Coumarin-suberoylanilide hydroxamic acid as a fluorescent probe for determining binding affinities and off-rates of histone deacetylase inhibitors.
Histone deacetylases (HDACs) are intimately involved in epigenetic regulation and, thus, are one of the key therapeutic targets for cancer, and two HDAC inhibitors, namely suberoylanilide hydroxamic acid (SAHA) and romidepsin, have been recently approved for cancer treatment. Because the screening and detailed characterization of HDAC inhibitors has been time-consuming, we synthesized coumarin-SAHA (c-SAHA) as a fluorescent probe for determining the binding affinities (K(d)) and the dissociation off-rates (k(off)) of the enzyme-inhibitor complexes. The determination of the above parameters relies on the changes in the fluorescence emission intensity (λ(ex)=325 nm, λ(em)=400 nm) of c-SAHA due to its competitive binding against other HDAC inhibitors, and such determination neither requires employment of polarization accessories nor is dependent on the fluorescence energy transfer from the enzyme's tryptophan residues to the probe. Our highly sensitive and robust analytical protocol presented here is applicable to most of the HDAC isozymes, and it can be easily adopted in a high-throughput mode for screening the HDAC inhibitors as well as for quantitatively determining their K(d) and k(off) values.
Singh
Raushan K
RK
Department of Chemistry and Biochemistry, North Dakota State University, Fargo, ND 58105, USA.
Mandal
Tanmay
T
Balasubramanian
Narayanaganesh
N
Cook
Gregory
G
Srivastava
D K
DK
D003374:Coumarins / Q000737:chemistry*; D047630:Depsipeptides / Q000737:chemistry / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000737:chemistry*; D056572:Histone Deacetylase Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D006655:Histone Deacetylases / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006877:Hydroxamic Acids / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D007700:Kinetics; D011485:Protein Binding; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D000077337:Vorinostat
D003374:Coumarins; D047630:Depsipeptides; D005456:Fluorescent Dyes; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D011994:Recombinant Proteins; D012097:Repressor Proteins; D000077337:Vorinostat; C030123:coumarin; C087123:romidepsin; C409470:HDAC8 protein, human; D006655:Histone Deacetylases
J Med Chem. 2005 Jul 28;48(15):5047-51
16033284
Biochemistry. 1998 Mar 10;37(10):3499-508
9521671
Anal Biochem. 2007 Mar 1;362(1):136-41
17250798
Nat Biotechnol. 2007 Jan;25(1):84-90
17211407
Anal Biochem. 2008 Jan 1;372(1):82-8
17920554
Nature. 2000 Jan 6;403(6765):41-5
10638745
Nat Chem Biol. 2010 Mar;6(3):238-243
20139990
Anal Biochem. 2003 Oct 15;321(2):202-8
14511685
J Neurochem. 2006 Jul;98(1):193-202
16805808
Mol Cell. 2003 Dec;12(6):1367-78
14690592
Drugs. 2009 Oct 1;69(14):1911-34
19747008
Nature. 1997 Sep 25;389(6649):349-52
9311776
Nat Rev Drug Discov. 2006 Sep;5(9):769-84
16955068
Mol Genet Metab. 2003 Sep-Oct;80(1-2):138-47
14567963
Biochem J. 2003 Mar 15;370(Pt 3):737-49
12429021
Anal Biochem. 1976 May 7;72:248-54
942051
J Am Chem Soc. 2004 Sep 8;126(35):10875-83
15339172
Anal Biochem. 1992 Aug 15;205(1):65-9
1332537
Annu Rev Biochem. 2001;70:81-120
11395403
Biochim Biophys Acta. 1969;185(2):269-86
4980133
Annu Rev Biochem. 2004;73:417-35
15189148
Structure. 2004 Jul;12(7):1325-34
15242608
Expert Rev Anticancer Ther. 2010 Jul;10(7):997-1008
20645688
FEBS Lett. 1995 Feb 27;360(2):111-4
7875313
Proc Natl Acad Sci U S A. 2004 Oct 19;101(42):15064-9
15477595
Expert Opin Investig Drugs. 2008 Feb;17(2):169-84
18230051
Proc Natl Acad Sci U S A. 1964 May;51:786-94
14172992
J Mol Biol. 2004 Apr 16;338(1):17-31
15050820
Chem Biol. 2003 Jan;10(1):61-8
12573699
J Biotechnol. 2006 Jun 25;124(1):258-70
16567013
Bioorg Med Chem Lett. 2009 Jul 1;19(13):3651-6
19457659
BMC Biotechnol. 2006 Mar 01;6:12
16509985
Biochemistry. 2004 Aug 31;43(34):11083-91
15323567
Curr Opin Struct Biol. 2005 Dec;15(6):673-80
16263263
Anal Biochem. 1996 Jun 1;237(2):260-73
8660575
Nat Rev Mol Cell Biol. 2001 Jun;2(6):422-32
11389466
Bioorg Med Chem Lett. 2008 May 1;18(9):2809-12
18430569