Mercoledì del PRIN n.7: Alberto Cassetta – Giuseppe Mangiatordi/Vittoria Nanna

Therapeutic opportunities in Lafora disease, a rare juvenile neurodegenerative epilepsy with no treatment

Dr. Alberto Cassetta

Lafora disease (LD) is a progressive and fatal epilepsy with teenage-onset. It is caused by the accumulation of insoluble polyglucosans in neurons, determined by an increased glycogen production. At the molecular level, genetic alterations cause missing degradation of the PTG protein (Protein Targeting to Glycogen), a therapeutic target in Lafora disease.

We aim at identifying small molecules halting and possibly reversing the course of Lafora disease.

A first drug repurposing approach will be explored by testing FDA-approved drugs in a newly developed Lafora disease model based on patients derived neural stem cells. This will be performed in high-throughput format using a phenotypic assay. In this context, we will also enlarge our biobank of LD patients stem cells.

A second, rational drug design pipeline will be applied to shape molecules on the PTG surface to interfere with its activation of glycogen synthesis. High-resolution three-dimensional structures of PTG in complex with carbohydrates and protein phosphatase 1, another protein regulating glycogen production, have already been determined by the collaborative network proposing the current project. PTG surface has been scanned, and compounds potentially interfering with its function have been identified by molecular docking. These rationally identified compounds will be tested for their binding to PTG together with a blind crystallographic screening of about 2000 fragments. The development of PROTACs (PROteolysis Targeting Chimeras), leading to the proteasomal degradation of PTG, is planned. As a third task, we will deepen our knowledge of the molecular basis of the disease by determining the crystallographic structures of LD-related macromolecular complexes, mainly focusing on the PTG/malin/laforin assembly. Malin and laforin are indeed the proteins mutated in LD.

In conclusion, the project will probe a new disease-modifying strategy to inform the development of novel therapies for LD, a severe pediatric epilepsy. A treatment option could then become available for LD patients in the short (drug repurposing) or medium term (new drug development with accelerated approval). The project will also make available to the scientific community a LD biobank, a LD phenotypic assay and the structure of the LD main molecular determinants.

Keywords

1. Lafora disease;
2. PTG protein (Protein Targeting to Glycogen);
3. Malin;
4. Laforin;
5. rational drug design;
6. PROTACs (PROteolysis Targeting Chimeras).

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Development of broad-spectrum coronavirus antiviral agents acting as allosteric modulators of the host protein sigma-1

Dr. Giuseppe Mangiatordi and Dr. Vittoria Nanna

S1R is a ligand-operated, membrane-bound chaperone normally located at the ER-mitochondria interface where it regulates the cross-talk between ER-Mitochondrion and ER-nucleus. Upon stress signals, the receptor translocates to ER and plasma membrane to regulate functional proteins. The recently identified interactions of s1R and s2R with viral proteins (NSP6 and Orf9c) and the strong anti-SARS-CoV-2 activity of several s1R ligands have prompted this collaborative project with the same group of scientists who revealed the SARS-CoV-2 interactome and that are obtaining ground-breaking results in the fight against SARS-CoV-2. Thanks to this collaboration, we showed that the s1R Knock Out (KO) or Knock down (KD) prevents the SARS-CoV-2 infection of cells (Caco-2 and A549-ACE2), while KO and KD of the s2R does not. Accordingly, s1R displays the same effect in SARS-CoV-1, with its KO completely abating the virus infection. These encouraging results prompted us to test a chemical library of PB28 analogues. These compounds were tested in vitro at the Pasteur Institute in Paris and while highly potent anti-SARS-CoV-2 compounds were found, a strict correlation between the s1R affinity and antiviral activity was not always observed. Preliminary docking data, obtained by applying the Induced Fit Protocol (IFD) into the s1R crystal structure (PDB code: 6DJZ), suggest that the antiviral activity could be linked to a specific binding mode. The clear s1R involvement in the coronavirus infections and the promising data from our s1R ligands encourage further characterization of the s1R role in these infections in order to develop novel highly potent ligands with the perspective of providing a therapeutic strategy also against the outbreak of mutants that might not respond to the developed vaccines and therapies addressed at viral components. The project will be based on a multidisciplinary approach utilizing up-to-date structural biology methodologies combined with computational and experimental techniques commonly used in drug discovery programs. All the compounds synthesized by will be tested for their affinity at the s1R. As a control, the anti-SARS-CoV-2 assays will be also performed at the Pasteur Institute together with the antiviral activity against other viruses such as orthomyxoviruses, flaviviruses, togaviruses and enteroviruses.

Keywords:

1. SIGMA-1 receptor
2. MD simulations
3. SAXS experiments
4. Allosteric mechanism
5. SARS-CoV-2