The effect of the antisickling compoundGBT1118 on the permeability of red blood cells from patients with sickle cell anemia

Abstract

Sickle cell anemia ( SCA ) is one of the commonest severe inherited disorders. Nevertheless, effective treatments remain inadequate and novel ones are avidly sought. A promising advance has been the design of novel compounds which react with hemoglobin S (HbS) to increase oxygen (O 2 ) affinity and reduce sickling. One of these, voxelotor ( GBT 440), is currently in advanced clinical trials. A structural analogue, GBT 1118, was investigated in the current work. As RBC dehydration is important in pathogenesis of SCA , the effect of GBT 1118 on RBC cation permeability was also studied. Activities of P sickle , the Gardos channel and the KC l cotransporter ( KCC ) were all reduced. Gardos channel and KCC activities were also inhibited in RBC s treated with Ca 2+ ionophore or the thiol reagent N ‐ethylmaleimide, indicative of direct effects on these two transport systems. Consistent with its action on RBC membrane transporters, GBT 1118 significantly increased RBC hydration. RBC hemolysis was reduced in a nonelectrolyte lysis assay. Further to its direct effects on O 2 affinity, GBT 1118 was therefore found to reduce RBC shrinkage and fragility. Findings reveal important effects of GBT 1118 on protecting sickle cells and suggest that this is approach may represent a useful therapy for amelioration of the clinical complications of SCA .

Pathophysiological Relevance of Renal Medullary Conditions on the Behaviour of Red Cells From Patients With Sickle Cell Anaemia

“…To promote sickling and lipid scrambling, red cells were incubated using three different conditions – hypoxia (30 and 0 mmHg oxygen tension), altered pH (6.0–8.0), and hypertonicity – in gently rotating Eschweiler tonometers at 1% Hct at 37°C for up to 80 min. To measure sickling, as defined in Al Balushi et al (2019) , red cell aliquots were then fixed using 0.3% glutaraldehyde, under the same conditions as incubation – these low levels of glutaraldehyde were chosen because of their minimal effect on saline osmolality. For each condition, 100 cells were assessed and sickling given as a percentage.…”

Inhibition of the Aquaporin-1 Cation Conductance by Selected Furan Compounds Reduces Red Blood Cell Sickling

“…Considering current drug discovery work for SCD, it is interesting to note that the compound GBT1118 is structurally related to Voxelotor, an approved drug recently shown in the HOPE Phase III clinical trial to be effective in increasing SCD patient hemoglobin concentrations, which in turn correlated with reduced incidence of vaso-occlusive crises ( Howard et al, 2021 ). GBT1118 reduces RBC sickling, increases oxygen affinity, stabilizes HbS structure, and decreases cation fluxes mediated by Psickle, Gardos and KCC pathways ( Al Balushi et al, 2019 ). Psickle (defined as the clotrimazole-insensitive component measured by radiolabelled Rb + entry in low oxygen) was the component most sensitive to block by GBT1118 (with an estimated IC 50 near 0.6 mM).…”

“…Psickle (defined as the clotrimazole-insensitive component measured by radiolabelled Rb + entry in low oxygen) was the component most sensitive to block by GBT1118 (with an estimated IC 50 near 0.6 mM). In the presence of GBT1118, an expected correlation between Psickle activity and Gardos activity was lost ( Al Balushi et al, 2019 ), perhaps suggesting the GBT1118-sensitive Psickle K + current is separate from the Ca 2+ permeation pathway. The possibility that AQP1 is an unrecognized secondary target of action for Voxelotor is currently being explored.…”

“…The involvement of a cation leak current in the sickling process has been appreciated for decades based on imbalances in K + and Na + levels in SCD cells ( Tosteson et al, 1952 ). However, a mystery concerning the molecular basis of Psickle has persisted, despite widespread interest in finding a Psickle inhibitor for expanding therapeutic strategies ( Lew et al, 1997 ; Hannemann et al, 2014 ; Al Balushi et al, 2019 ; Kaestner et al, 2020 ). Cation leak currents are activated in sickle cell RBCs during deoxygenation ( Joiner 1993 ; Joiner et al, 1988 ; Ma et al, 2012 ) and can be potentiated by membrane shear stress ( Johnson and Gannon 1990 ), such as that experienced in the microcirculation ( McMahon 2019 ).…”

Haptoglobin Therapeutics and Compartmentalization of Cell-Free Hemoglobin Toxicity

“…This seemingly minor modification facilitates the polymerization of Hb during tense state (T-deoxyHb) to relaxed state (R-oxyHb) transitioning, which promotes 'sickling' morphological changes and, ultimately, hemolysis of erythrocytes [80,81]. As a result, drug development efforts have targeted antipolymerization strategies to dilute intraerythrocytic HbS by increasing fetal Hb with oral hydroxyurea therapy [82,83] and to increase Hb oxygen affinity [84][85][86][87], using curative efforts such as gene therapy [88][89][90], as well as comprehensive strategies to address the acute and chronic progressive debilitating sequelae of SCD [91].…”