Development and validation of an oxygen dissociation assay, a screening platform for discovering, and characterizing hemoglobin–oxygen affinity modifiers

Mira P Patel,1 Vincent Siu,1 Abel Silva-Garcia,1 Qing Xu,2 Zhe Li,2 Donna Oksenberg1 1Biology Department, Global Blood Therapeutics Inc., South San Francisco, CA, USA; 2Chemistry Department, Global Blood Therapeutics Inc., South San Francisco, CA, USA Introduction: Hemoglobin (Hb) is a critical molecule necessary for all vertebrates to maintain aerobic metabolism. Hb–oxygen (O2) affinity modifiers have been studied to address various diseases including sickle cell disease, hypoxemia, tumor hypoxia, and wound healing. However, drug development of exogenous Hb modifiers has been hindered by the lack of a technique to rapidly screen compounds for their ability to alter Hb–O2 affinity. We have developed a novel screening assay based upon the spectral changes observed during Hb deoxygenation and termed it the oxygen dissociation assay (ODA). Methodology: ODA allows for the quantitation of oxygenated Hb at given time points during Hb deoxygenation on a 96-well plate. This assay was validated by comparing the ability of 500 Hb modifiers to alter the Hb–O2 affinity in the ODA vs the oxygen equilibrium curves obtained using the industry standard Hemox Analyzer instrument. Results: A correlation (R2) of 0.7 indicated that the ODA has the potential to screen and identify potent exogenous Hb modifiers. In addition, it allows for concurrent comparison of compounds, concentrations, buffers, or pHs on the level of Hb oxygenation. Conclusion: With a cost-effective, simple, rapid, and highly adaptable assay, the ODA will allow researchers to rapidly characterize Hb–O2 affinity modifiers. Keywords: hemox analyzer, in vitro assays, small-molecule screening, voxelotor