High throughput cell-based assay for identification of glycolate oxidase inhibitors as a potential treatment for Primary Hyperoxaluria Type 1.
Glycolate oxidase (GO) and alanine:glyoxylate aminotransferase (AGT) are both involved in the peroxisomal glyoxylate pathway. Deficiency in AGT function causes the accumulation of intracellular oxalate and the primary hyperoxaluria type 1 (PH1). AGT enhancers or GO inhibitors may restore the abnormal peroxisomal glyoxylate pathway in PH1 patients. With stably transformed cells which mimic the glyoxylate metabolic pathway, we developed an indirect glycolate cytotoxicity assay in a 1,536-well plate format for high throughput screening. This assay can be used to identify compounds that reduce indirect glycolate-induced cytotoxicity by either enhancing AGT activity or inhibiting GO. A pilot screen of 4,096 known compounds identified two membrane permeable GO inhibitors: dichromate salt and colistimethate. We also developed a GO enzyme assay using the hydrogen peroxide-Amplex red reporter system. The IC50values of potassium dichromate, sodium dichromate, and colistimethate sodium were 0.096, 0.108, and 2.3 μM in the GO enzyme assay, respectively. Further enzyme kinetic study revealed that both types of compounds inhibit GO activity by the mixed linear inhibition. Our results demonstrate that the cell-based assay and GO enzyme assay developed in this study are useful for further screening of large compound libraries for drug development to treat PH1.
Therapeutics for Rare and Neglected Diseases, National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD 20892, USA.|West China School of Public Health, Sichuan University, Chengdu, 610041, China.
Mengqiao
M
Wang
Therapeutics for Rare and Neglected Diseases, National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD 20892, USA.
Miao
M
Xu
Therapeutics for Rare and Neglected Diseases, National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD 20892, USA.
Yan
Y
Long
Department of Cell and Developmental Biology, Division of Biosciences, University College London, London WC1E 6BT, UK.
Sonia
S
Fargue
Therapeutics for Rare and Neglected Diseases, National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD 20892, USA.
Noel
N
Southall
Therapeutics for Rare and Neglected Diseases, National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD 20892, USA.
Xin
X
Hu
Therapeutics for Rare and Neglected Diseases, National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD 20892, USA.
John C
JC
McKew
Department of Cell and Developmental Biology, Division of Biosciences, University College London, London WC1E 6BT, UK.
Christopher J
CJ
Danpure
Therapeutics for Rare and Neglected Diseases, National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD 20892, USA.
Wei
W
Zheng
Protein Sci. 1997 May;6(5):1009-15
9144771
Arch Biochem Biophys. 2007 Sep 15;465(2):410-6
17669354
Arch Biochem Biophys. 1983 Apr 15;222(2):397-402
6342538
Plant Physiol. 1966 Dec;41(10):1623-31
16656449
Biochim Biophys Acta. 2006 Dec;1763(12):1776-84
17027096
Biochim Biophys Acta. 2013 Oct;1832(10):1776-83
23597595
Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10900-4
1961759
J Biol Chem. 2000 Apr 28;275(17):12590-7
10777549
Proc Natl Acad Sci U S A. 2014 Oct 7;111(40):14406-11
25237136
Proc Natl Acad Sci U S A. 1985 Oct;82(20):6855-9
16593616
J Biomol Screen. 1999;4(2):67-73
10838414
J Biomol Screen. 2014 Sep;19(8):1164-73
24907126
Am J Hum Genet. 1993 Aug;53(2):417-32
8101040
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2009 Dec 1;65(Pt 12):1246-53
20054120
J Cell Biol. 1989 Apr;108(4):1345-52
2925788
Drugs. 2014 Mar;74(3):377-87
24510624
Assay Drug Dev Technol. 2006 Oct;4(5):525-33
17115923
Int J Biol Macromol. 2011 Jul 1;49(1):62-70
21458484
Clin Investig. 1994 Sep;72(9):725-7
7849461
J Biomol Screen. 2009 Jul;14(6):708-15
19531665
Arabidopsis Book. 2010;8:e0130
22303256
Proc Natl Acad Sci U S A. 2006 Aug 1;103(31):11473-8
16864780
Nephrol Dial Transplant. 2012 May;27(5):1729-36
22547750
Sci Transl Med. 2011 Apr 27;3(80):80ps16
21525397
Proc Natl Acad Sci U S A. 2003 Jun 10;100(12):7277-82
12777626
FEBS Lett. 1975 May 15;53(3):292-6
1137956
Curr Chem Genomics. 2010 Oct 21;4:57-66
21331310
Drug Discov Today. 2013 Nov;18(21-22):1067-73
23850704
J Med Chem. 2008 Nov 13;51(21):6740-51
18834112
J Exp Bot. 2006;57(9):1899-908
16595582
PLoS One. 2014 Apr 09;9(4):e94338
24718375
Eur J Clin Pharmacol. 2013 Jul;69(7):1429-36
23508665
J Biol Chem. 2013 Jan 25;288(4):2475-84
23229545
Hum Mutat. 2009 Jun;30(6):910-7
19479957
J Chem Biol. 2009 Mar;2(1):1-9
19568788
Biochim Biophys Acta. 2015 Sep;1854(9):1212-9
25620715
J Biol Chem. 2014 Mar 28;289(13):8735-41
24515117
Trends Plant Sci. 2010 Jun;15(6):330-6
20403720
Biochem J. 2006 Mar 1;394(Pt 2):409-16
16309382