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Phthalides serve as potent modulators to boost fetal hemoglobin induction therapy for β-hemoglobinopathies

Wei-Ren Chen, Chia-Cheng Chou and Chia C. Wang

Data supplements

Article Figures & Data

Figures

  • Figure 1.

    Phthalides are more potent modulators than 2,3-BPG in lowering oxygen affinity of HbF. (A) Molecular structure of z-butylidenephthalide (z-But). (B) Molecular structure of z-ligustilide (z-Lig). (C) OEC curves of purified HbF (orange), purified HbA (gray), HbF treated with 0.6 mM 2,3-BPG (green), HbA treated with 0.6 mM 2,3-BPG (purple), HbF treated with 0.6 mM z-butylidenephthalide (pink), and HbF treated with 4.0 mM z-butylidenephthalide (blue). (C) Inset shows an enlarged view of the area specified by gray dashed lines. (D) P50 values for HbF treated with 2,3-BPG (gray columns), z-butylidenephthalide (orange columns) and z-ligustilide (purple columns) at varying levels of treatment ranging between 0.6 and 4.0 mM. (E) A comparison of P50 for pure HbA (i), HbA treated with 4 mM 2,3-BPG (ii), pure HbF (iii), HbF treated solely with 4 mM 2,3-BPG (iv), and HbF cotreated with 4 mM 2,3-BPG and 2.5 to 4 mM phthalides (v-vii), which explicitly show that additional phthalide treatments can help raise the P50 for HbF to a level similar to that of P50 for HbA treated solely with the same level of 2,3-BPG. (F) P50 evolution for HbF treated with 2,3-BPG only (gray curve) and cotreated with z-butylidenephthalide at 0.6 mM (green curve), 1.2 mM (orange curve), 2.5 mM (lime green curve), and 4.0 mM (purple curve), along with varying levels of 2,3-BPG. (G) P50 evolution for HbF treated with 2,3-BPG only (gray curve), and cotreated with z-ligustilide of 0.6 mM (green curve), 1.2 mM (orange curve), and 4.0 mM (purple curve) along with varying levels of 2,3-BPG.

  • Figure 2.

    Phthalides stabilize oxyHbF in the low-oxygen-affinity state. RR spectroscopy of oxyHbF treated with varying amounts of 2,3-BPG (A), z-butylidenephthalide (B), and z-ligustilide (C) and deoxyHbF treated with varying amounts of 2,3-BPG (D), z-butylidenephthalide (E), and z-ligustilide (F). Color code for panels A-F: pure HbF (gray), HbF treated with the specified effector of 1 mM (red), 4 mM (orange), 8 mM (green), and 12 mM (violet). (G) Percentages of the high-affinity state for oxyHbF treated with z-butylidenephthalide (purple curve), z-ligustilide (orange curve), and 2,3-BPG (gray curve) with increasing degrees of treatment. (H) Active sites of HbF upon treatment with z-butylidenephthalide, with the intermolecular interactions denoted as yellow dashed lines. (I) Active sites of HbF upon treatment with z-ligustilide, with the intermolecular interactions denoted as yellow dashed lines.

Tables

  • Table 1.

    Comparison of P50 values of HbF cotreated with 2,3-BPG and phthalides vs that of HbA treated solely with 2,3-BPG

    EffectorHbAHbFΔP50, mm Hg
    0.6 mM 2,3-BPG16.2415.05−1.19
    0.6 mM 2,3-BPG + 0.6 mM z-butylidenephthalide16.300.06
    4 mM 2,3-BPG18.8316.56−2.27
    4 mM 2,3-BPG + 2.5 mM z-butylidenephthalide18.630.20
    4 mM 2,3-BPG + 4 mM z-butylidenephthalide19.540.71
    4 mM 2,3-BPG + 4 mM z-ligustilide18.740.09
    • ▵P50 = (P50 of HbF cotreated with 2,3-BPG and chosen phthalide) – (P50 of HbA treated solely with 2,3-BPG at the same level).

  • Table 2.

    P50 and n50 values for HbF treated with phthalide and 2,3-BPG under various treatment conditions

    No phthalidePhthalide
    0.6 mM1.2 mM4.0 mM
    P50n50P50n50P50n50P50n50
    z-Butylidenephthalide
     No 2,3-BPG14.602.5915.872.6216.682.6217.062.56
     2,3-BPG
      0.60 mM14.912.5916.112.6516.352.717.862.56
      1.2 mM15.122.5816.422.6616.372.6818.112.52
      2.5 mM15.922.5916.622.6617.42.7118.632.52
      4.0 mM16.562.5917.372.6317.292.6819.542.54
    z-Ligustilide
     No 2,3-BPG14.602.5915.982.5416.162.5416.692.51
     2,3-BPG
      0.60 mM14.912.5916.032.6416.342.716.792.66
      1.2 mM15.122.5816.482.6516.582.716.842.67
      2.5 mM15.922.5917.012.6717.032.6817.932.69
      4.0 mM16.562.5917.392.6418.092.7118.742.7
    • P50 units are mm Hg.