MG149

histone acetyltransferase (HAT); CBP/p300; TIP60

MG 149（Tip60 HAT 抑制剂）在 200 μM 时可抑制大约 90% 的 Tip60 活性，但对 p300 和 PCAF 没有抑制作用。 MG 149 本质上与 Ac-CoA 竞争，但不与组蛋白底物竞争。 MG 149 的 HAT 抑制实验表明，两种药物均强烈抑制不同位置核提取物中的 HAT 活性 (p < 0.05) [1]。

MYST1抑制剂MG149的给药缓解了小鼠的AKI[2]。
MG149抑制MYST1可减轻小鼠急性肾损伤[2]
最后，我们试图解决系统性抑制小鼠Myst1活性是否会在AKI模型中产生有益作用的问题。为此，在诱导AKI之前，C57/BL6小鼠被腹膜注射MG149。MG149给药显著缓解了IR诱导的AKI，这可以从血浆BUN（图7A）和肌酐（图7B）水平的降低中得到证明。肾切片的H&E染色为MG149的保护作用提供了额外的支持，表明接受MG149的小鼠表现出更少的铸型和更少的广泛肾小管坏死（图7C）。DHE染色证实，MG149注射液抑制了ROS的产生（图7D），同时抑制了肾脏中Nox基因的表达水平（图7E，F）。我们再次在LPS诱导的AKI模型中测试了MG149给药的效果。与IR模型相似，注射MG149可显著减轻LPS相关的肾损伤，如血浆BUN（图S4A）和肌酐（图S4B）水平、ROS染色（图S4C）和Nox表达定量（图S4D、S4E）所示。综上所述，MG149对MYST1的抑制可能在体内保护急性肾损伤。

生化抑制试验[1]
在30°C下，在30µL的反应体积内进行放射性同位素标记的乙酰转移酶测定。反应缓冲液含有pH 8.0的50 mM HEPES、0.1 mM EDTA、50µg/mL BSA、1 mM二硫苏糖醇、0.1%Triton-X100和2%DMSO。14C标记的Ac-CoA用作乙酰供体。含有组蛋白H4的N端20个氨基酸序列的肽（即H4-20）用作p300和Tip60的底物，含有组蛋白H3的N端30个氨基酸序列（即H3-20）的肽用作PCAF的底物。在30°C下平衡其他组分（Ac-CoA、肽底物和抑制剂）5分钟后，用HAT酶启动HAT反应。速率测量基于初始条件（通常低于限制底物消耗的15%）。反应后，将混合物装载到Waterman P81滤纸上，然后用50mM碳酸氢钠（pH 9.0）洗涤三次。将纸风干，通过液体闪烁计数定量掺入肽底物的放射性量。在所有情况下，从总信号中减去背景乙酰化（在没有酶的情况下）。IC50被确定为抑制一半酶活性的抑制剂的浓度。对于IC50测定，测试了至少七种抑制剂浓度的范围，这些浓度在IC50附近变化至少20倍。所有检测都至少进行了两次，重复的检测结果通常在20%以内。IC50测量的条件为：；对于Tip60测定，反应含有10 nM Tip60、1µM Ac-CoA、100µM H4-20，反应时间为7分钟；对于PCAF测定，反应包含1 nM PCAF、1µM Ac-CoA和100µM H3-20，反应速度为3.5分钟；对于p300测定，反应含5 nM p300、1µM-Ac-CoA，100µM H4-20，反应时间为5分钟；对于MOF测定，反应涉及1 nM MOF、1µM-Ac-CoA或100µM H8-20，反应距离为5分钟。抑制剂筛选条件如图1的图例所示。

使用Dignam等人描述的程序从HeLa细胞或不同脑区的组织样本中制备核提取物。使用ELISA测定法测定核提取物中的HAT活性，其中使用steptavidin-biotin连接固定生物素化的组蛋白H3肽（aa 1至21，Anaspec–61702）或组蛋白H4肽（aa 2-24， 12-372）。如前所述进行ELISA。酶反应缓冲液含有0.01%Triton X-100、0.1 mM EDTA、50µg/mL BSA、1mM DTT和50 mM HEPES pH 7.4。核提取物根据蛋白质浓度进行标准化。酶反应中HeLa核提取物的最终蛋白质浓度为2.5µg/mL。对于脑组织样本的核提取物，浓度为40µg/mL。酶反应的反应时间为15分钟[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

57.53

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)

配方 1 中的溶解度: ≥ 2.75 mg/mL (8.08 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 27.5 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.75 mg/mL (8.08 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 27.5 mg/mL 澄清 DMSO 储备液加入900 μL 玉米油中，混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。