Mitapivat activates both recombinant PKLR and PKM2, increases protein thermal stability, and ameliorates morphology of β-thal erythroblasts. (A) In vitro activity of recombinant PKLR and PKM2 enzymes incubated with increasing concentrations of mitapivat. Data are mean ± standard deviation (SD; n = 3). (B) Dichroic signal (at 222 nm) of thermal denaturation of PKLR and PKM2 recorded in the absence or presence of saturating concentration of mitapivat (10 μM). Data are mean ± SD (n = 3). (C) Comparison between the calculated melting temperatures and statistical analysis. Data are mean ± SD (n = 3). t test significance, ∗∗∗∗P ≤ 0.0001. (D) Residual activity of PKLR and PKM2 enzymes in the absence or presence of mitapivat (10 μM) after incubation at 53°C. Values are reported as percentage of the initial activity assayed at 25°C. (E) Residual activity of PKLR and PKM2 at 45°C. All error bars are SDs. (F) May-Grunwald-Giemsa staining for erythroblast morphology (upper) of CD34⁺-derived erythroid precursors, on day 14 of culture, from patients with β-thal (cod β039) with or without mitapivat, showing improvement of irregular nuclear shape and chromatin condensation. One representative image from 5 with similar results is shown. Original magnification, 100×. Quantification of abnormal erythroblasts is shown in supplemental Figure 3A. Cell growth (lower) of the erythroid precursors, as in panel A. Data are mean ± SEM (n= 5). ^ˆP < .05 (compared with vehicle-treated cells).