A Triazole Disulfide Compound Increases the Affinity of Hemoglobin for Oxygen and Reduces the Sickling of Human Sickle Cells

Abstract

Sickle cell disease is an inherited disorder of hemoglobin (Hb). During a sickle cell crisis, deoxygenated sickle hemoglobin (deoxyHbS) polymerizes to form fibers in red blood cells (RBCs), causing the cells to adopt "sickled" shapes. Using small molecules to increase the affinity of Hb for oxygen is a potential approach to treating sickle cell disease, because oxygenated Hb interferes with the polymerization of deoxyHbS. We have identified a triazole disulfide compound (4,4'-di(1,2,3-triazolyl)disulfide, designated TD-3), which increases the affinity of Hb for oxygen. The crystal structures of carboxy- and deoxy-forms of human adult Hb (HbA), each complexed with TD-3, revealed that one molecule of the monomeric thiol form of TD-3 (5-mercapto-1H-1,2,3-triazole, designated MT-3) forms a disulfide bond with β-Cys93, which inhibits the salt-bridge formation between β-Asp94 and β-His146. This inhibition of salt bridge formation stabilizes the R-state and destabilizes the T-state of Hb, resulting in reduced magnitude of the Bohr effect and increased affinity of Hb for oxygen. Intravenous administration of TD-3 (100 mg/kg) to C57BL/6 mice increased the affinity of murine Hb for oxygen, and the mice did not appear to be adversely affected by the drug. TD-3 reduced in vitro hypoxia-induced sickling of human sickle RBCs. The percentage of sickled RBCs and the P₅₀ of human SS RBCs by TD-3 were inversely correlated with the fraction of Hb modified by TD-3. Our study shows that TD-3, and possibly other triazole disulfide compounds that bind to Hb β-Cys93, may provide new treatment options for patients with sickle cell disease.

Keywords: Bohr effect; P50; disulfide compound; hemoglobin; oxygen binding affinity; red blood cells; sickle cell disease; sickle hemoglobin.

Figure 1

(A) Structure of triazole disulfides (TD-1 and TD-3) and mercaptotriazoles (MT-1 and MT-3). (B) Left-shift of the oxygen dissociation curve (ODC) of purified HbA with increasing molar ratio of TD-3:Hb tetramer. The ODC for TD-3:Hb tetramer molar ratio of 6:1 overlaps with that of the molar ratio of 4:1. (C) The dose-dependent effect of TD-3 on the P₅₀ of HbA. The plots of TD-3 (circle) overlap with the plots of TD-1 (triangle). (D) Left-shift of the ODC of normal human blood with increasing molar ratio of TD-3 to Hb tetramer. (E) The dose-dependent effect of TD-3 on reducing the P₅₀ of normal RBCs. (F) Left-shift of the ODC of SS RBCs with increasing molar ratio of TD-3:Hb tetramer. (G) The dose-dependent effect of TD-3 on reducing the P₅₀ of SS RBCs. The ODCs were measured in triplicate at 37 °C. Each data point in panel C, E, and G represents the mean value of the P₅₀ determined from the ODCs. Error bars represent standard deviation (sd). Most error bars are too short to visualize because the sd was less than 1.2 mmHg.

Figure 2

Increase of the affinity of murine Hb for oxygen and covalently modified murine Hb by administration of TD-3 (100 mg/kg) to C57BL/6 mice. The oxygen dissociation curve of C57BL/6 murine RBCs (A) and murine hemolysate (B) measured at 37 °C before and 1 h after intravenous administration of TD-3 (100 mg/kg). The effect of time on the P₅₀ of murine Hb (C) and the percentage of modified Hb (D) before and after administration of TD-3. (E) Correlation between the percentage of modified Hb and the change in P₅₀ of the hemolysate. ΔP₅₀% = |(P₅₀ at a time point) – (baseline P₅₀)|/(baseline P₅₀). Each data point in C, D, and E represents the mean value, and error bars represent the standard deviation of the P₅₀ or modified Hb%. Four mice were used in each of the vehicle- and TD-3-treated group.

Figure 3

Effect of TD-3 on the oxygen affinity of NEM-modified Hb (A) and Hb C93A (B). NEM treatment blocked the ability of TD-3 to further decrease the P₅₀ of HbA. TD-3 did not decrease the P₅₀ of Hb C93A. Each data point represents the mean P₅₀, and error bars represent standard deviation (sd) of the P₅₀ measured at 37 °C in triplicate. Most error bars are too short to visualize because the sd was less than 0.7 mmHg.

Figure 4

Crystal structure of COHbA in a complex with TD-3. MT-3 formed a disulfide bond with the thiol of COHbA β-Cys93. (A) The binding sites of MT-3 in COHbA. Hb α and β subunits are shown as pale blue and brown, respectively. Carbon monoxide bound to heme is shown as a red and green sphere. The locations of MT-3, β-Cys93, and β-Ala142 are shown as sticks within the dashed rectangles. (B) The electron density of MT-3 and β-Cys93 indicated by the 2Fo-Fc map (gray mesh, contoured at 1.0σ). The N1 atom of MT-3 at β-Cys93 formed a hydrogen bond with the oxygen atom of β-Ala142, which is shown as a dotted line.

Figure 5

Crystal structure of deoxyHbA in a complex with TD-3. MT-3 formed a disulfide bond with the thiol of deoxyHbA β-Cys93. (A) The binding sites of MT-3 in deoxyHbA. Hb α and β subunits are shown in pale blue and brown, respectively. The locations of MT-3, β-Cys93, β-Lys144, and β-His146 are shown as sticks within the dashed rectangular areas. (B) The electron density of MT-3 and β-Cys93 indicated by the 2Fo-Fc map (gray mesh, contoured at 1.0σ). (C) β-subunit of the crystal structure of native deoxyHbA (PDB ID: 2DN2). β-Asp94 and β-His146 formed the characteristic salt-bridge to stabilize the T-state Hb. (D) β-subunit of the crystal structure of deoxyHbA in a complex with TD-3. The binding of MT-3 at the thiol of β-Cys93 disrupted the salt-bridge interaction between β-Asp94 and β-His146.

Figure 6

Inhibition of hypoxia-induced sickling of SS RBCs by TD-3 in vitro. The morphology of SS RBCs treated with vehicle (A) or with TD-3 (0.5–2 mM, B–E) and incubated with 4% oxygen at 37 °C for 2 h. (F) The effect of TD-3 on the percentage of sickled cells (sickled cells%) in cells exposed to hypoxia. (G) Representative oxygen dissociation curves (ODCs) of the hemolysate of SS RBCs with or without TD-3. The ODCs were measured in phosphate buffer (0.1 M phosphate, pH 7.0) at 25 °C. (H) HPLC chromatograms of hemolysates prepared from SS RBCs treated with TD-3 (0–2 mM). (I) The effect of treating SS RBCs with TD-3 on the percentage of modified Hb (modified Hb%). (J) The relationship between the modified Hb% and sickled cell% of SS RBCs treated with TD-3. Each data point represents the mean value of modified Hb% or sickled cells% measured in triplicate. Error bars represent standard deviation. Panel J was generated using the data from panels F and I.