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AG-348 enhances pyruvate kinase activity in red blood cells from patients with pyruvate kinase deficiency

Charles Kung, Jeff Hixon, Penelope A. Kosinski, Giovanni Cianchetta, Gavin Histen, Yue Chen, Collin Hill, Stefan Gross, Yaguang Si, Kendall Johnson, Byron DeLaBarre, Zhiyong Luo, Zhiwei Gu, Gui Yao, Huachun Tang, Cheng Fang, Yingxia Xu, Xiaobing Lv, Scott Biller, Shin-San Michael Su, Hua Yang, Janeta Popovici-Muller, Francesco Salituro, Lee Silverman and Lenny Dang

Data supplements

Article Figures & Data

Figures

  • Figure 1.

    Structure and function of PK-R and AG-348. (A) Simplified schematic of glycolysis. (B) Chemical structure of AG-348. (C) Activity of recombinant PK-R enzyme incubated with indicated concentrations of AG-348 (PEP = 0.065 mM). Mean, standard deviation, range, and number of experimental replicates are indicated in the column on right-hand side of panel. (D) Plot of activity of recombinant WT PK-R enzyme stimulated with PEP with or without preincubation by AG-348 (5 μM); the average of 3 technical replicates is shown. (E) Ribbon diagram of the cocomplex crystal structure of PK-R tetramer bound to AG-348 (shown as space-filling model in gray). PEP (blue) and FBP (brown) also shown as space-filling models. Diffraction statistics are shown below the diagram. (F) Model of AG-348 in PK-R allosteric- binding pocket illustrating key interactions. All error bars are standard deviations. ADP, adenosine diphosphate; PG, phosphoglyceric acid.

  • Figure 2.

    AG-348 activates WT PK-R in RBCs from healthy donors and in mice. (A) WT PK-R activity from RBCs from healthy donors incubated overnight with indicated concentrations of AG-348 (PEP = 0.1 mM). Mean, standard deviation, range, and number of experimental replicates are indicated in the column on right-hand side of panel. (B) ATP levels of healthy- donor RBCs incubated overnight with indicated concentrations of AG-348. (C) Concentration of AG-348 measured in plasma of mice dosed with AG-348 twice daily for 7 days at indicated dose levels. The bar graph depicts the calculated AUC0-12 hours at each dose level. (D) PK-R activity in RBCs from mice dosed with AG-348 as described in panel C (PEP = 0.1 mM). For clarity, only the 150 mg/kg dose level is shown here. See supplemental Figure 2 for full data plots. The bar graph depicts the calculated AUC0-12 hours at each dose level. (E) ATP levels and calculated AUC0-12 hours in whole blood from mice dosed with AG-348 as described in panel D. (F) 2,3-DPG levels and calculated AUC0-12 hours in whole blood from mice dosed with AG-348 as described in panel D. For clarity, only the 150 mg/kg dose level is shown in panels E and F. All error bars are standard deviations.

  • Figure 3.

    AG-348 activates a spectrum of recombinant mtPK-R enzymes. (A) PK-R tetramer with sites of mtPK-R enzymes tested in Figure 3B highlighted in red. For clarity, each mutation site is only shown in a single monomer. (B) Fold activation and AC50 values of AG-348 treatment of mtPK-R enzymes (AG-348 = 10 μM, PEP, as listed in the supplemental table). (C) Activity of recombinant R532W mtPK-R enzyme stimulated with PEP with or without preincubation with AG-348 (AG-348 = 5 μM). (D) Activity of recombinant R532W mtPK-R enzyme incubated with indicated concentration of FBP or AG-348 (PEP = 0.05 mM). Panels C and D show the average of 3 technical replicates. (E) Residual activity over time of WT or R510Q recombinant enzymes following incubation at 53°C (AG-348 = 5 μM, PEP = 2 mM). (F) Off-rate measurement of AG-348 or FBP (both at 5 μM final assay concentration, PEP = 2 mM) from recombinant R510Q enzyme. All error bars are standard deviations.

  • Figure 4.

    AG-348 activation of mtPK-R in RBCs from patients with PK deficiency. (A) Genotypes of patients with PK deficiency described in these studies. (B) PK-R protein levels measured in patients with PK deficiency or WT controls. Data were not available from this assay for patient C, but a Western blot showing approximate protein levels in this patient is shown in supplemental Figure 6C. (C) Levels of 2,3-DPG, PEP, and ATP in RBCs from patient A at indicated times following treatment with DMSO or AG-348. (D) PK-R activity in PK-deficient RBCs incubated with AG-348 for 24 h (PEP = 0.5 mM). All PK-R activity measurements used the coupled enzyme spectrometric assay, except in the case of patient B, for whom activity was assessed by direct measurement of pyruvate formation using liquid chromatography followed by tandem mass spectrometry as described in the supplemental Methods. (E) ATP levels in PK-deficient RBCs incubated with AG-348 for 24 h. For panels D and E, please note that patient samples E and F were received via overnight courier post–blood draw, whereas other patient samples were received on the same day as blood draw, but potentially up to 8 h post–blood draw (supplemental Figure 5B-C). (F) Table showing AG-348 AC50 and fold-change observed in PK-R activity and ATP levels from indicated patient samples. AC50 values shown are in nanomolar. Fold-change (in relation to DMSO control) PK-R activity and ATP graphs are shown in supplemental Figure 6B. (G) PK-R activity (left) and ATP levels (right) in RBCs from patient A or a WT control (both prepared ∼30 min post–blood draw), incubated with AG-348 for 24 h. Panels D, E, and G show the average of 3 technical replicates. All error bars are standard deviations.

Tables

  • Table 1.

    Kinetic parameters from recombinant PK-R enzymes measured with or without treatment with AG-348

    EnzymeNo compound5 μM AG-348
    Vmax, μmol/s/mgPEP KM, mMkcat, 1/skcat/KMVmax, μmol/s/mgPEP KM, mMkcat, 1/skcat/KM% of WT PK-R kcat/KM
    WT0.691.1342.537.60.740.3245.7142.0378
    R532W0.221.7313.67.90.210.4212.930.481
    R510Q1.601.4598.868.11.640.32101.5317.3844
    R479H0.501.6530.918.70.530.5532.458.5156
    R486W0.341.4521.214.60.390.2524.298.4262
    G332S0.142.738.33.00.151.119.48.523
    R490W0.371.5222.815.00.390.5124.247.6127
    G364D0.162.1410.14.70.220.5513.524.765
    T384M0.111.426.94.80.120.377.219.351
    • kcat, first-order rate constant; kcat/KM, measure of enzymatic efficiency39; KM, substrate concentration to achieve half maximal velocity; Vmax, maximum velocity.