https://orcid.org/0000-0002-5938-3048
Key Points
Activating pyruvate kinase improved red cell integrity in sickle cell disease by reducing tyrosine-phosphorylation of band 3 (Tyr-p-bd3)
ATP plays a critical role in preventing the membrane-destabilizing effects of Tyr-p-bd3 by improving activity of constitutive phosphatase
In a Phase 1 study (NCT04000165), we established proof-of-concept for activating pyruvate kinase (PK) in sickle cell disease (SCD) as a viable anti-sickling therapy. AG-348 (mitapivat), a PK activator, increased adenosine triphosphate (ATP) and decreased 2,3- diphosphoglycerate levels while patients were on treatment in line with the mechanism of the drug. We noted that the increased hemoglobin persisted for 4 weeks after stopping AG-348 until the end of study (EOS). Here, we investigated the pathways modulated by activating PK that may contribute to the improved red blood cell (RBC) survival after AG-348 cessation. We evaluated frozen whole blood samples taken at multiple timepoints from the patients in the Phase 1 study, from which RBC ghosts were isolated and analyzed by Western blotting for tyrosine-phosphorylation of band 3 (Tyr-p-bd3), ankyrin-1 and intact (active) protein tyrosine phosphatase 1B (PTP1B) levels. We observed a significant dose-dependent decrease in mean Tyr-p-bd3 from baseline in the subjects, accompanied by increase in levels of membrane-associated ankyrin-1 and intact PTP1B, all of which returned to near baseline by EOS. As PTP1B is cleaved (inactivated) by intracellular Ca2+-dependent calpain, we next measured the effect of AG-348 on ATP production and calpain activity, and the plasma membrane Ca2+ ATPase pump (PMCA)-mediated efflux kinetics in HbAA and HbSS erythrocytes. AG-348 treatment increased ATP levels, decreased calpain activity, and increased Ca2+ efflux. Altogether, our data indicate that ATP increase is a key mechanism underlying the increase in hemoglobin levels upon PK activation in SCD.
