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.