Periodic Reporting for period 1 - PRIGLUE (Protein-RNA interaction stabilization using molecular glues)
Período documentado: 2023-06-01 hasta 2025-11-30
In this project, we look at how RNA-binding proteins regulate the processing of messenger RNA (mRNA), the carrier of the instructions for the production proteins. In particular, we investigate splicing, which is the process in which the parts that do not carry information for the protein (introns) are removed from the parts that do contain relevant information (exons). Variation in the selection of which parts of the mRNA are used for the final product is known as alternative splicing and allows the production of multiple protein products from a single gene. Alternative splicing is one of the mechanisms that allows different cell types to emerge from the same genome, as well as the functioning of similar cells under different conditions. RNA-binding proteins that regulate this process are known as splicing factors and these exist in two forms: those that promote splicing close to where they bind to the mRNA and those that have the opposite effect. Such splicing factors are often found to be in abnormal states in cancer and various other diseases and therefore hold a lot of untapped potential as therapeutic targets.
A traditional way of interfering with splicing factors is to look for inhibitors that block their ability to bind to mRNA. However, inhibition can lead to undesired effects, since a single splicing factor regulates numerous alternative splicing events. Instead, in this project we aim to promote the interaction of a given splicing factor with a specific RNA sequence, so that we can direct splicing on a single (or small number of) event(s) in the desired direction. By doing so, we hope to force the formation of mRNA that produces protein with a beneficial function in a given disease. Such molecules we refer to as protein-RNA interaction molecular glues (PRIGLUEs).
A traditional way of interfering with splicing factors is to look for inhibitors that block their ability to bind to mRNA. However, inhibition can lead to undesired effects, since a single splicing factor regulates numerous alternative splicing events. Instead, in this project we aim to promote the interaction of a given splicing factor with a specific RNA sequence, so that we can direct splicing on a single (or small number of) event(s) in the desired direction. By doing so, we hope to force the formation of mRNA that produces protein with a beneficial function in a given disease. Such molecules we refer to as protein-RNA interaction molecular glues (PRIGLUEs).
The first part of the project aims at the splicing of the SMN2 gene which plays a role in the treatment of spinal muscular atrophy (SMA). Humans have two genes (SMN1 and SMN2) that could theoretically produce the SMN protein, but the SMN2 gene yields a non-productive mRNA. Patients with SMA also have a defective SMN1 gene and therefore have very low levels of the SMN protein leading to muscle degradation. It has already been shown that promoting a specific alternative splicing pattern in the SMN2 gene can convert it to a productive mRNA recovering the lost SMN protein levels. We aim to use this system to demonstrate the concept of PRIGLUEs. We have performed the initial screening to identify PRIGLUEs for the interaction between the splicing factor SRSF1 and the SMN2 gene. The interaction was previously reported to be weak, but to have a positive effect on the correct splicing of SMN2. A PRIGLUE would be able to strengthen this interaction and thereby promote the correct splicing pattern. Unfortunately, no hits were identified that could do so and neither could we confirm previously reported results on binding between SRSF1 and SMN2. Due to these issues, we are currently looking for alternative strategies.
In a second project, we investigate the splicing factor SRSF2, which is commonly mutated in myelodysplastic syndromes and leukemias. Wild-type SRSF2 regulates splicing by binding to both GGAG and CCAG sequences with equal affinity. However, the mutation (P95H) leads to a change in the affinities now strongly preferring CCAG. This change leads to a shift in the splicing events regulated by SRSF2 and contributes to the disease. We aim to identify molecules that stabilize the interaction between GGAG sequences and the mutant SRSF2. An increase in the affinity between these two partners could rebalance the selection of which mRNAs SRSF2 binds and restore correct splicing. Using various biochemical techniques we confirmed the previous findings that SRSF2 has a higher affinity for CCAG sequences and shed further light on the interaction. In parallel, we developed a screening assay to find PRIGLUEs, which is currently being optimized further and screening will commence shortly. In an attempt to create a control molecule, we designed oligonucleotides that can bind and inhibit SRSF2. These oligonucleotides bind mutant SRSF2 with high affinity while the affinity for wild-type SRSF2 is much lower. Preliminary experiments demonstrate that they are able to enter K562 leukemia cells and hint at correcting mutant SRSF2 driven splicing changes.
In a second project, we investigate the splicing factor SRSF2, which is commonly mutated in myelodysplastic syndromes and leukemias. Wild-type SRSF2 regulates splicing by binding to both GGAG and CCAG sequences with equal affinity. However, the mutation (P95H) leads to a change in the affinities now strongly preferring CCAG. This change leads to a shift in the splicing events regulated by SRSF2 and contributes to the disease. We aim to identify molecules that stabilize the interaction between GGAG sequences and the mutant SRSF2. An increase in the affinity between these two partners could rebalance the selection of which mRNAs SRSF2 binds and restore correct splicing. Using various biochemical techniques we confirmed the previous findings that SRSF2 has a higher affinity for CCAG sequences and shed further light on the interaction. In parallel, we developed a screening assay to find PRIGLUEs, which is currently being optimized further and screening will commence shortly. In an attempt to create a control molecule, we designed oligonucleotides that can bind and inhibit SRSF2. These oligonucleotides bind mutant SRSF2 with high affinity while the affinity for wild-type SRSF2 is much lower. Preliminary experiments demonstrate that they are able to enter K562 leukemia cells and hint at correcting mutant SRSF2 driven splicing changes.
Selective ligands of mutant SRSF2 are not yet known. Further development is still required, but these molecules might provide a new therapeutic strategy for patients that carry the mutation. In depth investigation into their effects on splicing is required as well as their effects on cancer cell growth. Nonetheless, oligonucleotides have various disadvantages as drugs and a small molecule would be preferred. Therefore, the results of the high-throughput screening campaign will still be highly valuable.