User Story: Prof. Alessandro Provenzani, University of Trento

User:

Prof. Alessandro Provenzani, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Italy

EU-OPENSCREEN screening partner site:

Prof. Petr Dzubak, Palacký University, Olomouc, Czech Republic

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease associated with the progressive loss of muscle control. Although most cases of ALS are sporadic, about 10% of cases have a strong genetic/familial component. In one subtype of familial ALS (C9ALS), a repetition of hexanucleotides generates an aberrant RNA that is translated via a non-canonical mechanism, called RAN translation, into a series of toxic dipeptide repeats (DPRs).

There is currently no cure for any form of ALS. However, as a potential therapeutic avenue, the research group of Prof. Alessandro Provenzani, CIBIO, University of Trento, Italy, has been working to identify small molecule hits that decrease C9ALS-associated DPR levels, which would thereby eliminate their toxic effects on neurons.

In a previous effort, Alessandro’s group successfully identified several small molecules and genetic pathways that modulate DPR levels whilst sparing normal protein synthesis (EMBO J. 2022 Jan 4;41(1):e105026). In this proof-of-principle work, they transfected cells with two reporter plasmids: one coded for a red fluorescent protein (representing normal protein synthesis), and one coded for DPRs attached to a green fluorescent protein (Figure 1). They successfully demonstrated the efficacy of this assay for identifying hits but, ultimately, could sample only limited chemical space due to the small size of the compound library available to them.

To expand the pool of potential hits, Alessandro ran a collaboration project with the group of Prof. Petr Dzubak, of the EU-OPENSCREEN screening partner Palacký University Olomouc, Czech Republic. The aim of this research project was to identify a larger number of hits with more potent and bioavailable small molecules. The project was funded by the EU Horizon 2020 research and innovation programme through the EU-OPENSCREEN-DRIVE project (Grant Agreement No. 823893).

Initially, Petr's group adapted the assay to a 384-well plate format, and its robustness was further validated. The EU-OPENSCREEN European Chemical Biology Library (ECBL), consisting of approximately 100,000 small molecules, was then used in single dose to select a restricted number of active small molecule hits. Petr’s group further validated these hits in dose-response, and the best compounds were sent to Alessandro for validation in orthogonal assays.

Alessandro’s group is now testing these hits in cells and animal models for efficacy, as some have demonstrated advantageous biodistribution profiles within organisms. Other molecules emerging from the screen are novel to the context of DPR modulation in familial ALS, thus they will require further chemical optimisation before they can be considered for application in animals.

With the help of Petr's group, and particularly Dr Jiri Rehulka, Palacký University Olomouc, Czech Republic, Alessandro highlights that he had the unique opportunity to perform an extensive screening campaign with high-quality laboratory practices. This EU-OPENSCREEN-DRIVE collaboration project therefore allowed him to crucially progress his research by identifying a novel class of small molecules which modulate his target.