Chemical compounds targeting MERCs: identification of their partners in physiological and pathological conditions

Informations projet

MERCURY

N° de convention de subvention: 896745

DOI

10.3030/896745

Projet clôturé

Date de signature de la CE 1 Avril 2020

Date de début 1 Août 2021

Date de fin 31 Juillet 2023

Financé au titre de

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Coût total

€ 183 473,28

Contribution de l’UE

€ 183 473,28

Coordonné par

UNIVERSITA DEGLI STUDI DI PADOVA
Italy

Periodic Reporting for period 1 - MERCURY (Chemical compounds targeting MERCs: identification of their partners in physiological and pathological conditions)

Période du rapport: 2021-08-01 au 2023-07-31

Résumé du contexte et des objectifs généraux du projet

Mitochondria and Endoplasmic Reticulum Contact Sites (MERCs) are sites where the surfaces of these two organelles are side by side. Recently, MERCs have received considerable attention because they play an important role in several cell processes, including regulating calcium and lipid homeostasis, mitochondria fission and fusion, and apoptosis. To execute and regulate these processes, MERCs must be dynamic and respond to the needs and metabolic state of the cell. Interestingly, the organelles remain separated by a narrow cleft, typically 10-50 nm apart. The size of the gap between the organelles and its width (i.e. the area of a membrane involved in the contacts) are the critical parameters that determine their cellular functions. Although the molecular nature of MERCs has been extensively studied, the composition of their tethering structures and the underlying processes that regulate their plasticity and functionality are not fully understood.
To clarify these “mysterious” contacts, the MERCURY project focused on the regulation of MERCs by small molecules. The project had two major objectives: first, to identify compounds that modulate MERCs. As a next step, selected molecules would be modified to create affinity probes. Second objective was to unravel the protein targets of the small molecules using the developed affinity probes. This would be of great relevance and importance to the field, as it is still not understood how metabolic changes “drive” MERCs to change their width and size. Since MERCs not only regulate organelle functions under physiological conditions, but may also play important roles in a number of diseases (e.g. type 2 diabetes and neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases), elucidation of the molecules that modulate their (patho)physiological functions may even be of clinical relevance (for drug repurposing or developing new drugs and using the identified molecules as the “lead compounds”).
I am convinced that the MSCA fellowship and the opportunity of a postdoctoral stay at the University of Padua has had a great impact on my future career. First of all, it allowed me to change my research area and learn new methods to complement my “research portfolio”. Second, it was a great stepping stone to becoming an independent scientist. I had many chances to present and discuss my project, to see and discuss the projects of my colleagues, and to learn from others. This opportunity has also allowed me to meet and discuss with world-class scientists and to find out more about the responsibilities of a senior position and to get to know the big picture of science. Third, I have learned not only scientific skills, but also management skills (managing orders, payments, deliveries) and new soft skills (leadership techniques, communicating with other people and companies).

Travail effectué depuis le début du projet jusqu’à la fin de la période considérée dans le rapport et principaux résultats atteints jusqu’à présent

The major goal the project was to develop a high-content screening assay and identify compounds that affect the contact sites between mitochondria and the endoplasmic reticulum. For this purpose, a high-content imaging system Operetta was available at the high-content screening facility of the host institution. Having screened the library, I analyzed the collected data using the R programming language and identified several promising hits that influence the mitochondria-ER contact sites. Finally, to validate the effect of the hit compounds, I tested their activity on the cells using an independent method, transmission electron microscopy.
Then we set to develop affinity probes from the identified hits and identify their protein targets. For this purpose, I planned to use the methodology developed previously (“iBodies”). In the first step of affinity probe preparation, the bioactive hits identified by phenotypic screening were first modified with a linker. Then, they will be conjugated to a hydrophilic copolymer decorated with biotin to form functional iBodies. We will then use the prepared iBodies to pull down the protein targets from the cell lysates.

I have given two presentations at the host lab summarizing the results to date. The host department also organizes a series of lectures (“MitoMeetings”) and my talk was presented in September 2023. I also had the opportunity to present my project at the lab retreat organized by several collaborating laboratories. I also shared my research to my former laboratory in Prague (February 2023). Finally, the International Research Office of my host institution (University of Padua) organized an interview with me to present my MSCA project to practically all researchers at the University and to share my experience with the MSCA application.
In addition to my colleagues and collaborators, I have presented my project to a wider scientific audience at several conferences that I have attended: EMBO Workshop: Mitochondrial Homeostasis and Human Disease (Girona, Spain, 2021); Cell Symposia: Multifaceted Mitochondria (Sevilla, Spain, 2022); EUROMIT 2023: International Meeting on Mitochondrial Pathology (Bologna, Italy, 2023); EMBO Workshop: Inter-organelle contacts biology (Fiuggi, Italy, 2023).
In parallel to disseminating the results to the scientific community, I also reached out to non-scientists. These activities were severely affected by the pandemics - the Researcher’s Night had to be held online as a video presentation (which, after some time, I found to be a new and interesting experience compared to the “usual” face-to-face meetings). This video, which is a brief explanation of my research, is available on the University of Padua’s YouTube channel. Similarly, meeting with students was quite problematic, especially during the first year of the fellowship. However, I did give a talk (April 2023) to high school students about science in general, my career, and my current MSCA project.
Fortunately, the publication of a popular science article on the topic of my research was not affected. In the article (published in the Czech journal “Vesmir” in April 2023) I described the field of organelle contact sites, in particular mitochondria-ER contacts, and briefly outlined my project.

Progrès au-delà de l’état des connaissances et impact potentiel prévu (y compris l’impact socio-économique et les conséquences sociétales plus larges du projet jusqu’à présent)

The project delivers a (semi-) automated screening methodology using high-content microscopy that can be applied to other projects not necessarily focused on the study of mitochondria-ER contact sites. Using this method, we identified several small compounds that modulate the contact sites between mitochondria and the endoplasmic reticulum. Then we synthesized the affinity probes to unravel the protein targets of these molecules.
I believe that the identification of protein targets may provide valuable insight into the regulation of MERCs, which would be the first step in subsequent research focused primarily on the (patho)physiological functions of MERCs. The project now opens up several avenues of exploration and I believe that in the end, the project will not only bring new knowledge to the field of mitochondria-ER contact sites, but will also establish a more general methodological concept for studying membrane contact sites.
Since dysregulation of the membrane contact sites has been implicated in several pathologies, including Alzheimer’s disease and diabetes, we were therefore aware of the potential pharmaceutical applications of the project. In fact, the third work package aimed to evaluate the identified MERCs modulators as potential drug candidates and to identify a lead compound for neurological disorders.

Overview of the high-content screening workflow.