Abstract
Background/Objectives: Sickle cell disease (SCD) is caused by a β-globin gene mutation (βGlu6Val) that produces sickle hemoglobin (HbS). When deoxygenated, HbS polymerizes, leading to red blood cell (RBC) sickling; therefore, hemoglobin is a central therapeutic target for SCD. Current strategies include increasing the levels of oxygenated HbS (which cannot polymerize) and/or directly destabilizing the deoxygenated HbS polymer. This study aimed to design and synthesize next-generation Michael acceptor antisickling hemoglobin modifiers (MMA-206, MMA-207, MMA-208, and MMA-209) and evaluate their antisickling efficacy. Methods: Four Michael acceptor compounds (MMA-206 to MMA-209) were synthesized and characterized. Their pharmacologic activities and modes of action were assessed in vitro using disulfide exchange reaction with normal hemoglobin, sickling inhibition assays with sickle red blood cells, and hemoglobin oxygen equilibrium curve analysis with normal and sickle red blood cells. Results: MMA-206 exhibited the strongest antisickling activity, outperforming previously studied Michael acceptor antisickling agents. All four MMA analogues bound to hemoglobin at βCys93, destabilizing the low-oxygen-affinity T-state and thereby preventing deoxygenation-induced HbS polymerization and RBC sickling. In addition, they appeared to directly destabilize the HbS polymer, indicating a second mechanism of action. Furthermore, time-dependent oxygen equilibrium measurements confirmed that their pharmacologic effect was sustained over time in vitro. Conclusions: The new Michael acceptor compounds, particularly MMA-206, demonstrated potent antisickling effects via dual mechanisms and showed sustained activity. These findings highlight Michael acceptor compounds' promise as hemoglobin oxygen-affinity modulators for the treatment of SCD.