High Throughput Kinetic Assay for Screening Potential Inhibitors of Sickle Hemoglobin PolymerizationAhmed S Mehanna*
Department of Pharmaceutical Sciences, School of Pharmacy, MCPHS University, Boston, MA, USA
- *Corresponding Author:
- Ahmed S Mehanna
Department of Pharmaceutical Sciences
School of Pharmacy, MCPHS University
179 Longwood Avenue, Boston, MA 02115, USA
E-mail: [email protected]
Received date: June 29, 2017; Accepted date: July 06, 2017; Published date: July 10, 2017
Citation: Mehanna AS (2017) High Throughput Kinetic Assay for Screening Potential Inhibitors of Sickle Hemoglobin Polymerization. Med Chem (Los Angeles) 7:193-196. doi: 10.4172/2161-0444.1000455
Copyright: © 2017 Mehanna AS. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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.