Abstract
There is a great deal of interest in neurotrophin therapy to prevent neuronal degeneration. However, the blood-brain barrier presents a major hurdle in the use of peptide therapeutics. The goal of this study was to identify small molecule, cell-permeable nerve growth factor (NGF) activators. Combinatorial libraries of asterriquinones (>300) and mono-indolyl-quinones (>60) were screened using a 96-well enzyme-linked immunosorbent assay that detects phosphorylated TrkA, the NGF receptor. The libraries were also screened for dose-dependent cytotoxicity. From these screens, we generated quantitative structure-activity relationship models for activity and toxicity, and then we selected two compounds, 2-(6-chloro-1H-indol-3-yl)-5-(2-cyclopropyl-1H-indol-3-yl)-3,6-dihydroxy-[1,4]benzoquinone (1H5) and 2,5-dimethoxy-3-(7-fluoro-1H-indol-3-yl)-[1,4]-benzoquinone (5E5), for further study based on high activity and low toxicity. Compound 1H5 (30 μM) is an asterriquinone that is a moderate TrkA activator (50% the activity of 100 ng/ml NGF), and it shows little toxicity at concentrations up to 100 μM. 1H5 can protect differentiated PC12 neurons from apoptotic cell death induced by NGF withdrawal. Compound 5E5 (30 μM) is a mono-indolyl-quinone that is a very strong activator of TrkA (>200% the activity of 100 ng/ml NGF), and it is nontoxic at concentrations up to 10 μM. Activation of TrkA can be detected at 1 μM 5E5, and 3 to 10 μM 5E5 activates TrkA and extracellular signal-regulated kinase as strongly as a maximal dose of NGF (100 ng/ml). A combination of a low dose of 5E5 (1 μM) with a submaximal dose of NGF (10 ng/ml) promotes neuronal differentiation of PC12 cells. These compounds represent a new class of TrkA activators that could have potential utility in the treatment of neurodegenerative diseases.
Footnotes
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This study was supported by grants from the American Diabetes Association (to M.C.P.), National Institute for Aging Grant R03 AG023191 (to N.J.G.W.), and the Alzheimer's Drug Discovery Foundation. The Foundation catalyzes and funds drug discovery and drug development for Alzheimer's disease and cognitive aging. To learn more about the Foundation, visit the website at www.alzdiscovery.org.
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N.J.G.W. is a faculty member of the University of California-San Diego Biomedical Sciences Graduate Program.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.106.118034.
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ABBREVIATIONS: NGF, nerve growth factor; BDNF, brain-derived neurotrophic factor; NT, neurotrophin; DAQ-B1, demethylasterriquinone-B1, 2-[2-(1,1-dimethyl-allyl)-1H-indol-3-yl]-3,6-dihydroxy-5-[7-(3-methyl-but-2-enyl)-1H-indol-3-yl]-[1,4]benzoquinone; IR, insulin receptor; ERK, extracellular signal-regulated kinase; CHO, Chinese hamster ovary; ELISA, enzyme-linked immunosorbent assay; TBS, Tris-buffered saline; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; SFM, serum-free medium; QSAR, quantitative structure-activity relationship; RMS, root mean square; CV, coefficient of variation; 1H5, 2-(6-chloro-1H-indol-3-yl)-5-(2-cyclopropyl-1H-indol-3-yl)-3,6-dihydroxy-[1,4]benzoquinone; 5E5, 2,5-dimethoxy-3-(7-fluoro-1H-indol-3-yl)-[1,4]-benzoquinone; MAPK, mitogen-activated protein kinase; PI3-kinase, phosphatidylinositol 3-kinase.
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↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
- Received December 1, 2006.
- Accepted April 25, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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