1Although the pathophysiology and molecular basis of sickle cell disease (SCD) were described more than half a century ago, an effective and safe therapy is not yet available. This may be explained by the lack of a suitable high-throughput technique that allows rapid screening of thousands of compounds for their antisickling effect. The authors have thus developed a novel high-throughput screening (HTS) assay based on detecting the ability of red blood cells (RBC) to traverse a column of tightly packed Sephacryl chromatography beads. When deoxygenated, sickle RBC are rigid and remain on the top of the column. However, when deoxygenated and treated with an effective antisickling agent, erythrocytes move through the Sephacryl media and produce a red dot on the bottom of the assay tubes. This approach has been adapted to wells in a 384-well microplate. Results can be obtained by optical scanning: The size of the red dot is proportional to the antisickling effect of the test molecule. The new assay is simple, inexpensive, reproducible, requires no special reagents, and should be readily adaptable to robotic HTS systems. It has the potential to identify novel drug candidates, allowing the development of new therapeutic options for individuals affected with SCD. (Journal of Biomolecular Screening. 2009:330-336) Key words: sickle cell disease, high-throughput screening, cell-based assay, assay development, drug discovery and has identified logical targets for pharmacological intervention aimed at reducing HbS polymerization and thus RBC sickling. To date, a limited number of drug candidates have been proposed, yet none of these agents are suitable for clinical use because of their low potency and/or high toxicity.3-14 It is only recently that the chemotherapeutic drug hydroxyurea (HU) has been shown to greatly reduce the frequency and severity of SCD complications for many patients. 4,6 Although it is the most effective treatment option available to date, its significant toxicity raises questions about its widespread and longterm clinical use.Earlier studies focusing on the pharmacologic therapy of SCD have typically evaluated 1 compound or a small series of structurally related molecules at a time. However, the identification of new drug candidates warrants the screening of a library containing tens of thousands of different molecules, a procedure referred to as high-throughput screening (HTS).
15,16Existing techniques are not necessarily well suited for automated HTS systems, inasmuch as they lack basic characteristics necessary to effectively screen for novel antisickling agents (i.e., high assay reliability and reproducibility, simple endpoints, short cycle times, and low per-sample cost). Therefore, our goal was to develop a simple, cell-based, HTS-compatible assay to screen for compounds with the ability to reduce or prevent RBC sickling under deoxygenated conditions and therefore the ability to inhibit the vaso-occlusive process. Our efforts were focused on developing the methodology to screen