A compound-based therapy approach for EBS (Magin 3)Completed
|Project lead||Prof Thomas Magin|
|Organisation||University of Leipzig, Leipzig, GERMANY|
|Project budget||EUR 253,100.00|
|Start date / Duration||01. Nov 2017 / 36 months|
|Funder(s) / Co-Funder(s)||DEBRA Austria, MSAP/EBEP Recommended|
|Research area||Molecular therapy, EB genetics, epigenetics & biology|
Short lay summary
The cytoskeleton maintains the shape of cells, protects them against force and helps cells to adhereto each other to form tissues such as the skin. Part of this system is a network of interlinked filamentous proteins called keratins, whose role is to provide resilience against mechanical forces and preserve cellular integrity. This network is of paramount importance for epithelial cells, which are constantly subject to stress. In epidermolysis bullosa simplex (EBS), the most recurrent mutations affect the genes coding for keratin 5 and keratin 14. When mutated, these proteins form clumps and are no longer functional, leading to fragile skin cells. The aim and vision of this project are to restore a functional keratin cytoskeleton by drugs that can be applied to the skin locally as a cream or solvent. The team led by Prof. Magin has proof-of-concept data based on a drug that has already been used to treat other diseases and can be considered safe. During this project, they will further characterize this drug and identify novel, more potent drugs for the treatment of EBS. To achieve these goals, the team led by Prof. Magin will analyze how the compound works in skin cells and what it does to remove protein aggregates. Next, they plan to investigate whether this molecule improves wound healing and helps re-establish the skin barrier, which protects the body against infections. In the last part of the project, they will screen for additional compounds that could restore a functional keratin network in EBS, laying the ground for future pre-clinical assays. Most of the experiments will be performed in EBS patients’ cells and will employ state of the art biochemistry and molecular biology techniques.
KRT5 and KRT14 mutations cause EBS, characterized by keratin aggregates that render the epidermis fragile upon trauma. The mechanisms by which such mutations cause keratin aggregation remain incompletely understood. The Magin lab has accumulated evidence that at least some mutations introduce secondary post-translational keratin modification (PTM). Together with EBS-associated mutations, such modifications appear to aggravate disease severity. If this is the case, drugs that interfere with keratin PTMs should reduce keratin aggregation. To build on this concept, they started a compound screen using a library of ~5.000 bioactive compounds, many of them representing re-purposed drugs, made available to them by Prof. J. Sexton, a colleague of Prof. M.B. Omary, their named collaborator (University of Michigan, Ann Arbor). The team has identified several compounds, which apparently reduce keratin clumping. The most promising compounds are currently under investigation regarding optimal concentration as well as application time. Furthermore, the team is developing function assays to assess whether reducing keratin clumping restores the formation of a stress-resilient cytoskeleton and ex vivo keratinocyte sheets. Those drugs that turn out to be the most effective will be validated further in preclinical models.
This team of researchers has identified compounds with promising profiles that could effectively help to stabilize otherwise fragile skin cells in EBS by modifying the cells keratin cytoskeleton. The concept my lead to therapeutics that can be locally applied to affected skin.
What did this project achieve?
In this project, the team followed the hypothesis that keratin aggregation can be reduced and cell adhesion be improved by drugs, aiming to make fragile skin of EBS sufferers more resilient. To test this, they screened a collection of ~5.000 drugs of which several were already in clinical use for the treatment of other diseases. They identified several drugs that reduced keratin aggregation in EBS patient-derived skin cells and focused on PKC412, a multi-kinase inhibitor because it showed the strongest effect in our cell-based assays. Detailed analysis revealed that PKC412 reduced phosphorylation of EBS-associated keratins and of desmoplakin, a major desmosome protein that anchors keratins at the plasma membrane. As a consequence, skin cells stick to each other much stronger than cells not treated with the inhibitor and maintained a cell sheet even when stressed. This project, helped by collaborators Prof. M. B. Omary (Rutgers University) and Prof. C. Has (Dermatology, Freiburg University) provides the basis to develop PKC412 in a drug for the localized treatment of EBS.