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J. Biol. Chem., Vol. 282, Issue 12, 9150-9161, March 23, 2007

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Pyrimethamine as a Potential Pharmacological Chaperone for Late-onset Forms of GM2 Gangliosidosis^*

Gustavo H. B. Maegawa^¶1, Michael Tropak, Justin Buttner, Tracy Stockley^||, Fernando Kok^**, Joe T. R. Clarke^¶, and Don J. Mahuran²

From the Division of Clinical and Metabolic Genetics, Departments of Pediatrics and ^||Pediatric Laboratory Medicine, Research Institute, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada, the ^¶Institute of Medical Sciences and Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5G 1L5, Canada and the ^**Department of Neurology, Faculdade de Medicina, University of Sao Paulo, 01246903 Brazil

Late-onset GM2 gangliosidosis is composed of two related, autosomal recessive, neurodegenerative diseases, both resulting from deficiency of lysosomal, heterodimeric -hexosaminidase A (Hex A, ). Pharmacological chaperones (PC) are small molecules that can stabilize the conformation of a mutant protein, allowing it to pass the quality control system of the endoplasmic reticulum. To date all successful PCs have also been competitive inhibitors. Screening for Hex A inhibitors in a library of 1040 Food Drug Administration-approved compounds identified pyrimethamine (PYR (2,4-diamino 5-(4-chlorophenyl)-6-ethylpyrimidine)) as the most potent inhibitor. Cell lines from 10 late-onset Tay-Sachs (11 -mutations, 2 novel) and 7 Sandhoff (9 -mutations, 4 novel) disease patients, were cultured with PYR at concentrations corresponding to therapeutic doses. Cells carrying the most common late-onset mutation, G269S, showed significant increases in residual Hex A activity, as did all 7 of the -mutants tested. Cells responding to PC treatment included those carrying mutants resulting in reduced Hex heat stability and partial splice junction mutations of the inherently less stable -subunit. PYR, which binds to the active site in domain II, was able to function as PC even to domain I -mutants. We concluded that PYR functions as a mutation-specific PC, variably enhancing residual lysosomal Hex A levels in late-onset GM2 gangliosidosis patient cells.

Received for publication, October 2, 2006 , and in revised form, January 7, 2007.

^* This work was supported in part by the Canadian Institutes of Health Research, the National Institutes of Health, and the Uger Estate and Life for Luke Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by a donation from Amicus Therapeutics USA.

² To whom correspondence should be addressed: Rm. 9146, 555 University Ave., Toronto, Ontario M5G 1X8, Canada. Tel.: 416-813-6161; Fax: 416-813-8700; E-mail: hex{at}sickkids.ca.

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