A-485   中文名称: A-485

A-485 对前列腺癌 PC-3 细胞处理三小时，导致 H3K27Ac 剂量依赖性减少，半数最大有效浓度 (EC50) 为 73 nM。 A-485 疗法对蛋白质 p300 或 CBP 的水平没有影响。 A-485 在大多数多发性骨髓瘤细胞系以及少数急性髓性白血病和非霍奇金淋巴瘤细胞系以及血液癌症中显示出强大的作用，其中发现了最大的敏感性。在所有五种前列腺癌细胞系中，A-485 都会导致 H3K27Ac 类似的减少[1]。

雄性 SCID 小鼠肿瘤生长后，每日两次腹膜内注射 A-485，治疗 21 天后可抑制 54% 的肿瘤生长（与载体对照相比，P<0.005）。此外，连续 7 天的 A-485 剂量会在给药后 3 小时导致 MYC 和 AR 依赖性基因 SLC45A3 的 mRNA 水平降低，以及 MYC 的蛋白质水平降低，这表明 A-485抑制体内 p300 介导的转录活性。这些结果是在携带肿瘤的动物中获得的。然而，在第七天，给药后 16 小时血浆和肿瘤中药物 A-485 的水平低于 3 小时。完成 A-485 剂量方案后，动物体重轻微下降 9%，并迅速恢复[1]。

p300和CBP生化活性测定[1]
通过使用TR-FRET测定检测组蛋白H4合成肽的赖氨酸残基的乙酰化，对p300 BHC和CBP-BHC结构域进行乙酰转移酶活性测定。使用含有100 mM HEPES的测定缓冲液在10μL体积内进行反应；pH 7.9，80μM EDTA，40μg/mL BSA，100 mM KCl，1 mM DTT，0.01%曲拉通X-100。鉴于EDTA在该测定中的使用，所用BHC蛋白的结构完整性可能会因该蛋白中存在多个含锌结构域（C/H3、PHD和RING）而受到影响。因此，p300 BHC活性测定也用在完全不存在EDTA的情况下纯化的p300BHC和在不存在EDTA时在测定缓冲液中纯化的p300-BHC进行。将每种感兴趣的化合物溶解在DMSO中，用Labcyte Echo在50 nL下以50μM至0.00075μM的3倍稀释度分配到白色384孔低容量板中。将0.6nM的p300 BHC或CBP-BHC蛋白与A-485或A-486预孵育30分钟。通过加入5μL 2μM的生物素化合成组蛋白-H4肽和0.5μM的乙酰辅酶a来引发反应。在室温下在加湿室中孵育1小时后，用10μL LANCE检测缓冲液中的3 nM LANCE Ultra Europium抗乙酰基组蛋白H4赖氨酸抗体、900 nM LANCE-ULight链霉抗生物素蛋白终止反应。TR-FRET测量使用Perkin-Elmer Envision获得，激光激发波长为335nm，发射波长为665nm和6201nm。对于乙酰辅酶A竞争实验，除了乙酰辅酶A浓度在0.078至10μM之间变化外，测定方法如上所述。使用Prism GraphPad 5对浓度/抑制反应曲线进行S形拟合，计算抑制的IC50值。

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p300αLISA肽结合分析[1]
使用AlphaLISA技术评估P300-BHC与组蛋白H4合成肽的结合。在40μL体积的白色384孔分析板中进行分析，使用含有100 mM HEPES、pH 7.9、80μM EDTA、40 ug/mL BSA、100 mM KCl、1 mM DTT、0.01%曲通X-100的分析缓冲液。将10μL生物素标记的H4肽加入10μL P300-BHC中（最终浓度分别为15 nM和115 nM），并在室温下孵育一小时。为了通过与生物素标记的肽竞争来证明AlphaLISA信号的降低，将10μL未标记的H4肽和生物素标签的H4肽的2倍混合物（终浓度分别为300μM和115 nM）加入20μL P300-BHC（终浓度为15nM）中，并在室温下孵育1小时。将20μL含有镍螯合物AlphaLISA受体珠和AlphaScreen链霉抗生物素蛋白供体珠的2倍混合物（终浓度各为20μg/mL）加入酶肽复合物中，并在室温下孵育1.5小时。使用Perkin-Elmer Envision在680nm激光激发和615nm发射下获得AlphaLISA计数。

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热位移分析[1]
在罗氏LightCycler 480仪器上使用在裂解缓冲液中用EDTA纯化的p300 BHC进行热位移测定（如上）。Sypro Orange染料以5000X的库存从Invitrogen购买。该测定在20mM HEPES、pH 7.5、50mM NaCl、1mM TCEP、2%DMSO和1:500稀释的染料中进行，蛋白质浓度为1.5 uM。制备50倍DMSO 1赖氨酸辅酶a和a-485储备样品，使最终DMSO浓度为2%（v/v）。所有样本均一式四份。

将细胞系铺板于 96 孔或 384 孔板中并粘附 24 小时。然后用 A-485 处理细胞 3、4 或 5 天。实验进行三次，并根据制造商的建议使用细胞活力测定来确定活细胞的分数。对于胸苷掺入测定，细胞用 A-485 处理 1、2、3 或 4 天。在该时间点之前 24 小时，添加氚化胸苷并将细胞再孵育 24 小时。然后在过滤板上分离基因组 DNA。

LuCap-77 CR xenograft efficacy studies.[1]
The LuCap-77 CR prostate PDX model was used. Donor tumors were dissociated and injected as a brie (1:2) into the right flank of 16 week old male C.B.-17 SCID mice on day 0 in a volume of 0.2 ml. Tumors were size matched on day 26 post-inoculation with a mean tumor volume of 211 ± 3 (SEM) mm3 with dosing beginning on day 28. No mice on study were excluded from the analysis. Mice were randomized into 1 treatment groups using Studylog software based on tumor volume. Tumor volume was calculated twice weekly. Measurements of the length (L) and width (W) of the tumor were taken via electronic caliper and the volume was calculated according to the following equation: V = L x W2/2 using Study Director version 3.1. Partial blinding was used. A different technician formulated and dosed compounds while the main investigator randomized and measured tumor volumes during the study. Tumor growth inhibition was calculated according to the following equation: TGI% = (mean tumor volume of the control group – mean tumor volume of the treated group) / mean tumor volume of the control group x 100.

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LuCap-77 CR xenograft PD studies [1]
LuCap-77 CR xenograft tumors were established in SCID mice and animals were dosed with A-485 as described above in “LuCap-77 CR xenograft growth studies” for 7 days. Three hours post the final dose, tumors were harvested and snap frozen on dry ice. For RNA isolation, tumors were homogenized in lysis solution from the 96 well RNA spin kit using a Precellys 24 homogenizer and further processed according to manufacturer’s instructions. For western blotting, tumors were homogenized in ice cold lysis buffer (as described in “Western blotting”) and centrifuged at 20,000 g for 20 min. The supernatant was then processed for western blotting as described above.

[1]. Lasko LM, et al. Discovery of a selective catalytic p300/CBP inhibitor that targets lineage-specific tumours. Nature. 2017 Oct 5;550(7674):128-132

The dynamic and reversible acetylation of proteins, catalysed by histone acetyltransferases (HATs) and histone deacetylases (HDACs), is a major epigenetic regulatory mechanism of gene transcription and is associated with multiple diseases. Histone deacetylase inhibitors are currently approved to treat certain cancers, but progress on the development of drug-like histone actyltransferase inhibitors has lagged behind. The histone acetyltransferase paralogues p300 and CREB-binding protein (CBP) are key transcriptional co-activators that are essential for a multitude of cellular processes, and have also been implicated in human pathological conditions (including cancer). Current inhibitors of the p300 and CBP histone acetyltransferase domains, including natural products, bi-substrate analogues and the widely used small molecule C646, lack potency or selectivity. Here, we describe A-485, a potent, selective and drug-like catalytic inhibitor of p300 and CBP. We present a high resolution (1.95 Å) co-crystal structure of a small molecule bound to the catalytic active site of p300 and demonstrate that A-485 competes with acetyl coenzyme A (acetyl-CoA). A-485 selectively inhibited proliferation in lineage-specific tumour types, including several haematological malignancies and androgen receptor-positive prostate cancer. A-485 inhibited the androgen receptor transcriptional program in both androgen-sensitive and castration-resistant prostate cancer and inhibited tumour growth in a castration-resistant xenograft model. These results demonstrate the feasibility of using small molecule inhibitors to selectively target the catalytic activity of histone acetyltransferases, which may provide effective treatments for transcriptional activator-driven malignancies and diseases.[1]

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In summary, we have overcome the long-standing challenge of developing a drug-like HAT inhibitor by identifying a first-in-class highly potent, selective, cell and in vivo active p300/CBP catalytic inhibitor, A-485. A similar approach also can be applied more broadly to develop inhibitors of other HATs. Furthermore, they underscore the value of therapeutically targeting the HAT activity of p300/CBP, providing a major advance in the road to evaluating the clinical utility of HAT inhibitors for multiple human diseases.[1]

C25H24F4N4O5

536.4755

536.168

C, 55.97; H, 4.51; F, 14.17; N, 10.44; O, 14.91

1889279-16-6

1889279-16-6

118958122

White to off-white solid powder

3.3

108Ų

C[C@@H](C(F)(F)F)N(CC1=CC=C(C=C1)F)C(=O)CN2C(=O)[C@]3(CCC4=C3C=CC(=C4)NC(=O)NC)OC2=O

VRVJKILQRBSEAG-LFPIHBKWSA-N

InChI=1S/C25H24F4N4O5/c1-14(25(27,28)29)32(12-15-3-5-17(26)6-4-15)20(34)13-33-21(35)24(38-23(33)37)10-9-16-11-18(7-8-19(16)24)31-22(36)30-2/h3-8,11,14H,9-10,12-13H2,1-2H3,(H2,30,31,36)/t14-,24+/m0/s1

N-(4-fluorobenzyl)-2-((R)-5-(3-methylureido)-2',4'-dioxo-2,3-dihydrospiro[indene-1,5'-oxazolidin]-3'-yl)-N-((S)-1,1,1-trifluoropropan-2-yl)acetamide

A-485; A 485; N-(4-Fluorobenzyl)-2-((R)-5-(3-methylureido)-2',4'-dioxo-2,3-dihydrospiro[indene-1,5'-oxazolidin]-3'-yl)-N-((S)-1,1,1-trifluoropropan-2-yl)acetamide; (1R)-N-[(4-Fluorophenyl)methyl]-2,3-dihydro-5-[[(methylamino)carbonyl]amino]-2',4'-dioxo-N-[(1S)-2,2,2-trifluoro-1-methylethyl]spiro[1H-indene-1,5'-oxazolidine]-3'-acetamide; CHEMBL4282264; A485.

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)

DMSO : ~125 mg/mL (~233.00 mM)

配方 1 中的溶解度: 11 mg/mL (20.50 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浮液；超声助溶。
*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: ≥ 2.08 mg/mL (3.88 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 20.8 mg/mL澄清的DMSO储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入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 3 中的溶解度: ≥ 2.08 mg/mL (3.88 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in 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 20.8 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 4 中的溶解度: ≥ 2.08 mg/mL (3.88 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 20.8 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。