Discovery of selective inhibitors against EBNA1 via high throughput in silico virtual screening.
BACKGROUND
Epstein-Barr Virus (EBV) latent infection is associated with several human malignancies and is a causal agent of lymphoproliferative diseases during immunosuppression. While inhibitors of herpesvirus DNA polymerases, like gancyclovir, reduce EBV lytic cycle infection, these treatments have limited efficacy for treating latent infection. EBNA1 is an EBV-encoded DNA-binding protein required for viral genome maintenance during latent infection.


METHODS
Here, we report the identification of a new class of small molecules that inhibit EBNA1 DNA binding activity. These compounds were identified by virtual screening of 90,000 low molecular mass compounds using computational docking programs with the solved crystal structure of EBNA1. Four structurally related compounds were found to inhibit EBNA1-DNA binding in biochemical assays with purified EBNA1 protein. Compounds had a range of 20-100 microM inhibition of EBNA1 in fluorescence polarization assays and were further validated for inhibition using electrophoresis mobility shift assays. These compounds exhibited no significant inhibition of an unrelated DNA binding protein. Three of these compounds inhibited EBNA1 transcription activation function in cell-based assays and reduced EBV genome copy number when incubated with a Burkitt lymphoma cell line.


CONCLUSIONS
These experiments provide a proof-of-principle that virtual screening can be used to identify specific inhibitors of EBNA1 that may have potential for treatment of EBV latent infection.
Li
Ning
N
Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China.
Thompson
Scott
S
Schultz
David C
DC
Zhu
Weiliang
W
Jiang
Hualiang
H
Luo
Cheng
C
Lieberman
Paul M
PM
D002051:Burkitt Lymphoma; D045744:Cell Line, Tumor; D003198:Computer Simulation; D004247:DNA / Q000378:metabolism; D055808:Drug Discovery / Q000379:methods*; D004353:Drug Evaluation, Preclinical / Q000379:methods; D020031:Epstein-Barr Virus Infections / Q000188:drug therapy*; D019309:Epstein-Barr Virus Nuclear Antigens / Q000187:drug effects*; D004854:Herpesvirus 4, Human; D006801:Humans; D011485:Protein Binding / Q000187:drug effects; D014158:Transcription, Genetic / Q000187:drug effects*; D014764:Viral Proteins / Q000037:antagonists & inhibitors*
D019309:Epstein-Barr Virus Nuclear Antigens; D014764:Viral Proteins; D004247:DNA; C417029:EBV-encoded nuclear antigen 1
Cell. 1996 Mar 8;84(5):791-800
8625416
Rev Med Virol. 2000 Mar-Apr;10(2):83-100
10713596
Chem Biol. 2006 Jul;13(7):745-51
16873022
Cell. 1995 Oct 6;83(1):39-46
7553871
Proteins. 1998 Oct 1;33(1):74-87
9741846
Cell. 1985 Oct;42(3):859-68
2996781
J Chem Inf Model. 2005 Jan-Feb;45(1):177-82
15667143
J Mol Biol. 1998 Dec 18;284(5):1273-8
9878348
N Engl J Med. 2004 Mar 25;350(13):1328-37
15044644
J Biol Chem. 2008 May 9;283(19):12819-30
18337247
J Virol. 1990 May;64(5):2369-79
2157891
Virology. 2006 Mar 15;346(2):385-93
16343579
Nature. 1985 Feb 28-Mar 6;313(6005):812-5
2983224
J Biol Chem. 2002 Jul 26;277(30):27401-11
12015325
Proc Natl Acad Sci U S A. 2003 Nov 25;100(24):14269-74
14603034
J Med Chem. 2003 Jun 19;46(13):2656-62
12801229
EMBO J. 2005 Apr 6;24(7):1406-17
15775975
Mol Cell Biol. 2006 Feb;26(3):1124-34
16428463
J Comput Aided Mol Des. 2002 Dec;16(12):883-902
12825621
J Med Chem. 2003 Jun 5;46(12):2287-303
12773034
Proc Natl Acad Sci U S A. 2007 May 1;104(18):7391-6
17463090
Proc Natl Acad Sci U S A. 2003 Sep 16;100(19):10989-94
12947043
Nat Rev Cancer. 2004 Oct;4(10):757-68
15510157
Anal Biochem. 2005 May 15;340(2):259-71
15840499
Bioorg Med Chem Lett. 2007 Oct 15;17(20):5652-5
17766109
Proc Natl Acad Sci U S A. 2006 Sep 19;103(38):14188-93
16966603