GTx011, an Anti-Sickling Compound, Improves SS Blood Rheology By Reduction of HbS polymerization Via Allosteric Modulation of O2 Affinity (2014)

Abstract Sickle cell disease is caused by a mutation in the β-globin gene leading to production of hemoglobin S (HbS). HbS polymerizes under hypoxic conditions, leading to changes in cytoplasmic viscosity and formation of rigid, non-deformable sickled red blood cells (RBC). Loss of RBC deformability leads to abnormal blood rheology, diminished oxygen (O2) delivery and contributes to vaso-occlusion. GTx011 is a novel orally bioavailable small molecule that has been shown to inhibit HbS polymerization in vitro by maintaining oxygenated HbS under hypoxic conditions. We investigated the mechanism by which GTx011 modulates O2 affinity. Although it binds to the N-terminal α chain of Hb via a reversible Schiff base, 1D 1H NMR studies indicated that the compound allosterically influenced the intra-dimer interface of Hb (α122His and α103His), and the distal valine surrounding heme pockets for both the α and β chains, suggesting a unique solution phase structure. GTx011’s unique allosteric action upon the heme pocket allows it to improve O2 affinity without sterically blocking the release of O2. This modulation of O2 affinity delays the onset of HbS polymers and therefore we hypothesized will improve the abnormal blood rheology observed in SS blood. We report here the beneficial effect of GTx011 on SSRBC deformability using four independent whole cell systems: 1) the profile of SSRBC during passage through a gel filtration column, 2) the ability and corresponding pressure required for SSRBCs to pass through a polycarbonate filter under hypoxic conditions, 3) the tension required to aspirate SSRBCs into a micropipette under low O2 and 4) changes to blood viscosity during deoxygenation. Under deoxygenating conditions, GTx011 dose dependently enabled RBC deformability required for migration of SSRBCs through a gel filtration column. The pressure required to pass SSRBCs through a polycarbonate filter and the tension required to aspirate SSRBCs into a micropipette under hypoxic conditions were also reduced (Table 1). We have established a novel blood viscosity assay, which employs an enzymatic reaction (ascorbic acid/ascorbic oxidase) for deoxygenation of blood, followed by traditional measurements in cone plate viscometers. This protocol enabled us to reduce O2 levels without the technical limitations of using sodium dithionite, laser induced deoxygenation or passive deoxygenation via N2. Time dependent measures of viscosity, at a fixed shear rate, allowed us to quantify GTx011-induced delay in the onset of polymerization, and the increase in cytoplasmic viscosity. As in polymerization experiments, GTx011 dose dependently delayed the onset of hyperviscosity of SS blood under deoxygenating conditions. HbS modification ranging from 10-30%, was sufficient to achieve an improvement in SS blood hyperviscosity (Table 2) These data suggest that GTx011 is a promising agent that has the potential to improve SSRBC deformability and blood viscosity by inhibiting HbS polymerization, the pathophysiologic hallmark of sickle cell disease. Table 1 Assay Migration Filterability Deformability unit (% deformable cells) (mm Hg) (mDynes/cm2) [HbS] 1 mM 1 mM 1 mM [GTx011] (mM) 0 0.5 1 0 0.5 1 0 0.5 1 Readout 16 42 89 358 162 129 101 32 25 Table 2 Assay Polymerization Viscosity [HbS] 50 μM 1.5 mM GTx011 modified HbS (%) 0 10 30 0 10 30 Delay time (min) 13 18 33 21 26 31 Disclosures Patel: Global Blood Therapeutics: Employment, Equity Ownership. Cabrales:Global Blood Therapeutics: Consultancy. Dufu:Global Blood Therapeutics: Employment, Equity Ownership. Metcalf:Global Blood Therapeutics: Consultancy, Equity Ownership. Sinha:Global Blood Therapeutics: Employment, Equity Ownership.