| Autor: |
Kaplan A; Department of Biological Sciences, Columbia University, New York, NY 10027;, Gaschler MM; Department of Chemistry, Columbia University, New York, NY 10027;, Dunn DE; Center for Drug Discovery and Department of Neurobiology, Duke University Medical Center, Durham, NC 27710;, Colligan R; Department of Biological Sciences, Columbia University, New York, NY 10027; Quantitative Proteomics Center, Columbia University, New York, NY 10027;, Brown LM; Department of Biological Sciences, Columbia University, New York, NY 10027; Quantitative Proteomics Center, Columbia University, New York, NY 10027;, Palmer AG 3rd; Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032; and., Lo DC; Center for Drug Discovery and Department of Neurobiology, Duke University Medical Center, Durham, NC 27710;, Stockwell BR; Department of Biological Sciences, Columbia University, New York, NY 10027; Department of Chemistry, Columbia University, New York, NY 10027; Howard Hughes Medical Institute and Department of Systems Biology, Columbia University, New York, NY 10027 bstockwell@columbia.edu. |
| Abstrakt: |
Protein disulfide isomerase (PDI) is a chaperone protein in the endoplasmic reticulum that is up-regulated in mouse models of, and brains of patients with, neurodegenerative diseases involving protein misfolding. PDI's role in these diseases, however, is not fully understood. Here, we report the discovery of a reversible, neuroprotective lead optimized compound (LOC)14, that acts as a modulator of PDI. LOC14 was identified using a high-throughput screen of ∼10,000 lead-optimized compounds for potent rescue of viability of PC12 cells expressing mutant huntingtin protein, followed by an evaluation of compounds on PDI reductase activity in an in vitro screen. Isothermal titration calorimetry and fluorescence experiments revealed that binding to PDI was reversible with a Kd of 62 nM, suggesting LOC14 to be the most potent PDI inhibitor reported to date. Using 2D heteronuclear single quantum correlation NMR experiments, we were able to map the binding site of LOC14 as being adjacent to the active site and to observe that binding of LOC14 forces PDI to adopt an oxidized conformation. Furthermore, we found that LOC14-induced oxidation of PDI has a neuroprotective effect not only in cell culture, but also in corticostriatal brain slice cultures. LOC14 exhibited high stability in mouse liver microsomes and blood plasma, low intrinsic microsome clearance, and low plasma-protein binding. These results suggest that LOC14 is a promising lead compound to evaluate the potential therapeutic effects of modulating PDI in animal models of disease. |
