Archives

  • 2018-07
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • 2021-06
  • 2021-07
  • 2021-08
  • 2021-09
  • 2021-10
  • 2021-11
  • 2021-12
  • 2022-01
  • 2022-02
  • 2022-03
  • 2022-04
  • 2022-05
  • 2022-06
  • 2022-07
  • 2022-08
  • 2022-09
  • 2022-10
  • 2022-11
  • 2022-12
  • 2023-01
  • 2023-02
  • 2023-03
  • 2023-04
  • 2023-05
  • 2023-06
  • 2023-08
  • 2023-09
  • 2023-10
  • 2023-11
  • 2023-12
  • 2024-01
  • 2024-02
  • 2024-03
  • Here we report the kinetics of inhibition of the MYST

    2022-08-03

    Here, we report the kinetics of inhibition of the MYST HAT family member KAT8 by MG149, and a calculation of the inhibitory constant Ki of MG149 for KAT8. The inhibition of HATs by MG149 could be correlated to inhibition of histone acetylation in murine PCLS upon MG149 treatment, as determined by a mass spectrometry based analysis. This inhibition was observed on histone H4 res 4–17, containing H4 K16 which is the target of KAT8. Finally, we report reduced pro-inflammatory gene expression upon treatment with MG149 in murine PCLS. Taken together, this indicates that 6-alkylsalicylates such as MG149 have potential for development towards applications in the treatment of inflammatory lung diseases.
    Materials and methods
    Results
    Discussion In this study the enzyme kinetics of inhibition of the MYST HAT family member KAT8 by the 6-alkylsalicylate MG149 were investigated. This study demonstrates that the inhibition of HATs by MG149 correlates with inhibition of histone acetylation as well as reduced pro-inflammatory gene expression in murine PCLS, representing an ex vivo model for inflammatory lung diseases. This suggests that 6-alkylsalicylates such as MG149 can be developed towards applications in the treatment of inflammatory lung diseases such as Midostaurin and COPD. Bacterial acute exacerbations of COPD or severe asthma, or acute lung injury, may also be relevant areas of application for MG149 based on the relevance of the employed LPS/IFNγ stimulus to these disease conditions. Here, we demonstrated that the 6-alkylsalicylate MG149 inhibits the MYST family HAT member KAT8 according to a similar mechanism as observed previously for the 6-alkylsalicylate AA [21]. These 6-alkylsalicylates bind to the EX form of the enzyme and stabilize this catalytically inactive conformation, thus inhibiting the acetylation of the histone H4 peptide. Knowledge of the catalytic mechanism of KAT8 as well as the binding kinetics allows for recalculating assay-dependent IC50 values for KAT8 inhibitors to assay-independent Ki values, based on an equation described by Cheng and Prusoff [21], [24]. Using this knowledge, an assay-independent Ki value of 39 ± 7.7 μM was determined for the inhibition of KAT8 by the 6-alkylsalicylate MG149. This can now be directly compared to the 6-alkylsalicylate AA from which MG149 was derived, for which a Ki value of 64 ± 8.9 μM was previously determined for KAT8 inhibition [21]. This indicates an improved potency for MG149 with respect to its Ki value for KAT8. In this study, MG149 demonstrated inhibition of histone acetylation. A small but significant inhibition of histone acetylation was observed upon treatment of the PCLS with 10 μM of MG149. Furthermore, this inhibition of histone acetylation was more pronounced upon combined treatment with SAHA compared to SAHA treatment alone. When studying HATi, a frequently applied method to enable investigation of inhibition of histone acetylation is the simultaneous treatment with a HDACi. This increases the histone acetylation level thus enlarging the window to observe inhibition of histone acetylation. The rationale for combined treatment with HDACi and HATi to increase the detection window to reveal the effect of the HATi under investigation could relate to the dynamics and stoichiometry of histone acetylation, as described previously [30][31]. In this study both methods were used and similar results were found in both cases. Taken together both methods confirm that the HATi MG149 inhibits histone acetylation in PCLS. Our observations are in line with literature in which it has been shown that co-treatments with MG149 and SAHA demonstrated inhibition of histone acetylation compared to SAHA treatment alone in a model using human cancer cell lines MOPL8 and K562 [32]. Similar results were obtained for another 6-alkylsalicylate analogue to MG149 in HepG2 cells [33]. In both cases western blot was used. This study demonstrates that application of mass spectrometry is more sensitive and enables detection of smaller changes in histone acetylation, which reduces the need to increase the detection window using HDACi. The fact that we did not observe inhibition of histone acetylation in RAW264.7 murine macrophages upon MG149 treatment indicates that this effect could be cell type specific, and could be explained by the presence of various cell types in PCLS, and possibly also due to effects on cell-cell interactions in the intact lung tissue samples.