This screen was conducted with two different people using one cytometer at a rate of 24 plates per day including analysis and took 26 days to screen the 195,000 compounds in the library at that time. on each bead set is easily obtained. An arbitrary cutoff of 40% inhibition was taken to indicate a screening hit or possibly active compound. This was a deliberate use of low stringency and assumed that further analysis would be done on many false-positive hits. The assay can also be performed at a series of compound concentrations to form a doseCresponse curve (diagrams a step-by-step protocol for this assay. Table 1. Summary of High-Throughput Screening Flow Cytometry Multiplex Assay Protocol shows an example of a doseCresponse curve for one of these compounds, with PubChem compound accession identifier (CID) number 650929, in multiplex on all six Bcl-2 family members. The weak response of the earlier preparation of Bcl-B has been corrected. It is clear that this compound inhibits the binding of F-Bim to Bcl-B much more than to any of the other family members. We emphasize that, for example, all beads with 10?5 M compound were in the same well. When the powders were tested, three of them had log EC50 values below ?5 (EC50 10?M), which we considered reliably active. They showed 20% inhibition of the other five antiapoptotic Bcl-2 family members at the highest concentration tested, 100?M. Active compounds were also identified for other antiapoptotic family members. FP Confirms the Flow Cytometry Active Compounds Two rounds of FP doseCresponse assays were performed to validate the flow cytometry Rabbit polyclonal to AnnexinA10 data, using not just the F-Bim probe, but also one with a different fluorophore, Cy5-Bim. shows an example of an FP doseCresponse curve for 10 compounds against GST-Bcl-B with F-Bim, in which only 3 compounds were declared active. Quantitative results for the three active compounds against Bcl-B are shown in shows an example of an ITC doseCresponse curve against Bcl-B, in which CID 650929 was declared active, and against Bcl-XL, in which the compound was declared inactive. shows that the ITC log EC50 values demonstrate significant binding of all 6-Thioguanine three compounds to Bcl-B, making it more plausible that our other measurements were in fact displaying competitive binding between F-Bim and the compounds to Bcl-B. The ITC demonstration of binding is particularly instructive, for the flow cytometry and FP measurements are prone 6-Thioguanine to similar optical artifacts. The compound CID 650929 appears particularly strong with respect to Bcl-B binding. Control experiments showed no binding of the compounds to Bcl-XL (data not shown), as a demonstration of specificity. The structures of these three active compounds are shown in em Figure 4 /em . PubChem reports many comparisons of the screening and follow-up data, including the number of assays in which a compound has been declared active and the number of assays in which it has been tested: for CID650929, these numbers are 9/513 (active/tested); for CID1243212, these are 3/371; and for CID666339, these are 11/488, making all three compounds attractive with respect 6-Thioguanine to specificity of interaction. Open in a separate window Fig. 4. Structures and compound identifier numbers of the three inhibitors of the binding of F-Bim to Bcl-B. Discussion This report demonstrates a multiplexed, bead-based flow cytometry screen that covers a small family of proteins, requiring 1?mg of each GST-fusion protein per family member, a fluorescent probe, and the GSH bead sets described herein. In general, the capture and display of molecules on microspheres has the potential of reducing the quantities of the captured reagent (the GST-fusion protein) and allows the less-expensive fluorescent reagent (the F-Bim peptide) to be used near its em K /em d. By this approach, a screen of a small library of compounds is within the reach of an individual laboratory with a single flow cytometer, but a larger screen would.