Abstract
Sickle cell disease is a genetic blood disorder caused by a single point mutation in the β globin gene where glutamic acid is replaced by valine at the sixth position of the β chain of hemoglobin (Hb). At low oxygen tension, the polymerization of deoxyHbS into fibers occurs in red blood cells (RBCs) leading to an impaired blood vessel transit. Sickle cell hemoglobin (HbS), when oxidized with hydrogen peroxide (H2O2), stays longer in a highly oxidizing ferryl (Fe4+) form causing irreversible oxidation of βCys93 to a destabilizing cysteic acid. We have previously reported that an antisickling drug can be designed to bind specifically to βCys93 and effectively protect against its irreversible oxidation by H2O2. Here, we report oxygen dissociation, oxidation, and polymerization kinetic reactions for four antisickling drugs (under different preclinical/clinical developmental stages) that either site-specifically target βCys93 or other sites on the HbS molecule. Molecules that specifically bind to or modify βCys93, such as 4,4′-di(1,2,3-triazolyl) disulfide (TD-3) and hydroxyurea (HU) were contrasted with molecules that target other sites on Hb including 5-hydroxymethyl-2-furfural (5-HMF) and L-glutamine. All reagents induced a left shift in the oxygen dissociation curve (ODC) except L-glutamine. In the presence of H2O2 (2.5:1, H2O2:heme), both TD-3 and HU reduced the ferryl heme by 22 and 37%, respectively, which corresponded to a 3- to 2-fold reduction in the levels of βCys93 oxidation as verified by mass spectrometry. Increases in the delay times prior to polymerization of HbS under hypoxia were in the following order: TD-3 > HU > 5-HMF = L-glutamine. Designing antisickling agents that can specifically target βCys93 may provide a dual antioxidant and antisickling therapeutic benefits in treating this disease.
Highlights
Single amino acid replacement in sickle cell disease (SCD) causes substantial reduction in the solubility of deoxyHbS leading to polymerization of tetramers into long fibers that decrease red blood cells (RBCs) membrane deformability
Similar reduction in P50 values was observed in the Oxygen dissociation curves (ODC) of Homozygous sickle RBCs (SS) cells that had been pre-incubated with TD-3 (2 mM) (Figure 2A); the P50 value of the SS cells was reduced from 34.2 to 9.6 mmHg
These results demonstrated the ability of TD-3 to permeate through RBC membranes and to interact with intracellular Sickle hemoglobin (HbS), consistent with early reports (Nakagawa et al, 2018)
Summary
Single amino acid replacement (glutamic acid → valine) in sickle cell disease (SCD) causes substantial reduction in the solubility of deoxyHbS leading to polymerization of tetramers into long fibers that decrease RBC membrane deformability. It is not surprising that most efforts directed toward finding a treatment have focused on the design of small antisickling molecular agents that can permeate RBCs and directly inhibit polymerization (Eaton and Bunn, 2017) It has been recognized for some time that the oxidative milieu within RBCs (in SCD patients) can be a source of toxic reactive oxygen pieces (ROS) (Mahaseth et al, 2005). Among these recently identified internal sources for ROS are NADPH oxidase subunits and mitochondria that are retained by mature SS RBCs (George et al, 2013; Jagadeeswaran et al, 2017). In addition to targeting its own β subunits ( the hotspot βCys93), ferrylHb and its associated radical (HbFe4+) actively interact with other biological molecules (Kassa et al, 2015)
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