Small Molecule Therapeutics for Sickle Cell Anemia

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Project Summary Sickle Cell Disease (SCD) is a common genetic condition that affects approximately 100,000 people of African heritage in the US, and more than 4 million worldwide. The disease is caused by a single amino acid substitution in b-globin that promotes a rigid sickle shape phenotype of red blood cells (RBCs) and eventually leads to numerous clinical manifestations such as vaso-occlusive crises, hemolysis, peripheral tissue damage, renal insufficiency, and premature death. Various therapeutic approaches have been approved or are in development for SCD, including bone marrow transplantation, gene therapy, induction of fetal hemoglobin, and anti-sickling agents. Clinically approved therapies often have limited effectiveness, significant side effects, or are too costly to reach the majority of SCD patients. Treatment of SCD would be advanced by development of novel and effective anti-sickling drugs. Our laboratory has developed an innovative imaging assay utilizing automated microscopy and customized algorithms that detect the sickled phenotype of RBCs. This assay has been miniaturized to the 1536-well plate format suitable for high throughput screening. Unlike previous approaches to identifying anti-sickling compounds – which are often limited to drugs that interact directly with hemoglobin – the new assay detects morphological changes in RBCs at very high throughput and provides identification of anti- sickling agents targeting pathways modulating hemoglobin polymerization or RBC morphology, including mechanisms that do not involve direct interaction with hemoglobin. This allows elucidation of new classes of potential therapeutics for SCD, as well as novel tools for investigating the underlying mechanisms of hemoglobin polymerization and RBC sickling. The overall goal of the R61 phase of this proposal is to generate anti-sickling candidates for future pre-clinical and clinical studies. We will carry out large scale, high throughput drug screening to identify molecules that prevent sickling of diseased RBCs. The resulting hits will be verified in dose response and a series of secondary assays. Lead compounds will be selected based on physiologically acceptable changes of hemoglobin oxygen affinity and RBC membrane deformability. Further optimization will be carried out in the R33 phase by medicinal chemistry. As part of the R33, lead scaffolds will be synthesized and evaluated for anti-sickling potency, hemoglobin oxygen affinity, and improved RBC membrane deformability to identify agents best suited for animal safety and efficacy studies. Townes HbSS mice expressing human globin genes will be used to test short term lethality, efficacy of anti-sickling activity, RBC oxygen affinity, and RBC half- life in vivo. As the final deliverable of this proposal, 3 or more drug candidates that are safe and efficacious in the mouse model will be selected for future pre-clinical studies.