High Throughput Kinetic Assay for Screening Potential Inhibitors of Sickle Hemoglobin Polymerization

Abstract

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.

Aromatic Aldehydes and Carboxylic Acids as inhibitors for Sickle Hemoglobin Polymerization

“…One of the approaches to develop therapeutic agents to treat the disease is to inhibit the polymerization process or delay its onset to allow deoxygenated blood to pass to the lung. In search of such agents that can inhibit or delay the polymerization, we have developed recently a high throughput assay screen organic compounds and natural products for anti-polymerization activities [2]. The assay helped in the identification of several thiol-containing compounds as potential anti-polymerization agents [3].…”