Department of Biology
Library of the School of Science

2024-12-19

2024

Giannakou Maria

Dimitris G. Hatzinikolaou, Professor of Microbial Biotechnology, Biology Dpt, University of Athens
Georgios Skretas, Director, Institute for Bio-innovation, Biomedical Sciences Research Center "Alex. Fleming"
Constantinos E. Vorgias, Professor of Biochemistry, Biology Dpt, University of Athens
Emmanuel Mikros, Professor of Pharmaceutical Chemistry, Pharmacy Dept, University of Athens
Vasileios Myrianthopoulos, Assistant Professor of Pharmaceutical Chemistry, Pharmacy Dept, University of Athens
Ieronymos-Jerome Zoidakis, Assistant Professor of Biochemistry-Molecular Biology, Biology Dpt, University of Athens
Evagelos Gikas, Professor of Analytical Chemistry, Chemistry Dept, University of Athens

«Εφαρμογή μεθόδων Συνθετικής Βιολογίας & Μοριακής εξέλιξης για την ανακάλυψη δυνητικών θεραπευτικών μορίων κατά νευροεκφυλιστικών ασθενειών που προκαλούνται από προβληματική πρωτεϊνική αναδίπλωση & συσσωμάτωση»

English

«Εφαρμογή μεθόδων Συνθετικής Βιολογίας & Μοριακής εξέλιξης για την ανακάλυψη δυνητικών θεραπευτικών μορίων κατά νευροεκφυλιστικών ασθενειών που προκαλούνται από προβληματική πρωτεϊνική αναδίπλωση & συσσωμάτωση»

Cu/Zn Superoxide Dismutase 1 (SOD1) is a 32-kDa homodimeric enzyme expressed predominantly in the cytosol, catalyzing the reaction of superoxide radicals O2- to harmless O2 and H2O2. It is a metalloenzyme with copper enabling the redox cycle responsible for its activity, zinc stabilizing the native dimeric conformation and an intramolecular disulfide bond stabilizing the native structure.
Mutations in the SOD1 are related to ALS disease, which is pathologically distinguished by atrophy and death of the affected motor neurons which lead to disease-associated mortality. ALS-associated SOD1 mutations are related to a mild reduction of the enzyme’s activity but their deleterious effect is mediated through a gain of function mechanism, rather than a loss of function effect, as proposed by cellular models and transgenic animal studies. Transgenic mice overexpressing ALS-associated SOD1 mutations develop symptoms of the disease while SOD1 knock-out mice are not susceptible to motor neuron loss. The mechanism by which SOD1 mutants cause selective motor neuron death still remains elusive. Cellular functions affected by mutations include: reduced dismutase activity, changes in the function of glutamate transporter’s related to excitotocixity, intracellular copper dyshomeostasis and increased protein’s propensity to midfold and aggregate, a mechanism which this thesis is dedicated.
Protein misfolding and aggregation is an important aspect of ALS since ubiquitinated protein inclusions containing SOD1 in the cytosol of motor neurons constitutes an indisputable finding and hallmark of the disease. The present work describes an attempt to discover compounds as rescuers of misfolding and aggregation of SOD1(A4V) with potentially therapeutic properties against ALS, using two different approaches.
The first approach exploits the drug repurposing advantages. We performed an in vitro screening of a well-characterized library of 1280 pharmacologically active compounds, LOPAC®. Each compound of the library was tested against SOD1(A4V) mutant and its ability to increase proteins thermal stability was assessed with Differential Scanning Fluorimetry Technique (DSF), in a high-throuput manner. DSF is a biophysical technique that quantifies thermal stability of a protein either alone or in the context of drug-protein interaction. LOPAC® screening led to the identification of one compound able to shift the protein-ligand Tm by 7oC, eleven compounds led to the appearance of a second Tm higher than that of the protein alone, while five compounds were found to reduce Tm even up to a difference of 18oC, suggesting a possible interaction and/or non-specific binding. Furthermore, the results of the molecular docking calculations support our hypothesis that the 12 compounds, which caused an increase in the Tm of this specific mutation, bind near the amino acid Cys111 (Pocket 1), an area critical to aggregation kinetics of the protein and important due to its involvement in covalent interactions with other compounds capable of stabilizing the protein.
In the second approach, an initial evaluation of selected cyclic peptides that previously emerged from the application of an engineered bacterial platform was performed. In this platform, combinatorial libraries of cyclic heptapeptides are biosynthesized inside E. coli cells and simultaneously screened for their ability to inhibit SOD1(A4V) aggregation. In this system approximately 192 million different cyclic heptapeptides comprising of the twenty natural aminoacids were produced and evaluated in a high-throughput manner in order to identify molecules that are able to prevent the aggregation of the pathogenic SOD1(A4V) variant.
Compounds emerged from forementioned screening approaches, were retested in secondary in vitro low-throughput experiments with purified SOD1(A4V) produced from E. coli cells. The subsequent experiments were designed to have as a read-out the inhibition of SOD1 aggregation and specifically kinetic experiments of SOD1 with and without the selected compounds and monitor if the addition of each compound is able to rescue the aggregation pathway. The ability to reduce amyloidogenic aggregation was monitored with ThT, which is an amyloidogenic dye able to shift its fluorescence upon binding on β-sheet structures, characteristic of amyloid (or amyloid-like) formation. In addition, by use of Dynamic Light Scattering (DLS), the distribution of SOD1 different aggregation species was assessed, giving us an idea on the size of the generated aggregates.
Heptapeptides P1 and P2 caused statistically significant reduction of ThT staining implying possible inhibition of SOD1(A4V) aggregation or formation of structures that are more amorphous rather than having amyloid component. Subsequent DLS experiments monitoring SOD1(A4V) size distribution suggest that possibly those two peptides may act as modulators of SOD1 aggregation rather than inhibitors.
The mechanism of their action could be further deciphered and validated by experiments using cells or small animal models, since in general in vitro techniques can not predict the effect of the differentiation of the pathogenesis pathways induced by the presence of the peptides.

Science

Health Sciences

ALS; SOD1; protein-misfolding diseases; drug discovery; drug repurposing; chemical library scanning; DSF technique; plasmid library, synthetic biology, microbial screen, cyclic peptides

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https://pergamos.lib.uoa.gr/uoa/dl/object/3447489