA. The latter also transports MccB17 into the cell cytoplasm. Comparison of MccJ25 and MccB17 revealed a tetrapeptide sequence (VGIG) common to both antibiotics. We speculated that this structural feature in MccJ25 could be a motif recognized by SbmA. To test this hypothesis, we used a MccJ25 variant in which the isoleucine in VGIG (position 13 in the MccJ25 sequence) was replaced by lysine (I13K). In experiments in which the FhuA receptor was bypassed, the substituted microcin showed an inhibitory activity similar to that of the wild-type peptide. Moreover, MccJ25 interfered with colicin M uptake by FhuA in a competition assay, while the I13K mutant did not. From these results, we propose that the Ile(13) residue is only required for interaction with FhuA, and that VGIG is not a major recognition element by SbmA.

The leucine-responsive regulatory protein, Lrp, modulates microcin J25 intrinsic resistance in Escherichia coli by regulating expression of the YojI microcin exporter.

Many Escherichia coli K-12 strains display an intrinsic resistance to the peptide antibiotic microcin J25. In this study, we present results showing that the leucine-responsive regulatory protein, Lrp, is involved in this phenotype by acting as a positive regulator of YojI, a chromosomally encoded efflux pump which expels microcin out of cells. Exogenous leucine antagonizes the effect of Lrp, leading to a diminished expression of the pump and an increased susceptibility to microcin J25.