CASSMedChem Lab

The portal for rare diseases and orphan drugs (https://www.orpha.net/) defines RDs as diseases that affects a small number of people compared to the general population. In Europe, a disease is rare if there is an incidence of 1 case per 2000 people. 

There are more than 6,000 different rare diseases in the EU, so while one rare disease may affect only a handful of patients, another may affect up to 250,000. Globally, there are up to 36 million people in the EU who are living with a RD. Around 80% of rare diseases are of genetic origin and of those 70% already start in childhood (EU Fact sheet https://health.ec.europa.eu/system/files/2023-05/ncd_2023_rare-diseases_factsheet_en.pdf)

Despite the big efforts done by EU action on rare diseases to improve the diagnosis, care, and treatment of patients, most RDs still lack specific treatments. Research on the treatment of RDs has focused on various modalities, including the use of antibodies, gene therapy, stem cell therapy and enzyme replacement therapy. However, the use of small molecules and drug repositioning remains a promising area of research, Figure 1.

One of the main limitations in finding cures for RDs is undoubtedly the low level of commercial investment. In our view, the development of rapid and cost-effective methods to assess the feasibility of RD drug development could be an important driver.

Our mission in RDs research is to support clinical geneticists in identifying specific mutations that result in the production of a mutated protein, which can serve as a target for small molecules capable of effectively restoring its function To this end, it is necessary to assess the druggability of protein products derived from mutant DNA by means of a structure-based method. 

We are developing a filtering process to select the most promising mutated proteins to be targeted with small molecules, which will then become potential candidates for a drug discovery program, Figure 2.

First, a panel of mutations arising from clinical data and related to the protein involved in a given RD is collected. The panel can be retrieved from the literature but is mainly provided by clinical geneticists collaborating with our group.

Then the protein is filtered on the basis of four criteria: 

• available information in the literature
• mRNA nonsense-mediated decay and pathogenicity scores
• protein WT 3D structure availability
• protein interactome features.

The four criteria contribute to define a score that allows to discard mutations that are poorly characterized or that probably produce instable or truncated proteins.

The mutated proteins with the highest scores are then modelled using experimental structures, when possible, otherwise using homology models obtained in house or downloaded by AlphaFold. Protein complexes arising from the interactome analysis are also modelled.

The following step includes the investigation of the protein structure in terms of stability, flexibility, surface properties and molecular interactions in order to characterize the potential pathogenic mechanism introduced by each mutation. Docking tools are finally used to find small molecules potentially able to bind to the proteins and restore the functionality of the mutated protein.

The method was successfully applied to mutations in the gene ALS2 which are responsible for rare motor neuron diseases, such as infantile onset ascending hereditary spastic paralysis (IAHSP) and juvenile primary lateral sclerosis (JPLS) and involved in some cases of amyotrophic lateral sclerosis (ALS). There are only ~50 reported cases of IAHSP, but it is estimated that ~150 children have this disease worldwide, for which no specific treatment is available.

Alsin is a protein essential for the development and maintenance of motor neurons through the endosomal/endocytic pathway, Figure 3A shows Alsin domains with the main known interacting proteins. To carry out its function, WT Alsin forms dimers by interaction of VPS9 domains and then tetramerizes; R1611W mutation responsible for IAHSP acts by destabilising the tetramer, Figure 3B.

We collected a panel of mutations of ALS2 suggested by several sources: the non-profit IAHSP patient organisation Help Olly (https://helpolly.it), the Paediatric neuropsychiatry unit specialising in rare genetic neurological disorders of IRCCS Stella Maris, and the literature. Among the mutations with a favourable score calculated with the procedure depicted above, Figure 2, we focused on R1611W. This was also the first mutation reported to our laboratory. R1611W occurs in the core of VPS9 and blocks the initial formation of the dimer, Figure 2 B. The domains of the Alsin structure involved in dimer formation and their potential reciprocal interactions were modelled for the WT and the W1611R mutation. These models suggested the possibility of searching for a small molecule that could bind to W1611R in the region affected by the mutation and mask the arginine charge. In principle, this would restore the hydrophobicity of the protein in VPS9, allowing the formation of the dimer.

To this aim, we looked for a binding pocket near R1611W, Figure 3C, and then the pocket was submitted to a structure-based virtual screening of 9815 approved molecules, retrieved from ZINC (v.v. 15) and Drug Bank (www.drugbank.com). 

The initial screening outputs were filtered with blood–brain barrier (BBB) permeability in silico models and after some refinement runs the virtual screening identified Menaquinone 4 (MK4, Figure 3D) as the most promising candidate. 

MK4 was approved for the treatment of osteoporosis in Japan and commercialized as supplements in Europe and USA. To confirm the in-silico predictions, we performed biochemical analyses in the presence/absence of MK4. Biochemical analysis suggested that in the presence of MK4, the mutated Alsin can tetramerise again and thus its function is restored, Figure 4E. MK4 is now administered to an IHASP patient under compassionate use.