histone acetyltransferase (HAT); CBP/p300; TIP60

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].

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.

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].

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 are important enzymes that regulate various cellular functions, such as epigenetic control of DNA transcription. Development of HAT inhibitors with high selectivity and potency will provide powerful mechanistic tools for the elucidation of the biological functions of HATs and may also have pharmacological value for potential new therapies. In this work, analogs of the known HAT inhibitor anacardic acid were synthesized and evaluated for inhibition of HAT activity. Biochemical assays revealed novel anacardic acid analogs that inhibited the human recombinant enzyme Tip60 selectively compared to PCAF and p300. Enzyme kinetics studies demonstrated that inhibition of Tip60 by one such novel anacardic acid derive, 20, was essentially competitive with Ac-CoA and non-competitive with the histone substrate. In addition, these HAT inhibitors effectively inhibited acetyltransferase activity of nuclear extracts on the histone H3 and H4 at micromolar concentrations.[1]

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A host of pathogenic factors induce acute kidney injury (AKI) leading to insufficiencies of renal function. In the present study we evaluated the role of myocardin-related transcription factor A (MRTF-A) in the pathogenesis of AKI. We report that systemic deletion of MRTF-A or inhibition of MRTF-A activity with CCG-1423 significantly attenuated AKI in mice induced by either ischemia-reperfusion or LPS injection. Of note, MRTF-A deficiency or suppression resulted in diminished renal ROS production in AKI models with down-regulation of NAPDH oxdiase 1 (NOX1) and NOX4 expression. In cultured macrophages, MRTF-A promoted NOX1 transcription in response to either hypoxia-reoxygenation or LPS treatment. Interestingly, macrophage-specific MRTF-A deletion ameliorated AKI in mice. Mechanistic analyses revealed that MRTF-A played a role in regulating histone H4K16 acetylation surrounding the NOX gene promoters by interacting with the acetyltransferase MYST1. MYST1 depletion repressed NOX transcription in macrophages. Finally, administration of a MYST1 inhibitor MG149 alleviated AKI in mice. Therefore, we data illustrate a novel epigenetic pathway that controls ROS production in macrophages contributing to AKI. Targeting the MRTF-A-MYST1-NOX axis may yield novel therapeutic strategies to combat AKI.[2]

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.)