High Throughput Kinetic Assay for Screening Potential Inhibitors of Sickle Hemoglobin Polymerization
The current manuscript describes a high throughput assay designed to identify organic compounds with potential inhibitory effects on sickle hemoglobin polymerization. The assay is fast, economic and reproducible. In just 20-minutes, a test compound can be screened for anti-polymerization activity at five different concentrations; each in quadruple; using as little as 10 mg of purified sickle hemoglobin. The assay was conducted in high phosphate buffer concentration (1.5M), a concentration that allows sickle hemoglobin to polymerize at a very low concentration of 50.0 μM. The new assay was validated by evaluating the inhibitory effects of the amino acid phenylalanine, a standard control used in all gelation assays, and hydroxyl urea, the only FDA approved drug to treat sickle cell anemia. Phenylalanine showed a reproducible and concentration-dependent delay of sickle hemoglobin polymerization at a concentration range of 25-75mM. Hydroxyurea; although its action is thought to be through promoting fetal hemoglobin formation, was found to have direct inhibitory effects on sickle hemoglobin polymerization but at a high concentration range of 64-500mM. The assay was applied for random screening of several organic compounds and 2-thio-salicylic acid was identified to be a powerful inhibitor for polymerization at much lower concentration range of 0.1-1.6 mM.