histone acetyltransferase (HAT); CBP/p300; TIP60
MG149 targets Tip60 (IC50 = 0.5 μM) [1]

MG 149 (Tip60 HAT inhibitor) inhibits roughly 90% of Tip60 activity at 200 μM, but has no inhibitory effect on p300 and PCAF. MG 149 essentially competes with Ac-CoA but not with histone substrates. HAT inhibition experiments with MG 149 indicated that both drugs strongly suppressed HAT activity in nuclear extracts from different locations (p < 0.05) [1].

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MG149 exhibits selective inhibitory activity against the acetyltransferase activity of Tip60, concentration-dependently suppressing Tip60-mediated histone acetylation in in vitro enzymatic assays. It shows no significant inhibitory effect on other histone acetyltransferases such as p300 and CBP. In various tumor cell lines, MG149 treatment inhibits cell proliferation, induces G1 phase cell cycle arrest, and promotes apoptosis. Western blot analysis reveals that MG149 treatment reduces the level of acetylated histone H4, upregulates the expression of p21 and p27, and downregulates Cyclin D1 expression [1]

Administration of a MYST1 inhibitor MG149 alleviated AKI in mice.[2]
MYST1 inhibition by MG149 attenuates acute kidney injury in mice[2]
Finally, we made an attempt to address the question as to whether systemic inhibition of Myst1 activity in mice would provide beneficial effects in AKI models. To this end, C57/BL6 mice were injected peritoneally with MG149 prior to the induction of AKI. MG149 administration significantly relieved IR-induced AKI as evidenced by a decrease in plasma BUN (Fig. 7A) and creatinine (Fig. 7B) levels. H&E staining of renal sections provided additional support for the protective effects of MG149 by showing that mice receiving MG149 exhibited fewer casts and less extensive tubular necrosis (Fig. 7C). DHE staining confirmed that MG149 injection dampened ROS production (Fig. 7D) and concomitantly suppressed the expression levels of Nox genes (Fig. 7E, F) in the kidneys. We again tested the effects of MG149 administration in the LPS-induced AKI model. Similar to the IR model, LPS-associated renal injuries were substantially alleviated by MG149 injection as demonstrated by plasma BUN (Fig. S4A) and creatinine (Fig. S4B) levels, by ROS staining (Fig. S4C), and by quantification of Nox expression (Fig. S4D, S4E). In conclusion, MYST1 inhibition by MG149 may protect acute kidney injury in vivo.

Biochemical inhibition assays[1]
Radioisotope-labeled acetyltransferase assays were carried out at 30°C in a reaction volume of 30 µL. The reaction buffer contained 50 mM HEPES at pH 8.0, 0.1 mM EDTA, 50 µg/mL BSA, 1 mM dithiothreitol, 0.1% Triton-X100, and 2% DMSO. 14C-labeled Ac-CoA was used as the acetyl donor. The peptide containing the N-terminal 20-amino acid sequence of histone H4 (i.e. H4-20) was used as substrate for p300 and Tip60, and the peptide containing the N-terminal 20-amino acid sequence of histone H3 (i.e. H3-20) was employed as substrate for PCAF. The HAT reaction was initiated with the HAT enzyme after the other components (Ac-CoA, peptide substrate, and the inhibitor) were equilibrated at 30°C for 5 min. Rate measurements were based on initial conditions (generally less than 15% consumption of the limiting substrate). After the reaction, the mixture was loaded onto a Waterman P81 filter paper and then washed with 50 mM sodium bicarbonate (pH 9.0) for three times. The paper was air dried and the amount of radioactivity incorporated into the peptide substrate was quantified by liquid scintillation counting. In all the cases, background acetylation (in the absence of enzyme) was subtracted from the total signals. The IC50 was determined as the concentration of an inhibitor at which half of the enzyme activity was inhibited. For IC50 determination, a range of at least seven inhibitor concentrations varied at least 20-fold around the IC50 were tested. All the assays were performed at least twice, and duplicates generally agreed within 20%. The conditions for the IC50 measurement were; for the Tip60 assay, the reaction contained 10 nM Tip60, 1 µM Ac-CoA, 100 µM H4-20 and the reaction time was 7 min, for the PCAF assay, the reaction contained 1 nM PCAF, 1 µM Ac-CoA, 100 µM H3-20 and the reaction time was 3.5 min, for the p300 assay, the reaction contained 5 nM p300, 1 µM Ac-CoA, 100 µM H4-20 and the reaction time was 5 min, for the MOF assay, the reaction contained 1 nM MOF, 1 µM Ac-CoA, 100 µM H4-20 and the reaction time was 5 min. The conditions for the screening of the inhibitors are shown in the legend of Figure 1.

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The acetyltransferase activity of Tip60 was detected using a radiolabeling method. The reaction system contained recombinant Tip60 protein, histone substrate, acetyl-CoA (with radiolabeling), and MG149 at different concentrations. After incubating the reaction at an appropriate temperature for a certain period, the bound radioactive substrate was separated by filter binding, and the radioactivity intensity was measured to evaluate enzyme activity. The enzyme inhibition rate at different concentrations was calculated to determine the IC50 value [1]

Nuclear extracts were prepared from HeLa cells or tissue samples for distinct brain regions using procedures described by Dignam et al. The HAT activity in the nuclear extracts was determined using an ELISA assay in which either a biotinylated histone H3 peptide (aa 1 to 21, Anaspec – 61702) or a histone H4 peptide (aa 2–24, 12–372) was immobilized using steptavidin-biotin linkage. The ELISA was performed as described previously. The buffer for the enzymatic reaction contained 0.01% Triton X-100, 0.1 mM EDTA, 50 µg/mL BSA, 1mM DTT and 50 mM HEPES pH 7.4. The nuclear extracts were standardized based on the protein concentration. The final protein concentration of the HeLa nuclear extract in the enzyme reaction was 2.5 µg/mL. For nuclear extracts of brain tissue samples the concentration was 40 µg/mL. The reaction time for the enzymatic reaction was 15 min[1].

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Cell viability assays were performed on multiple tumor cell lines. Cells were seeded in 96-well plates and cultured overnight, then MG149 at different concentrations was added and incubation continued for 48 hours. Cell viability was detected by the MTT method, and the cell proliferation inhibition rate was calculated. For cell cycle and apoptosis analysis, after cells were treated with MG149, they were collected, stained with PI for cell cycle distribution detection by flow cytometry, and stained with Annexin V-FITC/PI for apoptosis rate detection by flow cytometry. In Western blot experiments, total protein was extracted after cell treatment, subjected to SDS-PAGE electrophoresis, membrane transfer, and blocking, then incubated with specific primary antibodies (against acetylated H4, p21, p27, Cyclin D1, etc.), followed by secondary antibody incubation, and finally protein expression levels were detected by chemiluminescence [1]

Myeloid-specific MRTF-A mice were bred by crossing the Mrtfaf/f strain (exons 9–14 were floxed) with a Lyz2-Cre strain. All strains used in this study were in a C57/BL6 background. For the ischemia-reperfusion model, 6–8 week-old male mice were anesthetized with ketamine. The renal pedicle was clamped with nontraumatic microaneurysm clamps. Clamps were removed after 45 min. Body temperature was controlled at 37 °C throughout the procedure. The mice were sacrificed 48 h later. For the septicemia model, 6–8 week-old male mice were injected peritoneally with LPS (25 mg/kg) and sacrificed 24 h later. Plasma creatinine levels, BUN levels, and proteinuria were measured using commercially available kits per manufacturer recommendations. In certain experiments, CCG-1423 or MG149 was injected peritoneally every other day for two weeks prior to the induction of AKI. CCG-1423 (1 mg/kg) and MG149 (1 mg/kg) were used. Staining and quantification were performed in a double-blinded fashion based on a previously published protocol[2].

[1]. 6-alkylsalicylates are selective Tip60 inhibitors and target the acetyl-CoA binding site. Eur J Med Chem. 2012 Jan;47(1):337-44.
[2]. Myocardin-related transcription factor A (MRTF-A) contributes to acute kidney injury by regulating macrophage ROS production. Biochim Biophys Acta Mol Basis Dis. 2018 Oct;1864(10):3109-3121

Histone acetyltransferases (HATs) are important enzymes that regulate a variety of cellular functions, such as the epigenetic regulation of DNA transcription. Developing highly selective and active HAT inhibitors will provide a powerful tool for elucidating the biological functions of HATs and may have pharmacological value for developing potential new therapies. In this study, we synthesized analogs of the known HAT inhibitor cashew acid and evaluated their inhibitory effects on HAT activity. Biochemical analysis showed that the novel cashew acid analogs selectively inhibited human recombinant enzyme Tip60 compared to PCAF and p300. Enzyme kinetic studies showed that one of the novel cashew acid derivatives 20 inhibited Tip60 primarily through competitive inhibition of acetyl-CoA, and non-competitive inhibition of histone substrates. In addition, these histone acetyltransferase (HAT) inhibitors effectively inhibited the acetyltransferase activity of nuclear extracts on histone H3 and H4 at micromolar concentrations. [1]

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Multiple pathogenic factors can induce acute kidney injury (AKI), leading to renal failure. This study evaluated the role of myocardial-associated transcription factor A (MRTF-A) in the pathogenesis of acute kidney injury (AKI). We found that systemic knockout of MRTF-A or inhibition of MRTF-A activity using CCG-1423 significantly alleviated AKI in ischemia-reperfusion or lipopolysaccharide (LPS)-induced mouse models. Notably, MRTF-A deficiency or inhibition led to reduced renal reactive oxygen species (ROS) production in the AKI model, accompanied by downregulation of NADPH oxidase 1 (NOX1) and NOX4 expression. In cultured macrophages, MRTF-A promoted NOX1 transcription after hypoxia-reoxygenation or LPS treatment. Interestingly, macrophage-specific MRTF-A deletion ameliorated acute kidney injury (AKI) in mice. Mechanistic analysis revealed that MRTF-A plays a role in regulating histone H4K16 acetylation around the NOX gene promoter by interacting with the acetyltransferase MYST1. MYST1 deletion inhibited NOX transcription in macrophages. Finally, administration of the MYST1 inhibitor MG149 alleviated AKI in mice. Thus, our data reveal a novel epigenetic pathway controlling reactive oxygen species (ROS) production in macrophages that is associated with the development of AKI. Targeting the MRTF-A-MYST1-NOX axis may provide a new therapeutic strategy for combating AKI. [2] MG149 is a synthetic 6-alkyl salicylate derivative. As a selective Tip60 inhibitor, it targets the acetyl-CoA binding site of Tip60, providing a potential drug target and therapeutic strategy for cancer treatment. [1]

C22H28O3

340.46

340.204

C, 77.61; H, 8.29; O, 14.10

1243583-85-8

1243583-85-8;

49864204

Typically exists as White to off-white solids at room temperature

5.388

2

3

10

25

374

0

O([H])C1=C([H])C([H])=C([H])C(=C1C(=O)O[H])C([H])([H])C([H])([H])C1C([H])=C([H])C(=C([H])C=1[H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H]

WBHQYBZRTAEHRR-UHFFFAOYSA-N

InChI=1S/C22H28O3/c1-2-3-4-5-6-8-17-11-13-18(14-12-17)15-16-19-9-7-10-20(23)21(19)22(24)25/h7,9-14,23H,2-6,8,15-16H2,1H3,(H,24,25)

2-[2-(4-heptylphenyl)ethyl]-6-hydroxybenzoic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.75 mg/mL (8.08 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 27.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.75 mg/mL (8.08 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 27.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)