AG-348 (mitapivat), an allosteric activator of red blood cell pyruvate kinase, increases enzymatic activity, protein stability, and adenosine triphosphate levels over a broad range of PKLR genotypes

Pyruvate kinase (PK) deficiency is a rare hereditary disorder affecting red blood cell (RBC) glycolysis, causing changes in metabolism including a deficiency in adenosine triphosphate (ATP). This affects red cell homeostasis, promoting premature removal of RBC from the circulation. In this study, we characterized and evaluated the effect of AG-348, an allosteric activator of PK that is currently in clinical trials for treatment of PK deficiency, on RBC and erythroid precursors from PK-deficient patients. In 15 patients, ex vivo treatment with AG-348 resulted in increased enzymatic activity in all patients' cells after 24 hours (h) (mean increase: 1.8-fold; range: 1.2-3.4). ATP levels increased (mean increase: 1.5-fold; range: 1.0-2.2) similar to control cells (mean increase: 1.6-fold; range: 1.4-1.8). Generally, PK thermostability was strongly reduced in PK-deficient RBC. Ex vivo treatment with AG-348 increased residual activity from 1.4- to >10-fold more than residual activity of vehicle-treated samples. Protein analyses suggest that a sufficient level of PK protein is required for cells to respond to AG- 348 treatment ex vivo, as treatment effects were minimal in patient cells with very low or undetectable levels of PK-R. In half of the patients, ex vivo treatment with AG-348 was associated with an increase in RBC deformability. These data support the hypothesis that drug intervention with AG- 348 effectively up-regulates PK enzymatic activity and increases stability in PK-deficient RBC over a broad range of PKLR genotypes. The concomitant increase in ATP levels suggests that glycolytic pathway activity may be restored. AG-348 treatment may represent an attractive way to correct the underlying pathologies of PK deficiency. (AG-348 is currently in clinical trials for the treatment of PK deficiency. Registered at clinicaltrials.gov identifiers: NCT02476916, NCT03853798, NCT03548220, NCT03559699).