DNA-linked Inhibitor Antibody Assay (DIANA) for sensitive and selective enzyme detection and inhibitor screening.
Human diseases are often diagnosed by determining levels of relevant enzymes and treated by enzyme inhibitors. We describe an assay suitable for both ultrasensitive enzyme quantification and quantitative inhibitor screening with unpurified enzymes. In the DNA-linked Inhibitor ANtibody Assay (DIANA), the target enzyme is captured by an immobilized antibody, probed with a small-molecule inhibitor attached to a reporter DNA and detected by quantitative PCR. We validate the approach using the putative cancer markers prostate-specific membrane antigen and carbonic anhydrase IX. We show that DIANA has a linear range of up to six logs and it selectively detects zeptomoles of targets in complex biological samples. DIANA's wide dynamic range permits determination of target enzyme inhibition constants using a single inhibitor concentration. DIANA also enables quantitative screening of small-molecule enzyme inhibitors using microliters of human blood serum containing picograms of target enzyme. DIANA's performance characteristics make it a superior tool for disease detection and drug discovery.
Gilead Sciences and IOCB Research Centre, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, 166 10, Czech Republic vanavratil@gmail.com.|Department of Biochemistry, Faculty of Science, Charles University in Prague, Prague, 128 43, Czech Republic.
Václav
V
Navrátil
Gilead Sciences and IOCB Research Centre, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, 166 10, Czech Republic.|Department of Biochemistry, Faculty of Science, Charles University in Prague, Prague, 128 43, Czech Republic.
Jiří
J
Schimer
Gilead Sciences and IOCB Research Centre, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, 166 10, Czech Republic.|Department of Biochemistry, Faculty of Science, Charles University in Prague, Prague, 128 43, Czech Republic.
Jan
J
Tykvart
Gilead Sciences and IOCB Research Centre, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, 166 10, Czech Republic.|Department of Biochemistry, Faculty of Science, Charles University in Prague, Prague, 128 43, Czech Republic.
Tomáš
T
Knedlík
Department of Urology, Thomayer Hospital in Prague, Prague, 140 59, Czech Republic.
Viktor
V
Vik
Gilead Sciences and IOCB Research Centre, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, 166 10, Czech Republic.
Pavel
P
Majer
Gilead Sciences and IOCB Research Centre, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, 166 10, Czech Republic konval@uochb.cas.cz.|Department of Biochemistry, Faculty of Science, Charles University in Prague, Prague, 128 43, Czech Republic.
Jan
0000-0003-0695-9266
J
Konvalinka
Gilead Sciences and IOCB Research Centre, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, 166 10, Czech Republic pavelsacha@gmail.com.|Department of Biochemistry, Faculty of Science, Charles University in Prague, Prague, 128 43, Czech Republic.
Pavel
P
Šácha
D001681:Biological Assay; D004247:DNA; D055808:Drug Discovery; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004798:Enzymes / Q000378:metabolism*; D006801:Humans; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D004791:Enzyme Inhibitors; D004798:Enzymes; D004247:DNA
Nucleic Acids Res. 1995 Feb 11;23(3):522-9
7885849
Br J Pharmacol. 2011 Mar;162(6):1239-49
21091654
Cancer Res. 2000 Dec 15;60(24):7075-83
11156414
Proc Natl Acad Sci U S A. 2009 Sep 22;106(38):16233-8
19805286
EMBO J. 2006 Mar 22;25(6):1375-84
16467855
Schizophr Res. 2012 Apr;136(1-3):160-1
22300789
Cancer Res. 2001 Aug 15;61(16):6029-33
11507047
Angew Chem Int Ed Engl. 2016 Feb 12;55(7):2356-60
26749427
J Med Chem. 1996 Jan 19;39(2):619-22
8558536
Nat Rev Drug Discov. 2008 Feb;7(2):168-81
18167490
Nat Med. 1999 Dec;5(12 ):1396-402
10581082
J Neurochem. 2005 Oct;95(2):314-23
16190866
Genome Res. 2005 Oct;15(10):1388-92
16204192
J Clin Oncol. 2012 Feb 10;30(5):e62-3
22203757
Cancer Res. 1997 Sep 1;57(17):3629-34
9288760
FEBS J. 2009 Aug;276(16):4448-62
19678840
Nucleic Acids Res. 2011 Aug;39(15):e102
21646338
Int J Cancer. 2003 Jul 20;105(6):873-81
12767076
Prostate. 2014 Dec;74(16):1674-90
25262926
Nat Biotechnol. 2008 Jan;26(1):127-32
18183025
Urology. 2010 Feb;75(2):257-61
19963243
Proc Natl Acad Sci U S A. 2009 Nov 3;106(44):18437-42
19841273
Clin Chem. 2005 Dec;51(12):2415-8
16179424
J Med Chem. 2011 Mar 24;54(6):1896-902
21361354
J Neurochem. 2002 Feb;80(3):477-87
11905994
Nat Methods. 2007 Apr;4(4):327-9
17369836
Prostate. 2014 May;74(7):768-80
24647901
Br J Cancer. 2003 Sep 15;89(6):1067-71
12966427
Bioorg Med Chem. 2014 Aug 1;22(15):4099-108
24954515
J Med Chem. 2001 Feb 1;44(3):298-301
11462970
Br J Cancer. 2012 Sep 25;107(7):1131-7
22918393
Angew Chem Int Ed Engl. 2013 Dec 16;52(51):13760-3
24307504
Bioorg Med Chem Lett. 2004 May 3;14(9):2351-6
15081039
Cancer Res. 2009 Jan 1;69(1):358-68
19118021
Chem Soc Rev. 2014 May 21;43(10):3324-41
24643375
J Med Chem. 2009 Jan 22;52(2):347-57
19111054
Br J Cancer. 2000 Jun;82(11):1808-13
10839295
Immunochemistry. 1971 Sep;8(9):871-4
5135623
Nat Clin Pract Urol. 2009 Mar;6(3):164-7
19265858
Cancer Res. 2011 May 1;71(9):3364-76
21415165
J Urol. 2016 Aug;196(2):382-91
26964917
PLoS One. 2015 Jul 07;10(7):e0131861
26151906
Nat Chem Biol. 2007 Aug;3(8):466-79
17637779
World J Surg. 2006 Apr;30(4):628-36
16555021
Biochemistry. 1985 Jun 18;24(13):3165-73
3927973
Biochemistry. 2005 Apr 19;44(15):5561-71
15823014
Br J Clin Pharmacol. 2005 Aug;60(2):128-36
16042665
Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5381-3
1608946