A High-Content Screening Assay for Small Molecules That Stabilize Mutant Triose Phosphate Isomerase (TPI) as Treatments for TPI Deficiency
| Autor: | Michael J. Palladino, Stacy L. Hrizo, Tracey D. Myers, E. Michael Meyer, Samantha L. Eicher, Laura L. Vollmer, Andreas Vogt |
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| Rok vydání: | 2021 |
| Předmět: |
0301 basic medicine
Phenotypic screening Mutant Drug Evaluation Preclinical Biology medicine.disease_cause Biochemistry Article Analytical Chemistry Green fluorescent protein Triosephosphate isomerase Small Molecule Libraries 03 medical and health sciences 0302 clinical medicine Genes Reporter Drug Discovery Enzyme Stability parasitic diseases medicine Humans Mutation Drug discovery HEK 293 cells High-Throughput Screening Assays HEK293 Cells 030104 developmental biology High-content screening Molecular Medicine 030217 neurology & neurosurgery Triose-Phosphate Isomerase Biotechnology |
| Zdroj: | SLAS Discov |
| ISSN: | 2472-5552 |
| DOI: | 10.1177/24725552211018198 |
| Popis: | Triose phosphate isomerase deficiency (TPI Df) is an untreatable, childhood-onset glycolytic enzymopathy. Patients typically present with frequent infections, anemia, and muscle weakness that quickly progresses with severe neuromusclar dysfunction requiring aided mobility and often respiratory support. Life expectancy after diagnosis is typically ~5 years. There are several described pathogenic mutations that encode functional proteins; however, these proteins, which include the protein resulting from the "common" TPIE105D mutation, are unstable due to active degradation by protein quality control (PQC) pathways. Previous work has shown that elevating mutant TPI levels by genetic or pharmacological intervention can ameliorate symptoms of TPI Df in fruit flies. To identify compounds that increase levels of mutant TPI, we have developed a human embryonic kidney (HEK) stable knock-in model expressing the common TPI Df protein fused with green fluorescent protein (HEK TPIE105D-GFP). To directly address the need for lead TPI Df therapeutics, these cells were developed into an optical drug discovery platform that was implemented for high-throughput screening (HTS) and validated in 3-day variability tests, meeting HTS standards. We initially used this assay to screen the 446-member National Institutes of Health (NIH) Clinical Collection and validated two of the hits in dose-response, by limited structure-activity relationship studies with a small number of analogs, and in an orthogonal, non-optical assay in patient fibroblasts. The data form the basis for a large-scale phenotypic screening effort to discover compounds that stabilize TPI as treatments for this devastating childhood disease. |
| Databáze: | OpenAIRE |
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