Abstract

Sickle cell disease (SCD) is an inherited disorder caused by a point mutation in the β-globin gene leading to formation of hemoglobin S (HbS). A primary and obligatory event in the molecular pathogenesis of SCD is the polymerization of deoxygenated HbS leading to sickling of red blood cells (RBCs). Prolonging the oxygenation of HbS should in principle delay polymerization relative to transit time through microcirculation, thus avoiding sickling. Discovered by Global Blood Therapeutics, GTx011 is a novel orally available small molecule that demonstrates desirable pharmacokinetics in multiple species. In addition, GTx011 binding to HbS produces concentration-dependent left shifts in partial oxygen pressure (p50) of oxygen equilibrium curves, indicating an increase in oxygen affinity. We report here that GTx011 delays in vitro polymerization and prevents sickling.

GTx011 was evaluated in an adapted version of the polymerization assay described by Adachi et al. [Blood Cells (1982) 8:213-224]. Purified HbS (pre-incubated with GTx011) in 1.8 M potassium phosphate was passively de-oxygenated, followed by an induction of polymerization via temperature jump from 4°C to 37°C. Polymerization was quantified by measuring turbidity of the HbS solution under continued hypoxia. For sickling experiments, RBCs pre-incubated with GTx011 were subjected to hypoxia (pO2 of ∼30 mmHg) for 0.5 hr and subsequently imaged using a light microscope. In de-sickling experiments, RBCs were first sickled under hypoxia and then treated with GTx011 under continued hypoxia (2 hr) before acquiring images of cells. The percentage of sickled cells in each image was quantified using CellVigene software.

GTx011 delayed HbS polymerization in a dose-dependent manner. The delay profile was similar to that of CO-liganded HbA, a well characterized intracellular inhibitor of HbS polymerization. Moreover, GTx011 prevented sickling of RBCs under hypoxia suggesting that GTx011 has the ability to prevent intracellular HbS polymerization. It should be noted that in this experimental system, RBCs were exposed to hypoxia for a relatively longer period of time than typical RBC transit times through microcirculation. Interestingly, GTx011 also reversed sickling of pre-sickled RBCs. The ability of GTx011 to de-sickle RBCs suggests that it may promote transition from the low affinity HbS (T-state HbS) into its high affinity conformer (R-state HbS), a form that is not incorporated into HbS polymers. Thus, by stabilizing the oxy (R) conformation of HbS, GTx011 has the potential to be a therapeutic agent for prevention of HbS polymerization and sickling in SCD patients.

Table 1
Assay Polymerization Sickling De-sickling 
Unit DTcpd-DTHbS(min) (% sickled) (% sickled) 
[HbS] 50 µM ∼1 mM (20% HCT) ∼1 mM (20% HCT) 
[Cpd] 25 µM 50 µM 100 µM 1 mM 2 mM 5 mM 1 mM 2 mM 5 mM 
GTx011 3.9 12.8 22.9 41 33 28 57 34 31 
5-HMF 1.8 6.3 76 65 50 97 96 89 
No Cpd 85 85 85 96 96 96 
Assay Polymerization Sickling De-sickling 
Unit DTcpd-DTHbS(min) (% sickled) (% sickled) 
[HbS] 50 µM ∼1 mM (20% HCT) ∼1 mM (20% HCT) 
[Cpd] 25 µM 50 µM 100 µM 1 mM 2 mM 5 mM 1 mM 2 mM 5 mM 
GTx011 3.9 12.8 22.9 41 33 28 57 34 31 
5-HMF 1.8 6.3 76 65 50 97 96 89 
No Cpd 85 85 85 96 96 96 
Table 2
Assay Polymerization 
Unit DTHbS/HbA-DTHbS(min) 
[Hb] 50 µM 
% HbA-CO 20% 30% 40% 
HbA-CO 2.3 11.8 16.8 
Assay Polymerization 
Unit DTHbS/HbA-DTHbS(min) 
[Hb] 50 µM 
% HbA-CO 20% 30% 40% 
HbA-CO 2.3 11.8 16.8 

Disclosures:

Dufu:Global Blood Therapeutics: Employment, Equity Ownership. Sinha:Global Blood Therapeutics: Employment, Equity Ownership.

Author notes

*

Asterisk with author names denotes non-ASH members.