High-Throughput Assay to Screen Small Molecules for Their Ability to Prevent Sickling of Red Blood Cells

Abstract

Sickle cell disease (SCD) is an inherited disorder of hemoglobin (Hb); approximately 300,000 babies are born worldwide with SCD each year. In SCD, fibers of polymerized sickle Hb (HbS) form in red blood cells (RBCs), which cause RBCs to develop their characteristic “sickled” shape, resulting in hemolytic anemia and numerous vascular complications including vaso-occlusive crises. The development of novel antisickling compounds will provide new therapeutic options for patients with SCD. We developed a high-throughput “sickling assay” that is based on an automated high-content imaging system to quantify the effects of hypoxia on the shape and size of RBCs from HbSS SCD patients (SS RBCs). We used this assay to screen thousands of compounds for their ability to inhibit sickling. In the assay, voxelotor (an FDA-approved medication used to treat SCD) prevented sickling with a z ′-factor > 0.4, suggesting that the assay is capable of identifying compounds that inhibit sickling. We screened the Broad Repurposing Library of 5393 compounds for their ability to prevent sickling in 4% oxygen/96% nitrogen. We identified two compounds, SNS-314 mesylate and voxelotor itself, that successfully prevented sickling. SNS-314 mesylate prevented sickling in the absence of oxygen, while voxelotor did not, suggesting that SNS-314 mesylate acts by a mechanism that is different from that of voxelotor. The sickling assay described in this study will permit the identification of additional, novel antisickling compounds, which will potentially expand the therapeutic options for SCD.

Evaluation of Current Clinical Research Spectrum of Sickle Cell Anemia to Approach New Innovative Developmental Techniques for Enhancing Sudanese Health Systems

“…Several techniques and assays are used for the detection and monitoring of the sickle disease (54). These methods can be separated into two focal groups: (1) presently used systems in the analysis of SCD; and…”

Modulation of the allosteric and vasoregulatory arms of erythrocytic oxygen transport

“…Investigating other potential benefits of voxelotor in SCD patients, Mendelson et al (2024) used a mouse model to show that of voxelotor could replace the several months of RBC transfusion currently required prior to gene therapy for SCD. New assays will allow the future identification of novel anti-sickling compounds ( Nakagawa et al, 2022 ). These new roles for voxelotor, as well as the discovery of novel compounds, could increase the overall availability and use of disease-modifying therapy (DMT) for SCD, as rates of DMT use remain low even with the introduction of newer therapies ( Newman et al, 2023 ).…”

Drug discovery by a basic research scientist

“…My reaction to his suggestion was to think about using a kinetic assay, which is the most relevant to the pathophysiology, to carry out large scale screening for discovering new drugs, rather than attacking a specific target. With very few exceptions ( Nakagawa et al, 2022 ), all of the very large number of drug discovery projects for sickle cell disease based on inhibiting polymerization of HbS, the root cause of the pathology in sickle cell disease, were focused on a specific target ( Mehanna, 2001 ; Olubiyi et al, 2019 ). The most frequent targeting approach has been to bind a drug to the non-polymerizing R quaternary conformation of HbS ( Sunshine et al, 1982 ; Eaton and Hofrichter, 1990 ; Henry et al, 2020 ) to reduce the population of the polymerizing T quaternary conformation ( Nakagawa et al, 2014 ; Safo and Kato, 2014 ; Pagare et al, 2022 ).…”