FIGURE 5.

High throughput primary screening for inhibitors of a3-B2 interaction, using an ELISA-based binding assay. The Chembridge DIVERSet collection of 10,000 synthetic chemical compounds was screened using the ELISA-based binding assay described and executed on a robotics platform (see “Experimental Procedures”). A, B-score statistics are shown for the compounds, where ELISA absorbances were analyzed in the presence of test compounds. The B-score statistic smoothes systematic machine error and takes into account positive and negative controls. Small black diamonds indicate results for individual compounds. White dashed line indicates mean (B-score = 0), and dotted black lines indicate ± 3 S.D. from the mean. Outliers with a B-score less than ± 3 S.D. were regarded as hits. Hits were retested twice in the primary screen and ranked according to deviation from the mean. The highest ranking hits (with lowest B-scores) were picked for secondary screening. The hit discussed here, KM91104, is indicated with a heavy circle. The only analog of KM91104 in the library (KM91201) is indicated with a light circle. It was also identified as a hit in the primary screen but failed in downstream validation screens (unacceptably high IC₅₀; see also Fig. 9). B shows the chemical structure of compound KM91104, 3,4-dihydroxy-N′-(2-hydroxybenzylidene)benzohydrazide (left panel). This compound was the best hit, overall, in terms of effectiveness in inhibiting osteoclast resorption without effect on osteoclast viability or differentiation, as described in subsequent figures. The inactive analog, KM91201 (N′,N″-(1,4-phenylenedimethylidene)bis(3,4-dihydroxybenzohydrazide)), is shown in the right panel.