Activation of Glycolytic Enzymes in Red Blood Cells for Therapeutic Purposes

Abstract

Background: Glucose-6-phosphate dehydrogenase (G6PD) is a crucial enzyme in the pentose phosphate pathway and pivotal for maintaining redox balance in red blood cells (RBCs). Deficiency in G6PD can lead to hemolytic anemia, particularly under oxidative stress. While therapeutic strategies are primarily preventive, recent advancements propose the activation of G6PD as a therapeutic approach. Aim: This study investigated the activation of the glycolytic enzyme G6PD in RBCs using a small molecule activator, AG1, to evaluate its potential in mitigating oxidative stress and increasing cell survival. Methods: RBCs from healthy donors were isolated and incubated with AG1 and Bay-11. Bay-11 was utilized to mimic G6PD deficiency. Oxidative stress was induced using diamide. The impact on G6PD and PK enzyme activity, RBC deformability, and hemolysis was assessed using spectrophotometric assays, RoxyScans and Osmoscans. Results: AG1 did not significantly alter G6PD enzyme activity in healthy RBCs. Incubation with Bay-11 significantly decreased G6PD activity, but AG1 failed to protect or rescue this depletion. RoxyScan analysis revealed no significant protective effects of AG1 against oxidative stress induced by diamide. However, Osmoscan and spectrophotometric assay results indicated that AG1-incubated samples show slightly improved deformability and hydration status after prolonged storage and a notable reduction in hemolysis. Conclusion: While AG1 showed potential in reducing hemolysis during RBC storage, it did not significantly increase G6PD activity or protect against oxidative stress in healthy RBCs. These findings suggest that AG1 might stabilize RBCs under storage conditions but is not effective in activating G6PD in the context of oxidative stress. Further research is necessary to elucidate its mechanism and potential therapeutic applications.