Absorbance-based biochemical primary high throughput screening assay to identify inhibitors of Methionine sulfoxide reductase A (MsrA)
Name: Absorbance-based biochemical primary high throughput screening assay to identify inhibitors of Methionine sulfoxide reductase A (MsrA). ..more
BioActive Compounds: 2709
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Herbert Weissbach, Florida Atlantic University
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1R03DA032473-01
Grant Proposal PI: Herbert Weissbach, Florida Atlantic University
External Assay ID: MSRA_INH_ABS_1536_1X%INH
Name: Absorbance-based biochemical primary high throughput screening assay to identify inhibitors of Methionine sulfoxide reductase A (MsrA).
Oxidative damage, resulting from the production of reactive oxygen species (ROS) within cells, is believed to be a major factor in age-related diseases and the aging process. One of the mechanisms by which this damage occurs is via oxidation of methionine residues to methionine sulfoxide (Met(O)) derivatives in cellular proteins, which can lead to protein inactivation (1). These Met(O) species can be repaired/reduced by the thioredoxin (Trx)-dependent (2, 3) action of Methionine sulfoxide reductase A (MsrA) (4, 5). MsrA can reduce both free and protein-bound Met(O), and is highly expressed in oxidant-sensitive tissues such as kidney (6), neurons (7), liver(8), retinal epithelial cells (9), and macrophages (10). Each round of methionine oxidation and reduction by the MsrA system destroys one equivalent of ROS (11). Importantly, the action of MsrA has been shown to prevent irreversible oxidative protein damage and extend life span of both flies and yeast (12-14). As a result, the identification of compounds that modulate MsrA activity could have therapeutic value for cardiovascular (15, 16), neurodegenerative, lung (17), and eye diseases (18) involving oxidative damage. Similarly, because MsrA is found in virtually all species (19), and the catalytic mechanism has been elucidated (1, 20, 21), the identification of chemical tools that modulate MsrA would help elucidate its function and activation in cells, and may lead to useful tools to extend lifespan and reduce aging-related diseases (11).
1. Lowther, W.T., N. Brot, H. Weissbach, and B.W. Matthews, Structure and mechanism of peptide methionine sulfoxide reductase, an "anti-oxidation" enzyme. Biochemistry, 2000. 39(44): p. 13307-12.
2. Lowther, W.T., N. Brot, H. Weissbach, J.F. Honek, and B.W. Matthews, Thiol-disulfide exchange is involved in the catalytic mechanism of peptide methionine sulfoxide reductase. Proc Natl Acad Sci U S A, 2000. 97(12): p. 6463-8.
3. Sagher, D., D. Brunell, J.F. Hejtmancik, M. Kantorow, N. Brot, and H. Weissbach, Thionein can serve as a reducing agent for the methionine sulfoxide reductases. Proc Natl Acad Sci U S A, 2006. 103(23): p. 8656-61.
4. Weissbach, H., L. Resnick, and N. Brot, Methionine sulfoxide reductases: history and cellular role in protecting against oxidative damage. Biochim Biophys Acta, 2005. 1703(2): p. 203-12.
5. Weissbach, H., F. Etienne, T. Hoshi, S.H. Heinemann, W.T. Lowther, B. Matthews, G. St John, C. Nathan, and N. Brot, Peptide methionine sulfoxide reductase: structure, mechanism of action, and biological function. Arch Biochem Biophys, 2002. 397(2): p. 172-8.
6. Moskovitz, J., H. Weissbach, and N. Brot, Cloning the expression of a mammalian gene involved in the reduction of methionine sulfoxide residues in proteins. Proc Natl Acad Sci U S A, 1996. 93(5): p. 2095-9.
7. Yermolaieva, O., R. Xu, C. Schinstock, N. Brot, H. Weissbach, S.H. Heinemann, and T. Hoshi, Methionine sulfoxide reductase A protects neuronal cells against brief hypoxia/reoxygenation. Proc Natl Acad Sci U S A, 2004. 101(5): p. 1159-64.
8. Vougier, S., J. Mary, and B. Friguet, Subcellular localization of methionine sulphoxide reductase A (MsrA): evidence for mitochondrial and cytosolic isoforms in rat liver cells. Biochem J, 2003. 373(Pt 2): p. 531-7.
9. Marchetti, M.A., G.O. Pizarro, D. Sagher, C. Deamicis, N. Brot, J.F. Hejtmancik, H. Weissbach, and M. Kantorow, Methionine sulfoxide reductases B1, B2, and B3 are present in the human lens and confer oxidative stress resistance to lens cells. Invest Ophthalmol Vis Sci, 2005. 46(6): p. 2107-12.
10. Moskovitz, J., N.A. Jenkins, D.J. Gilbert, N.G. Copeland, F. Jursky, H. Weissbach, and N. Brot, Chromosomal localization of the mammalian peptide-methionine sulfoxide reductase gene and its differential expression in various tissues. Proc Natl Acad Sci U S A, 1996. 93(8): p. 3205-8.
11. Brunell, D., H. Weissbach, P. Hodder, and N. Brot, A high-throughput screening compatible assay for activators and inhibitors of methionine sulfoxide reductase A. Assay Drug Dev Technol, 2010. 8(5): p. 615-20.
12. Moskovitz, J., E. Flescher, B.S. Berlett, J. Azare, J.M. Poston, and E.R. Stadtman, Overexpression of peptide-methionine sulfoxide reductase in Saccharomyces cerevisiae and human T cells provides them with high resistance to oxidative stress. Proc Natl Acad Sci U S A, 1998. 95(24): p. 14071-5.
13. Moskovitz, J., B.S. Berlett, J.M. Poston, and E.R. Stadtman, The yeast peptide-methionine sulfoxide reductase functions as an antioxidant in vivo. Proc Natl Acad Sci U S A, 1997. 94(18): p. 9585-9.
14. Ruan, H., X.D. Tang, M.L. Chen, M.L. Joiner, G. Sun, N. Brot, H. Weissbach, S.H. Heinemann, L. Iverson, C.F. Wu, and T. Hoshi, High-quality life extension by the enzyme peptide methionine sulfoxide reductase. Proc Natl Acad Sci U S A, 2002. 99(5): p. 2748-53.
15. Haenold, R., R. Wassef, N. Brot, S. Neugebauer, E. Leipold, S.H. Heinemann, and T. Hoshi, Protection of vascular smooth muscle cells by over-expressed methionine sulphoxide reductase A: role of intracellular localization and substrate availability. Free Radic Res, 2008. 42(11-12): p. 978-88.
16. Shao, B., G. Cavigiolio, N. Brot, M.N. Oda, and J.W. Heinecke, Methionine oxidation impairs reverse cholesterol transport by apolipoprotein A-I. Proc Natl Acad Sci U S A, 2008. 105(34): p. 12224-9.
17. Ogawa, F., K. Shimizu, T. Hara, E. Muroi, K. Komura, M. Takenaka, M. Hasegawa, M. Fujimoto, K. Takehara, and S. Sato, Autoantibody against one of the antioxidant repair enzymes, methionine sulfoxide reductase A, in systemic sclerosis: association with pulmonary fibrosis and vascular damage. Arch Dermatol Res, 2010. 302(1): p. 27-35.
18. Brennan, L.A., W. Lee, T. Cowell, F. Giblin, and M. Kantorow, Deletion of mouse MsrA results in HBO-induced cataract: MsrA repairs mitochondrial cytochrome c. Mol Vis, 2009. 15: p. 985-99.
19. Delaye, L., A. Becerra, L. Orgel, and A. Lazcano, Molecular evolution of peptide methionine sulfoxide reductases (MsrA and MsrB): on the early development of a mechanism that protects against oxidative damage. J Mol Evol, 2007. 64(1): p. 15-32.
20. Boschi-Muller, S., S. Azza, S. Sanglier-Cianferani, F. Talfournier, A. Van Dorsselear, and G. Branlant, A sulfenic acid enzyme intermediate is involved in the catalytic mechanism of peptide methionine sulfoxide reductase from Escherichia coli. J Biol Chem, 2000. 275(46): p. 35908-13.
21. Taylor, A.B., D.M. Benglis, Jr., S. Dhandayuthapani, and P.J. Hart, Structure of Mycobacterium tuberculosis methionine sulfoxide reductase A in complex with protein-bound methionine. J Bacteriol, 2003. 185(14): p. 4119-26.
primary, PRUN, Enzyme, MsrA, Methionine sulfoxide reductase A, reductase, oxidoreductase, peptide Met(O) reductase, kinetic, biochemical, enzymatic, oxidation, oxidative, NADPH, methionine, Absorbance, Abs, inhibit, inhibitor, inhibition, bovine, HTS, high throughput screen, 1536, Scripps, Scripps Florida, MLSMR, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this assay is to identify inhibitors of Methionine sulfoxide reductase A (MSRA). For most MSRA's there are 3 cysteines (Cys) involved in the catalytic reaction. The Cys located nearest the N-terminal is at the catalytic center and attacks the sulfur of the Met(o), followed by release of Met and formation of a sulfenic acid moiety on the enzyme. Subsequent loss of water, followed by a thioldisulfide interchange, leaves the enzyme in an oxidized state. The enzyme must then be reduced in order to function catalytically. The thioredoxin (Trx) system has been shown to be the physiological reducing system for MSRA. However, the enzyme can also be reduced by a variety of chemical reducing agents including dithiothreitol and selenocysteamine (reduced selenocystamine). In addition to Met(o), MSRA is also capable of reducing a variety of methyl sulfoxides including DMSO.
In this biochemical assay, recombinant MSRA enzyme is incubated in the presence of purified thioredoxin reductase (TrxB), reduced nicotinamide adenine dinucleotide phosphate (NADPH), selenocystamine (SeCm) and test compounds. MSRA reduces the DMSO used as solvent for the test compounds to dimethylsulfide. TrxB catalyzes the transfer of hydrogen from NADPH to SeCm, and the reduced SeCm in turn reduces oxidized MSRA. The reactions are carried out at room temperature in black, 1536-well, clear-bottom plates and are read at 340 nm absorbance. As the reaction proceeds, absorbance at 340 nm will decrease as NADPH is oxidized to NADP. High and low control wells contain either 2 mM N-Ethylmaleimide (NEM) in DMSO or DMSO alone. NEM acts as an inhibitor by alkylating the active site of MSRA. Wells containing NEM or a functional inhibitor from the test compounds will show diminished oxidation of NADPH, resulting in stable well absorbance over the course of the assay. Compounds are tested at a nominal concentration of 15.5 uM.
Prior to the start of the assay, 4 uL of assay buffer (50 mM Tris pH 7.4) containing a cocktail of 940 nM MSRA, 358 nM TrxB, 100 uM SeCm and 2 mM NADPH were dispensed into 1536 well microtiter plates.
The assay is then started by transferring test compounds in DMSO, or controls in DMSO (1.5% final concentration). Plates were centrifuged and after 40 minutes of incubation at 25 C, absorbance was read on a EnVision microplate reader (PerkinElmer, Turku, Finland) using a 340 nm filter and reading in optimized absorbance mode.
The percent inhibition for each compound was calculated as follows:
%_Inhibition = 100 * ( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median_Low_Control ) )
Low_Control is defined as wells containing the MSRA protein cocktail with 100 uM SeCm and DMSO.
Test_Compound is defined as wells containing MSRA protein cocktail with 100 uM SeCm in the presence of test compound in DMSO.
High_Control is defined as wells containing the MSRA protein cocktail, 100 uM SeCm and 2 mM NEM in DMSO.
PubChem Activity Outcome and Score:
A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Two values were calculated: (1) the average percent inhibition of all samples tested and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter for each plate, i.e. any compound that exhibited greater % inhibition than that particular plate's cutoff parameter was declared active.
The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % Inhibition values are reported as activity score zero.
The PubChem Activity Score range for active compounds is 100-4, and for inactive compounds 4-0.
List of Reagents:
Bovine MSRA protein (supplied by Assay Provider)
E. coli TrxA (supplied by Assay Provider)
E. coli TrxB (supplied by Assay Provider)
1.5M TRIS pH 7.4 (Assay Provider)
NADPH (Sigma, part N7505)
1536 well assay plates (Corning part 7338)
Selenocystamine (Sigma Aldrich S0520)
N-Ethylmaleimide (Sigma Aldrich E3876)
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well absorbance or fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)