Development of a Triazolyldisulfide Compound That Increases the Affinity of Hemoglobin for Oxygen and Reduces Hypoxic Sickling of Sickle Cells
Authors: Nakagawa Akito , Michele Ferrari , Liu Chen , Berra Lorenzo , Klings Elizabeth S. , Safo Martin K. , Bloch Donald B. , Abdulmalik Osheiza , Zapol Warren M. 
Published: October 2019

Abstract
Introduction: In patients with sickle cell disease,during a vasoocclusive crisis, deoxygenated sickle hemoglobin (HbS) polymerizes forming fibers of HbS in red blood cells (SS RBCs). HbS fibers distort SS RBCs, causing microvascular occlusion, increased thrombosis and inflammation, and severe pain for patients. Increasing the affinity of Hb for oxygen reduces sickling of SS RBCs, because oxygenated HbS does not form polymers. It has been reported that modification of the thiol group of Hb β-Cys93 increases Hb's affinity for oxygen by destabilizing the T-state and stabilizing the R-state. In addition, modification of HbS β-Cys93 might prevent interaction between HbS molecules and thereby reduce sickling. Herein we report a novel triazolyldisulfide compound (4,4'-Di(1,2,3-triazolyl)disulfide, designated "TD-3"), which increases the affinity of Hb for oxygen and reduces the sickling of hypoxic human SS RBCs in vitro. Intravenous administration of TD-3 to mice increases the affinity of murine Hb for oxygen. The effects of TD-3 on the affinity of Hb for oxygen may be a result of interaction with HbA-Cys93.
Methods: TD-3 hydrate was dissolved in a mixture of Dulbecco's phosphate buffered saline and 30% polyethylene glycol 400. The oxygen dissociation curve (ODC) of Hb was measured at 37°C (pH 7.3) using a HEMOX analyzer. The partial oxygen pressure at which 50% of Hb is oxygenated was determined as P50 from the ODC and the P50 was used to assess the affinity of Hb for oxygen. SS whole blood was incubated with TD-3 (Hb tetramer/TD-3 = 1/1 mol/mol) for 10 min at 37°C and the P50 of TD-3 treated RBCs was determined. To evaluate the ability of TD-3 to reduce sickling in vitro, SS RBCs (Hct ≈ 20%) were incubated with TD-3 in a gas mixture of 4% oxygen and 96% nitrogen and the percentage of sickled RBCs was determined after incubation. To determine the effect of TD-3 on time-dependent change of P50 in vitro, normal human whole blood (Hct ≈ 45-50%) was incubated with TD-3 (Hb tetramer/TD-3 = 1/1 mol/mol) for 10 min and 8 h at 37°C. At both times, the P50 of RBCs was determined. To determine the effect of TD-3 on the time-dependent change of P50 in vivo, TD-3 (100 mg/kg) was administered intravenously to C57BL/6 mice and the P50 of murine Hb was determined (as hemolysate) before, and 1 and 24 h after TD-3 treatment. As a first step to investigate the mechanism of action of TD-3, normal adult hemoglobin (HbA) was treated with N-ethylmaleimide (NEM) to block HbA β-Cys93 and the P50 of NEM-treated HbA was determined. Either HbA or NEM-treated HbA was incubated with TD-3 at 37°C for 10 min (Hb tetramer/TD-3 = 1/6 mol/mol). After the incubation, the P50 of HbA and NEM-treated HbA was determined.
Results: Incubation of TD-3 with SS RBCs reduced the P50 of SS RBCs from 29 mmHg to 24 mmHg. Incubation of SS RBCs with TD-3 (2 mM) in 4% oxygen decreased the percentage of sickled RBCs from 94% (without TD-3) to 22%.
The P50 of TD-3-treated human normal RBCs was decreased from 25 mmHg (prior to adding TD-3) to 21 mmHg at 10 min. At 8 h, the P50 of TD-3-treated human normal RBCs was the same as that of RBCs treated with vehicle alone. The P50 of TD-3-treated murine Hb was reduced from 21 mmHg (before treatment with TD-3) to 18 mmHg at 1 h. At 24 h, the P50 of TD-3-treated murine Hb was the same as that of mice treated with vehicle alone. At 24 h, all of the mice that were treated with either TD-3 or vehicle alone were alive and appeared normal.
Treatment of HbA with NEM reduced the P50 from 17 mmHg to 8 mmHg. Incubation of TD-3 with either HbA or NEM-treated HbA reduced the P50 of HbA from 17 mmHg to 4 mmHg, but did not alter the P50 of NEM treated HbA (8 mmHg).
Conclusions: 4,4'-Di(1,2,3-triazolyl)disulfide (TD-3) increased the affinity of human normal and SS RBCs for oxygen and reduced the sickling of hypoxic human SS RBCs in vitro. Administration of TD-3 to healthy mice increased the affinity of murine Hb for oxygen and the compound was well tolerated by the mice. The effect of TD-3 on the affinity of HbA for oxygen was impaired by blocking the thiol group of HbA β-Cys93 with NEM, suggesting that the mechanism by which TD-3 increases the affinity of Hb for oxygen and reduces sickling may be through interaction with Hb β-Cys93. Our data demonstrate that TD-3 has the potential to prevent and treat sickle cell disease.

Disclosures
Nakagawa: Massachusetts General Hospital: Patents & Royalties: MGH filed a patent application on the use of heteroaryl disulfide compounds including TD-3 to increase the oxygen-binding affinity of hemoglobin and treat sickle cell disease and other uses of these compounds. The patent hasn't issued or licensed yet.. Zapol:Massachusetts General Hospital: Patents & Royalties: MGH filed a patent application on the use of heteroaryl disulfide compounds including TD-3 to increase the oxygen-binding affinity of hemoglobin and treat sickle cell disease and other uses of these compounds. The patent hasn't issued or licensed yet..