Pyrimethamine, an antimalarial drug with well documented pharmacokinetics, was confirmed as a β-hexosaminidase pharmacological chaperone and compared favorably with our best carbohydrate-based pharmacological chaperone in patient cells with various mutant genotypes. These observations lead us to screen the NINDS library of 1040 Food and Drug Administration approved compounds for pharmacological chaperones. Two of these inhibitors had derivatives that had been tested in humans for other purposes. To expand the repertoire of pharmacological chaperones to more ‘drug-like’ compounds, we screened the Maybridge library of 50 000 compounds using a real-time assay for non-carbohydrate-based β-hexosaminidase inhibitors and identified several that functioned as pharmacological chaperones in patient cells. Each of the known β-hexosaminidase inhibitors (low μM IC 50) increased mutant enzyme levels to ≥ 10% in chronic Tay-Sachs fibroblasts and also attenuated the thermo-denaturation of β-hexosaminidase.
Pharmacological chaperones are enzyme enhancement therapy-agents that are competitive inhibitors of the target enzyme. Our long experience in working with the β-hexosaminidase (EC 3.2.1.52) isozymes system and its associated deficiencies (Tay-Sachs and Sandhoff disease) lead us to search for possible enzyme enhancement therapy-agents that could treat the chronic forms of these diseases which express 2–5% residual activity. Many lysosomal storage diseases are candidates for enzyme enhancement therapy and have the additional advantage of requiring only 5–10% of normal enzyme levels to reduce and/or prevent substrate accumulation. Candidate diseases are those associated with a mutant protein that has difficulty folding and/or assembling into active oligomers in the endoplasmic reticulum. One of the foremost strength of this project is the outstanding composition of six project partners, which makes up a cross-disciplinary team with representatives from both industry and academia.Enzyme enhancement therapy is an emerging therapeutic approach that has the potential to treat many genetic diseases. The project also investigates the importance of various laminas during the differentiation and culturing of the hiPSCs. Novel in this project is the combination of co-culture, a 3D system and hiPSCs for development of a scalable in vitro BBB model. To induce differentiation of endothelial cells to brain microvascular endothelial cells (BMECs) present in the BBB, a co-culturing of various combinations of neural cells are performed. To improve the performance of this system the project will apply both 2D and 3D culturing systems in the effort to develop an in vitro BBB model that mimics the in vivo structural organisation in tissue. The culture environment plays a key role both for differentiation and preservation of morphological characteristics of cells. Such system will be exceptionally useful for toxicity testing and for studies of neural disorders.
The overall goal is to provide a platform that demonstrates high level of robustness and reproducibility for screening brain-penetrating therapeutics, which will be of great value for the pharmaceutical industry. To address these shortcomings, this project use human induced pluripotent stem cells (hiPSCs) as an infinitive human cell source for developing a BBB model system. However, these models have been hampered by cell availability and model fidelity, restricting their use in large-scale screens. This has led investigators to develop in vitro BBB model systems to enable detailed mechanistic studies and drug screens. Due to its barrier characteristics, the BBB significantly hampers development of neuropharmaceuticals by preventing the up-take of small molecules in the brain tissue. He blood-brain barrier (BBB) plays an important role in brain health and is often comprised in neurological disorders. The project aims at developing a human in vitro model based on human pluripotent stem cells that can mimic important aspects of the blood-brain-barrier. The pharmaceutical industry has an urgent need for in vitro model systems with high human relevance that can be used for toxicity testing, drug development, and disease modelling.
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