Screen of the ReFRAME Compound Library for Therapeutic Agents to Prevent Red Blood Cell Sickling Using an Improved High Throughput Sickling Assay

Abstract

Sickle cell disease (SCD) is an autosomal recessive disorder of blood characterized by a mutation in the β chain of hemoglobin (Hb), leading to the production of sickle Hb (HbS). In SCD, under low oxygen conditions, red blood cells (RBCs) containing HbS form a characteristic "sickle" shape, resulting in chronic hemolytic anemia and acute vaso-occlusive crises. Current therapies for SCD have limitations in efficacy or availability, highlighting the need for new anti-sickling drugs. To facilitate the discovery of new anti-sickling compounds, we previously developed a high throughput sickling assay, which permits rapid screening of thousands of compounds for the ability to inhibit RBC sickling. In this study, we improved the sickling assay by optimizing the assay condition and expanded our screening efforts by evaluating the Repurposing, Focused Rescue, and Accelerated Medchem (ReFRAME) compound library, which contains approximately 2.5 times more compounds than previously screened. We were able to increase the number of blood samples that were adequate for identifying anti-sickling compounds in the improved sickling assay and identified voxelotor and SNS-314 as compounds that successfully prevented sickling. The improved sickling assay will increase access to valuable blood samples from SCD volunteers, providing more opportunities to develop anti-sickling compounds for treating SCD.

Figure 1

Sickle cell percent of 20 SS blood samples in the buffer and the one diluted with water at 9:1 volume ratio in the sickling assay. The bar graphs represent the mean percentage of sickle cells, with error bars showing the mean ± SEM (n = 20 biological replicates). A two-sided paired t-test was performed using R 4.4.1 to compare the sickle cell percents between the two groups, and differences were considered statistically significant if the p-value was less than 0.05. The dashed line indicates 40% of sickle cells, above which a blood sample was considered adequate for screening compounds for their anti-sickling activity.

Figure 2

A flowchart to screen 13,736 compounds for the ability of the compounds to prevent sickling while maintaining the normal size and shape of normal RBCs. S_compound: percentage of sickle cells in a compound-treated sample. μ_DMSO: mean percentage of sickle cells in the DMSO-treated samples tested in the same plate. A_compound: apparent cell area of a compound-treated sample. μ_voxelotor′: mean average cell area in the voxelotor-treated samples tested in the same plate. O_compound: percentage of other cells in a compound-treated sample. μ_voxelotor″: mean percentage of other cells in the voxelotor-treated samples tested in the same plate.

Figure 3

Representative fluorescent image of SS RBCs treated with DMSO (A), voxelotor 30 μM as the positive control of the assay (B), compound 7 30 μM (C), compound 8 30 μM (D), compound 9 30 μM (E), and compound 10 30 μM (F) in 4% oxygen. The bars in the images indicate 20 μm. Dose–response curves of compounds 7 (G), 8 (H), 9 (I), and 10 (J) on the sickle cell percentage in three measurements (measurements 1–3). The upper and lower dotted lines in panels (G–J) indicate the average sickle cell percent of DMSO-treated samples and voxelotor-treated samples in all the tests, respectively. The structure of compounds 7 (voxelotor, K), 8 (SNS-314, L), 9 (bismuth ethanedithiol, M), and 10 (cetrorelix acetate, N).