Phenotypic screening of the ReFRAME drug repurposing library to discover new drugs for treating sickle cell disease.

Stem cell transplantation and genetic therapies offer potential cures for patients with sickle cell disease (SCD), but these options require advanced medical facilities and are expensive. Consequently, these treatments will not be available for many years to the majority of patients suffering from this disease. What is urgently needed now is an inexpensive oral drug in addition to hydroxyurea, the only drug approved by the FDA that inhibits sickle-hemoglobin polymerization. Here, we report the results of the first phase of our phenotypic screen of the 12,657 compounds of the Scripps ReFRAME drug repurposing library using a recently developed high-throughput assay to measure sickling times following deoxygenation to 0% oxygen of red cells from sickle trait individuals. The ReFRAME library is a very important collection because the compounds are either FDA-approved drugs or have been tested in clinical trials. From dose-response measurements, 106 of the 12,657 compounds exhibit statistically significant antisickling at concentrations ranging from 31 nM to 10 µM. Compounds that inhibit sickling of trait cells are also effective with SCD cells. As many as 21 of the 106 antisickling compounds emerge as potential drugs. This estimate is based on a comparison of inhibitory concentrations with free concentrations of oral drugs in human serum. Moreover, the expected therapeutic potential for each level of inhibition can be predicted from measurements of sickling times for cells from individuals with sickle syndromes of varying severity. Our results should motivate others to develop one or more of these 106 compounds into drugs for treating SCD.

Kinetic assay shows that increasing red cell volume could be a treatment for sickle cell disease.

Although it has been known for more than 60 years that the cause of sickle cell disease is polymerization of a hemoglobin mutant, hydroxyurea is the only drug approved for treatment by the US Food and Drug Administration. This drug, however, is only partially successful, and the discovery of additional drugs that inhibit fiber formation has been hampered by the lack of a sensitive and quantitative cellular assay. Here, we describe such a method in a 96-well plate format that is based on laser-induced polymerization in sickle trait cells and robust, automated image analysis to detect the precise time at which fibers distort ("sickle") the cells. With this kinetic method, we show that small increases in cell volume to reduce the hemoglobin concentration can result in therapeutic increases in the delay time prior to fiber formation. We also show that, of the two drugs (AES103 and GBT440) in clinical trials that inhibit polymerization by increasing oxygen affinity, one of them (GBT440) also inhibits sickling in the absence of oxygen by two additional mechanisms.