title: The use of small molecule high-throughput screening to identify inhibitors of the proteinase 3-NB1 interaction.
abstract: Anti-neutrophil cytoplasmic antibodies (ANCA) to proteinase 3 (PR3) are found in patients with small-vessel vasculitis. PR3-ANCA bind strongly to membrane PR3 (mPR3) that is presented by the NB1 receptor. We performed high-throughput screening using a small molecule library to identify compounds that inhibit PR3-NB1 binding. We established a human embryonic kidney (HEK293) cell-based system, where approximately 95 +/- 2% of the NB1-transfected cells expressed the NB1 receptor on the cell surface. Addition of 0.1 microg/ml human PR3 to 10(4) NB1-expressing HEK293 cells resulted in PR3 binding that was detected by immunofluorescence using a fluorescence plate reader assay. We identified 13 of 20 000 molecules that inhibited PR3 binding by >70%. Seven of 13 substances showed reproducible inhibition in four additional validation experiments. Two selected compounds (27519 and 27549) demonstrated a dose-dependent inhibition over a range from 6.25 to 100 microM as measured by the plate reader assay. We used flow cytometry as a second assay, and found that both compounds reproducibly inhibited PR3 binding to NB1-transfected HEK293 cells at 50 microM (inhibition to 42 +/- 4% with compound 27519 and to 47 +/- 6% with compound 27549 compared to the dimethylsulphoxide control). Furthermore, compounds 27519 and 27549 also inhibited binding of exogenous PR3 to human neutrophils. In contrast, the compounds did not decrease mPR3 expression on resting neutrophils, but reduced the tumour necrosis factor-alpha-mediated mPR3 increase on NB1(pos) neutrophils when present continuously during the assay. The findings suggest that small inhibitory compounds provide a potential therapeutic tool to reduce mPR3 by preventing its binding to NB1.
authors.affiliation: Experimental and Clinical Research Center, Max-Delbrück Center for Molecular Medicine and Charité Medical Faculty, Berlin, Germany. mira.choi@charite.de
authors.forename: M
authors.initials: M
authors.lastname: Choi
authors.forename: C
authors.initials: C
authors.lastname: Eulenberg
authors.forename: S
authors.initials: S
authors.lastname: Rolle
authors.forename: J P
authors.initials: JP
authors.lastname: von Kries
authors.forename: F C
authors.initials: FC
authors.lastname: Luft
authors.forename: R
authors.initials: R
authors.lastname: Kettritz
mesh_terms: D019268:Antibodies, Antineutrophil Cytoplasmic / Q000276:immunology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005434:Flow Cytometry; D058851:GPI-Linked Proteins; D006801:Humans; D007519:Isoantigens / Q000235:genetics / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D015394:Molecular Structure; D053146:Myeloblastin / Q000276:immunology / Q000378:metabolism*; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism; D004364:Pharmaceutical Preparations / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D011956:Receptors, Cell Surface / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D019268:Antibodies, Antineutrophil Cytoplasmic; C407242:CD177 protein, human; D058851:GPI-Linked Proteins; D007519:Isoantigens; D008562:Membrane Glycoproteins; D004364:Pharmaceutical Preparations; D011956:Receptors, Cell Surface; D014409:Tumor Necrosis Factor-alpha; D053146:Myeloblastin
references.citation: Br J Haematol. 1992 Jul;81(3):336-45
references.pmid: 1382544
references.citation: Clin Exp Immunol. 2000 May;120(2):392-8
references.pmid: 10792393
references.citation: Kidney Int. 2001 May;59(5):1729-38
references.pmid: 11318943
references.citation: Am J Pathol. 1990 Nov;137(5):1113-20
references.pmid: 2240162
references.citation: Blood. 1990 Jan 1;75(1):242-50
references.pmid: 2153032
references.citation: Blood. 2007 May 15;109(10):4487-93
references.pmid: 17244676
references.citation: Br J Haematol. 1996 Jun;93(3):707-13
references.pmid: 8652399
references.citation: J Leukoc Biol. 2007 Feb;81(2):458-64
references.pmid: 17077162
references.citation: Clin Exp Immunol. 2008 Jun;152(3):508-16
references.pmid: 18462208
references.citation: Am J Pathol. 2009 Apr;174(4):1212-20
references.pmid: 19264905
references.citation: Blood. 1989 Nov 1;74(6):1888-93
references.pmid: 2679910
references.citation: Curr Opin Rheumatol. 2010 Jan;22(1):1-7
references.pmid: 19881350
references.citation: Am J Pathol. 2000 Jul;157(1):211-20
references.pmid: 10880391
references.citation: Am J Pathol. 2005 Jul;167(1):39-45
references.pmid: 15972950
references.citation: Blood. 1999 Oct 1;94(7):2487-96
references.pmid: 10498622
references.citation: J Biol Chem. 2008 Dec 19;283(51):35976-82
references.pmid: 18854317
references.citation: Kidney Int. 2002 Feb;61(2):502-15
references.pmid: 11849390
references.citation: Proteins. 2008 Jun;71(4):1655-69
references.pmid: 18076025
references.citation: J Am Soc Nephrol. 1999 Jun;10(6):1224-33
references.pmid: 10361860
references.citation: J Leukoc Biol. 2004 Jan;75(1):87-98
references.pmid: 14525959
references.citation: Arthritis Rheum. 2009 May;60(5):1548-57
references.pmid: 19404956
references.citation: Proc Natl Acad Sci U S A. 1990 Jun;87(11):4115-9
references.pmid: 2161532
references.citation: J Am Soc Nephrol. 2002 Sep;13(9):2232-8
references.pmid: 12191967
references.citation: J Am Soc Nephrol. 2005 Jul;16(7):2216-24
references.pmid: 15901769
references.citation: J Biol Chem. 2009 Sep 11;284(37):25211-9
references.pmid: 19605350
references.citation: J Biol Chem. 2008 May 30;283(22):15152-9
references.pmid: 18367444
references.citation: Shock. 2006 Aug;26(2):128-33
references.pmid: 16878019
references.citation: J Am Soc Nephrol. 2007 Aug;18(8):2330-9
references.pmid: 17634439
references.citation: Mol Cell Proteomics. 2005 Oct;4(10):1503-21
references.pmid: 15985654
references.citation: Blood. 1997 May 15;89(10):3503-21
references.pmid: 9160655
references.citation: Blood. 2007 Dec 1;110(12):4086-95
references.pmid: 17712045
references.citation: Proc Natl Acad Sci U S A. 2008 May 20;105(20):7275-80
references.pmid: 18480264
references.citation: J Am Soc Nephrol. 2006 Dec;17(12):3355-64
references.pmid: 17108314
references.citation: J Clin Invest. 2002 Oct;110(7):955-63
references.pmid: 12370273
references.citation: Kidney Int. 2004 Jun;65(6):2172-83
references.pmid: 15149330
references.citation: J Am Soc Nephrol. 2003 Jan;14(1):68-75
references.pmid: 12506139