FB23-2

FTO (IC50 = 2.6 μM)

FTO是一种信使核糖核酸N6-甲基腺苷（m6A）去甲基化酶，据报道可促进白血病的发生。采用基于结构的合理设计，我们开发了两种有前景的FTO抑制剂，即FB23和FB23-2，它们直接与FTO结合并选择性抑制FTO的m6A去甲基酶活性。FB23-2模拟FTO耗竭，在体外显著抑制人急性髓系白血病（AML）细胞系细胞和原代母细胞AML细胞的增殖并促进其分化/凋亡。[1]
FB23-2 直接连接到 FTO，以特定方式抑制其 m6A 去甲基酶活性。人急性髓系白血病 (AML) 细胞系细胞和原代母细胞 AML 细胞的增殖受到显着抑制，并在体外被鼓励分化/凋亡，模拟 FTO 耗尽。 [1]

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FTO/FB23复合物的结构表明，对FB23羧酸的优化不会干扰FTO的亲和力和特异性。为了提高FB23的渗透性，我们根据生物异构原理合成了苄基羧酸衍生物。苯甲羟肟酸，称为FB23-2（图1H和S1B），对NB4和MONOMAC6细胞的抗增殖活性显著提高，IC50为0.8-1.5μM（图1I），并在体外保持对FTO去甲基化的抑制活性（图1J）。为了确定绝对构型，我们确定了FB23-2的X射线晶体结构，该结构明确地显示了羟肟酸的氨基氢和羰基之间的分子内氢键（图1H，右图）。此外，我们使用核奥弗豪瑟效应（NOE）分析了溶液中FB23-2的相对构型，NOE是核自旋极化通过空间而不是化学键的转移。NOESY光谱中H-1和H-10之间强烈的NOE相关性也支持分子内氢键（图S1E）。有了这些证据，FB23-2与FTO的对接导致FB23-2在与结合到FTO的抑制剂FB23的晶体学确定的结合模式完美重叠的位置上非常吻合（图S1F）。接下来，我们通过LC-MS/MS定量检测了细胞对FB23-2的摄取（图1K）。值得注意的是，在MONOMAC6和NB4细胞中检测到FB23-2约为0.05-0.2 nmol/百万细胞，这比FB23的细胞摄取高出几倍（见图1G）。同时，在FB23-2处理的AML细胞中也检测到少量的FB23，这可能是FB23-2的水解产物。FB23-2细胞内浓度的增加可能有助于其改善AML细胞的抗增殖作用。

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FB23-2增加一组AML细胞中的RNA甲基化[1]
研究人员检查了用FTO抑制剂处理AML细胞时RNA上m6A的变化。用FB23-2处理NB4和MONOMAC6细胞后，m6A斑点印迹法检测到转录组中m6A丰度大幅增加（图2A）。LC-MS/MS定量进一步证实了暴露于FB23-2后AML细胞mRNA中细胞m6A的增加（图2B）。m6Am是FTO的另一种底物，我们观察到暴露于FB23-2的AML细胞中m6Am丰度也有类似的增加（图2B）。然而，m6Am的整体水平远低于m6A。由于最近的报告独立显示，FTO对m6Am开始的mRNA表达水平的影响最小（Akichika等人，2019；Sun等人，2019，Wei等人，2018），因此m6A而不是m6Am很可能是AML细胞中FTO的主要底物（Su等人，2019）。

FB23-2对从健康供体分离的人类正常骨髓（BM）细胞的增殖影响最小（图2C）。与FTO KD对MLL-AF9（MA9）和FLT3ITD/NPM1 AML小鼠细胞增殖的影响一致（Li等人，2017b），FB23-2以剂量依赖的方式显著抑制了这两种模型中BM细胞的增殖（图2D），同时m6A丰度增加（图2E）。此外，我们在一组具有不同遗传背景的其他AML细胞系中确定了FB23-2的抗白血病作用，包括MA9.3ITD（具有MLL-AF9和FLT3ITD突变）、MA9.3RAS（具有ML-L-AF9和NRAS突变）、U937（具有t（10；11）易位）、ML-2（具有t。正如预期的那样，FB23-2有效地抑制了这些AML细胞系的增殖，IC50范围为1.9μM至5.2μM，并增加了这些细胞系中m6A的丰度（图2E和2F）。这些结果共同表明，FB23-2具有广泛的FTO抑制和抗白血病作用。

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FB23-2对FTO显示出高选择性[1]
研究人员对FTO抑制剂的选择性进行了分析。与MA类似，FB23和FB23-2在体外均不抑制ALKBH5去甲基化（图S2A）。如药物亲和力反应靶稳定性（DARTS）测定所示（Lomenick等人，2009），FB23-2不能直接与AML细胞裂解物中的ALKBH5结合（图S2B），并且对ALKBH5的转录水平和蛋白质稳定性仅显示出微弱的影响（图S2C和S2D）。此外，我们检查了FTO抑制剂对AML和/或其他癌症中涉及的表观遗传靶标的抑制作用，包括组蛋白脱乙酰基酶（HDAC）、端粒沉默1样阻断剂（DOTL1）、含溴胺的“阅读者”蛋白（BRD）、赖氨酸特异性脱甲基酶1（LSD1）和含Jumonji结构域的组蛋白脱甲基酶（Shortt等人，2017）。FB23和FB23-2在体外略微减弱了这些靶点的活性，而阳性对照抑制剂显示出显著的活性（表S2）。同样，20μM FB23-2在体外对TET1蛋白的抑制作用最小（图S2E），并且不会改变AML细胞中5mC或5hmC的丰度（图S2F）。FB23-2未改变NB4细胞中的主要组蛋白甲基化（图S2G）。此外，我们对FB23-2进行了更广泛的酶特异性测试。10μM FB23-2对405种人类激酶活性的抑制作用被绘制在激酶系统发育树上（图S2H）。进一步评估了抑制率超过40%的激酶。FB23-2抑制了六种激酶，IC50约为3.0-13.4μM。FB23-2对这些激酶的抑制效率远低于公认的激酶抑制剂。FB23-2也几乎不抑制致癌蛋白酶（表S3）。尽管MA在不同程度上抑制COX-1和COX-2（Vane等人，1998），但即使在50μM的浓度下，FB23和FB23-2也没有观察到显著抑制环氧化酶（图S2I）。综上所述，这些结果表明我们的抑制剂对FTO显示出很高的酶选择性。

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FB23-2表现出FTO依赖性的抗增殖活性，促进髓系分化和凋亡[1]
与FTO对AML细胞中ASB2和RARA表达的负调控一致（Li等人，2017b），FB23和FB23-2处理显著增加了NB4和MONOMAC6细胞中mRNA和蛋白质水平的丰度（图3A和3B）。FTO以m6A修饰依赖的方式正向调节MYC和CEBPA（Su等人，2018）。与shRNA诱导的FTO KD类似，FB23或FB23-2确实抑制了NB4和MONOMAC6细胞中MYC和CEBPA的表达（图3C）。

为了进一步确定FB23-2对AML细胞增殖的抑制作用是否依赖于FTO，我们使用CRISPR-Cas9生成了稳定的FTO KO NB4 AML细胞。FB23-2显著抑制了AML细胞的增殖，但对具有稳定FTO KO的AML细胞的影响要温和得多（图3D），这表明FB23-2对AML细胞增殖的抑制作用取决于对激活的FTO信号的抑制。一致地，我们发现FTO KD和60μM FB23或3μM FB23-2在NB4细胞中具有相当的效果（图3E）。为了验证FB23-2和FTO之间的直接相互作用，我们进行了DARTS测定。正如预期的那样，在FB23-2存在的情况下，FTO蛋白变得对蛋白酶具有抗性（图3F），表明FB23-2确实与细胞裂解物中的FTO结合。

研究人员进一步描述了FB23-2对AML细胞的影响。与FTO对AML细胞髓系分化和凋亡的抑制作用一致（Li等人，2017b），FB23-2以剂量依赖的方式显著加速了全反式维甲酸（ATRA）诱导的NB4和MONOMAC6细胞的髓系分化（图4A和4B）。此外，FB23-2诱导AML细胞凋亡（图4C和4D）和G1期细胞周期阻滞（图4E和4F）。总的来说，这些结果表明FB23-2在AML细胞中表现出FTO依赖性活性。

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FB23和FB23-2在AML细胞中靶向与FTO KD相似的信号通路[1]
为了研究哪些基因和信号通路负责FTO抑制剂的抗白血病功能，研究人员对FTO KD、FB23处理或FB23-2处理的NB4 AML细胞以及对照细胞进行了转录组全RNA测序（RNA-seq）分析。通过对三种不同比较的独立分析，我们发现FTO KD、FB23治疗和FB23-2治疗都显著抑制了MYC靶点、E2F靶点和G2M检查点信号级联，这可能有助于FTO抑制剂和FTO KD对细胞周期和增殖的抑制作用（图5A和S3A-S3D）。此外，所有三种治疗均持续激活细胞凋亡和p53通路（图5A）。全球基因集富集分析（GSEA）表明，FTO-KD和FB23或FB23-2治疗在调节一组功能重要的信号通路方面显示出相似的效果（图S3E-S3G）。值得注意的是，FTO-KD增加的绝大多数途径（43个中的41条，95.3%）也可以被FB23-2富集（图5B和S3H）；同样，FB23-2抑制的大多数信号通路也受到FTO KD的抑制（图5C和S3H）。这些结果强烈表明，FTO抑制剂，尤其是FB23-2，对AML细胞中控制细胞周期、细胞增殖和细胞存活的关键信号通路具有与FTO KD相同的作用。

在异种移植小鼠中，FB23-2显著减缓人类AML细胞系和原代细胞的发育。[1]
FB23-2抑制白血病进展并提高白血病小鼠的存活率。[1]
FB23-2在治疗患者来源的异种移植（PDX）AML小鼠模型中显示出疗效[1]
FB23-2在小鼠中是安全的，并且显示出良好的药代动力学特征。[1]

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FB23-2抑制白血病进展并提高白血病小鼠的存活率[1]
接下来，我们在异种移植白血病模型中评估了FB23-2的体内治疗效果。将NOD/LtSz scid IL2RG-SGM3（NSGS）小鼠（Wunderlich等人，2010）异种移植MONOMAC6 AML细胞，异种移植后10天，每天将FB23-2（2mg/kg）或载体对照注射到单个小鼠体内，持续10天。值得注意的是，FB23-2注射显著延迟了全面白血病症状的发作，并通过使中位生存期几乎翻一番显著延长了生存期（图7C）。与载体相比，FTO抑制剂治疗抑制了白血病恶性肿瘤，包括脾肿大和肝肿大减少（图7D）。FACS分析证实，FB23-2注射抑制了受体小鼠中人类AML细胞的丰度（图7E和S5C）。为了进一步解释FB23-2对体内AML细胞分化的影响，我们收集了FB23-2和载体对照处理的异种移植物小鼠的外周血（PB）、骨髓和脾脏样本，并用抗人CD15和抗人CD11b对其进行染色。通过流式细胞仪测定，FB23-2治疗促进了AML细胞在体内的分化（图7F和7G）。PB涂片的Wright Giemsa染色显示，FTO抑制剂治疗的AML小鼠的白血病母细胞受到抑制并部分分化；一致地，脾脏和肝脏的H&E染色也显示，FB23-2治疗的小鼠AML细胞扩散较少（图7H）。综上所述，我们的数据表明，FB23-2对FTO的药理学抑制可显著抑制白血病进展并延长生存期。

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FB23-2在治疗患者来源的异种移植（PDX）AML小鼠模型中显示出治疗效果[1]
我们评估了FB23-2在治疗人类原代AML细胞方面的治疗潜力。对四名具有不同细胞遗传学的AML患者进行了检测（表S8）。FB23-2抑制了所有四组原代AML细胞的增殖，IC50值范围为1.6μM至16μM（图8A）。FB23-2还诱导了这些原代AML细胞的细胞凋亡（图S6A），降低了集落形成单位（CFU）容量（图8B），并加速了ATRA介导的髓系分化（图S6B）。此外，FB23-2治疗还上调了FTO的两个直接靶点ASB2和RARA的表达（图8C），并提高了全局mRNA m6A丰度（图8D），从而支持了我们的结论，即FB23-2通过直接靶向患者来源的原代AML细胞中的FTO信号传导显示出治疗效果。

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最后，我们在PDX AML小鼠模型中测试了FB23-2的体内治疗效果。将原代AML细胞异种移植到亚致死照射的NSGS小鼠体内。我们通过FACS分析受体小鼠PB中供体AML细胞的百分比来监测AML白血病细胞的体内植入。当受体小鼠有3-5%的供体来源的AML细胞时，受体小鼠用FB23-2或DMSO治疗17天。与对照组小鼠（中位存活时间为48天）相比，FB23-2治疗小鼠的疾病潜伏期（中位生存时间为58天）显著延长（图8E）。此外，受体小鼠移植AML细胞的FACS分析显示，FB23-2治疗后，PB（图8F）和BM（图8G）中AML母细胞的比例显著降低。与我们的研究结果一致，即FB23-2在体外诱导AML细胞系的分化，我们发现FB23-2处理的小鼠体内存在更多分化的髓系细胞，细胞质/细胞核比例增加（图8H）。来自FB23-2处理的PDX小鼠的白血病细胞产生的CFU明显更少，集落大小明显减小，而来自DMSO处理的PDX-小鼠的白血病细胞则更少（图8I和8J），因此表明FB23-2治疗的AML细胞的白血病恶性程度明显受损。值得注意的是，在接受治疗的小鼠体内，FB23-2不仅影响了大量AML细胞，而且显著消除了白血病干细胞（LSCs，由CD34+CD38-定义）（图8K）。

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为了进一步评估原代PDX小鼠中功能性LSCs的数量，我们进行了二次移植。与来自原代FB23-2处理的PDX小鼠的AML细胞的次级受体小鼠相比，来自原代DMSO处理的PDX小鼠的AML细胞的次级受体（对照组）具有明显更高的植入率（图8L）。所有对照组、继发性PDX小鼠在66天内死亡，而50%的携带FB23-2处理的AML细胞的继发性PDX-小鼠在100天后仍然存活（图8M），这表明在初次受体小鼠中接受FB23-2治疗后，能够在继发受体体内再生白血病的功能性LSCs的数量显著减少。综上所述，我们的数据表明，FB23-2诱导的AML细胞分化显著减少了体内功能性原发性AML LSCs的数量。

激酶和蛋白酶谱[1]
激酶和蛋白酶分析由Eurofins Pharma Discovery Services进行。分别在1μM和10μM的FB23-2存在下，对405种激酶进行了抑制激酶谱分析。以Met（h）激酶为例进行激酶谱分析，将Met（小时）与8 mM MOPS（pH 7.0）、0.2 mM EDTA、250μM KKKGQEEEYVFIE、1 mM原钒酸钠、5 mM 6-甘油磷酸钠、10 mM Mg（OAc）2和[γ-33P]-ATP（所需的比活性和浓度）一起孵育。通过加入Mg（OAc）2/ATP混合物引发反应。在室温下孵育40分钟后，通过加入磷酸至终浓度为0.5%来终止反应。然后将10μl反应物点样到P30滤垫上，在0.425%磷酸中洗涤四次4分钟，在甲醇中洗涤一次，然后干燥和闪烁计数。结果用方程计算，抑制率（%）=（最大信号）/（最大最小）×100%。不含酶但所有其他成分的反应为Min，与DMSO的反应为Max。将10μM对405种激酶的抑制百分比绘制到激酶系统发育树上。每组有两次重复。对于IC50测定，获得不同浓度下FB23-2的抑制百分比，并使用GraphPad Prism 5中的方程通过非线性回归分析计算每种测试激酶的IC50值。[1]
以Caspase2为例，将1μM或10μMFB23-2与人重组Caspase2在50 mM HEPES、pH 7.4、100 mM NaCl、0.1%CHAPS、1 mM EDTA、10%甘油、10 mM DTT的反应缓冲液中在37°C下预孵育15分钟，然后加入25μM底物Z-VDVAD-AFC。孵育1小时后，用荧光分光光度法定量AFC信号，DMSO组按100%处理。每组有两次重复。

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FTO抑制剂对COX-1和COX-2酶的影响[1]
按照制造商的方案，使用COX荧光抑制剂筛查试剂盒评估FTO抑制剂FB23和FB23-2对COX1和COX2酶的抑制作用。简而言之，COX-1和COX-2分别与受试化合物在室温下孵育5分钟，然后将10μl ADHP（10-乙酰基-3,7-二羟基吩恶嗪）加入样品和背景孔中（不含COX酶）。通过快速加入10μl花生四烯酸引发反应，并在室温下孵育2分钟。使用535nm的激发波长和595nm的发射波长来获得信号。

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基于HPLC的RNA中m6A去甲基化抑制作用的测定[1]
对已报道的测定方法进行了一些修改，进行了体外ssRNA去甲基化（Huang et al.，2015）。将含有0.25μM FTOΔN31或3μM ALKBH5ΔN66、5μM 15聚体ssRNA（5′-AUGUCA（m6A）CAGCAGC-3′）、300μΜ2OG、280μΜ（NH4）2Fe（SO4）2、2 mM L-抗坏血酸和50 mM Tris-HCl（pH 7.5–8.0）中所需浓度的抑制剂的反应在25°C下孵育30分钟。通过在90°C下加热5分钟终止反应，然后用核酸酶P1和碱性磷酸酶消化混合物。使用GraphPad Prism 5.0™上的非线性回归、剂量-反应拟合，根据指定浓度抑制剂存在下m6A去甲基化的抑制百分比对IC50值进行定量。所有反应一式三份进行

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FTO/FB23配合物的结晶及结构测定[1]
结晶采用悬滴蒸汽扩散法在18°C下进行。将8mg/ml的FTOΔN31蛋白与5倍FB23孵育，并与含有100mM柠檬酸钠（pH 5.4）、11.5%（w/v）聚乙二醇3350和8%异丙醇的储液混合。使用额外的20%（v/v）甘油对晶体进行冷冻保护。衍射数据是在上海同步加速器研究设施（SSRF）的BL18U1和BL17U1光束线上收集的。所有X射线数据均使用HKL2000程序（Otwinowski和Minor，1997）进行处理，并在CCP4程序中转换为结构因子（Collaborative Computational Project，1994）。以FTO/MA配合物（PDB编码4QKN）的结构为搜索模型，在Phaser中通过分子置换求解该结构。用REFMAC5程序对结构复合体FTO/FB23的模型进行了计算精化。

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核磁共振（NMR）滴定法[1]
使用磷酸盐缓冲液（20mM磷酸钠（pH 7.4）、100mM NaCl、5%DMSO）在配备有25°C低温冷却探针的Bruker Avance III-600MHz光谱仪上进行NMR数据采集。实验样品分别含有200μM FB23和0μM、1μM、2μM和3μM的FTO蛋白

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细胞热位移测定法（CETSA）[1]
CETSA是根据先前描述的方案进行的（Martinez-Molina等人，2013）。收集NB4和MONOMAC6细胞，并在50mM Tris-HCl（pH 7.5）、150mM NaCl和2mM DTT中裂解。将50μM FB23或DMSO加入上清液中，并在25°C下孵育25分钟。在不同温度下变性5分钟后，离心样品，并通过蛋白质印迹分析上清液。所有实验一式三份进行。

当从AML小鼠分离的MA9和FLT3/NPM1原代细胞、五种人AML细胞（MA9.3ITD、MA9.3RAS、U937、ML2和MV4-11）和人原代AML细胞用DMSO或5μM FB23-2处理72小时进行点印迹分析时，NB4和MONOMAC6细胞用不同浓度的DMSO或FB23-2进行处理。[1]

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细胞增殖测定5000个细胞/孔NB4、FTO KO NB4和MONOMAC6 AML细胞接种并用DMSO或FTO抑制剂处理72小时。根据制造商的说明，用CellTiter 96®AQueous非放射性细胞增殖测定法测定细胞增殖。接种10000个细胞/孔的人AML细胞（MA9.3ITD、MA9.3RAS、U937、ML2和MV4-11）和来自AML患者的四个原代细胞，并如所示进行FTO抑制剂处理96小时。将从AML小鼠分离的10000个细胞/孔MA9和FLT3/NPM1原代细胞以及5000个细胞/孔shNS和shFTO NB4细胞接种并用FTO抑制剂处理24小时、48小时、72小时和96小时以进行增殖测定。[1]

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AML细胞NB4和MONOMAC6细胞中FB23和FB23-2的定量分别用10μM FB23或FB23-2处理24小时。用0.1%台盼蓝区分活细胞，计数，然后用PBS通过多次洗涤收获。用50%（v/v）水/甲醇将细胞稀释到100μl中，然后进行几个冲击冻融循环。收集上清液进行分析。应用与TSQ Quantiva质谱仪耦合的Ultimate 3000系统来测定化合物FB23和FB23-2的细胞浓度。在XSELECT™HSS T3柱（100 mm×3.0 mm，2.5μm；Waters，USA）上分离分析物。用于洗脱的流动相为（A）0.1%（v/v）甲酸/水和（B）0.1%（v/v）甲酸/乙腈。质谱仪在负MRM模式下操作。母公司到产品的转换为m/z 375.1→339.1, 375.1→FB23为298.1，m/z为390.3→318.0, 390.3→FB23-2分别为289.9。[1]

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m6A点印迹分析NB4和MONOMAC6细胞用不同浓度的DMSO或FB23-2处理72小时，而从AML小鼠分离的MA9和FLT3/NPM1原代细胞、5个人AML细胞（MA9.3ITD、MA9.3RAS、U937、ML2和MV4-11）和人原代AML细胞用DMSO或5μM FB23-2进行72小时的点印迹分析。根据制造商的说明，用miRNeasy Mini试剂盒分离总RNA，并用PolyATract mRNA分离系统IV进一步富集poly（A）+RNA。将RNA样品在RNA结合缓冲液中稀释，在65°C下变性5分钟。然后将一体积的20倍SSC缓冲液加入RNA样品中，然后用Bio-Dot仪器点在Amersham Hybond-N+膜上。通过紫外线照射将RNA样品交联到膜上。用0.02%亚甲基蓝（MB）作为负载对照对膜进行染色。紫外线交联和MB染色后，用PBST洗涤膜，在室温下用5%脱脂奶粉封闭1小时，并与m6A抗体（1:2000）在4°C下孵育过夜。最后，将膜与HRP缀合的山羊抗兔IgG孵育，并用Amersham ECL Prime Western印迹检测试剂显影。[1]

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LC-MS/MS定量AML细胞中的m6A和m6Am NB4和MONOMAC6细胞用二甲基亚砜或20μM FB23-2培养72小时。根据斑点印迹法分离信使核糖核酸，然后用Ribo-Minus转录组分离试剂盒去除受污染的rRNA。用5个单位的RppH用热塑性缓冲液将300 ng mRNA去帽，然后用核酸酶P1在42°C下消化产物1小时。随后，加入1单位碱性磷酸酶和NH4HCO3（100mM），并在37°C下再孵育1小时。Ultimate 3000系统与TSQ Quantiva质谱仪相结合，用于定量a、m6A和m6Am的细胞水平。将样品离心并加载到XSELECT™CSH™C18柱（100 mm×3.0 mm，2.5μm）上，并用梯度甲醇洗脱。A、m6A和m6Am的母体到产物的转变分别为268.1/136.1、282.1/150.1和296.2/150.1。

Animal model[1]
The NSGS mice were bred and subjected to the xeno-transplantation model. For the AML mouse model, 0.2 × 106 MONOMAC6 cells were directly transplanted into NSGS mice via tail vein. After 10 days, FB23-2 (2 mg/kg/day) and DMSO vehicle were intraperitoneally injected into the mice for a continuous 10 days. The mice were euthanized by CO2 inhalation if they exhibited classical AML symptoms including hunched posture, paralysis, and reduced body weight. Meanwhile, the PB, spleen, and liver samples were collected for further analysis.
PDX models were generated by injecting primary BM cells from AML patient Pt 2017_63 (2 X 106 per mouse) into the tail veins of 6- to 8-week-old sublethally irradiated (2.5 Gy) NSGS mice (NOD.Cg-PrkdcscidIl2rgtm1WjlTg(CMV-IL3, CSF2, KITLG)1Eav/MloySzJ), which were purchased from The Jackson Laboratory. When recipient mice had 3–5% donor-derived AML cells in PB, 6 mg/kg/day FB23-2 was delivered by i.p. for 17 days, vehicle DMSO was administrated as control. Mice were weighed daily during treatment and doses were recalculated to make sure the mice received a consistent dose of 6 mg/kg/day. One day after the 17-day full treatment, mice were randomly picked up, and then PB cells were collected and analyzed for the engraftment of leukemia cells by FACS using anti-human-CD45 and anti-mouse-CD45. When the mice became moribund, BM cells were collected and analyzed for the engraftment of leukemia cells by FACS using anti-human-CD45 and anti-mouse-CD45. In addition, the LSCs population was determined as the human CD34+CD38−population. For second transplantation, the patient AML cells, collected from the spleen of primary NSGS mice which were transplanted with BM cells from AML patients and received FB23-2 or DMSO treatment, were transplanted into NSGS mice irradiated at 2.5 Gy. 8 weeks post transplantation, PB cells were collected for FACS analysis using anti-human-CD45 and anti-mouse-CD45 and the mice were continued to monitor for survival.

FB23-2 displays a favorable pharmacokinetic profile [1]
Next, a single dose of 3 mg/kg FB23-2 was i.p. administrated to Sprague Dawley (SD) rats for the pharmacokinetic profile (Figure 7B and Table S7). The Cmax and Tmax value of FB23-2 were 2421.3 ± 90.9 ng/ml and 0.08 hr, respectively. FB23-2 elimination half-life, T1/2 was 6.7 ± 1.3 hr, and the AUC0–24 was 2184 ± 152 hr × ng/ml. Meanwhile, FB23 was also detected, with Cmax and Tmax as 142.5 ± 26.1 and 0.4 ± 0.1 hr, respectively. The metabolic stability of FB23-2 in the SD rat liver microsome was also determined, with an estimated T1/2 of 128 min, and an intrinsic clearance of 19.7 ml/min/kg. Lastly, we measured the degree of protein binding by FB23-2. Nearly 100% FB23-2 inhibitor was bound to plasma proteins. In summary, FB23-2 displayed a favorable pharmacokinetic profile for in vivo study.

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Pharmacokinetics [1]
Inhibitor FB23-2 was formulated in DMSO at 3 mg/ml. SD rat (male, 7 – 8 weeks old, n = 3) were treated intraperitoneally with 1 ml/kg formulated compound. Blood samples were collected by retro-orbital bleeding at 5 min, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hr after the intraperitoneal administration. Blood was collected into EDTA-containing tubes and plasma was obtained by centrifugation at 2,000 g for 5 min. FB23-2 and its hydrolysis metabolite FB23 concentrations in plasma were quantitated by LC-MS/MS method. Noncompartmental analysis with Phoenix 1.4 (Pharsight, USA) was used for all analytical measurements. Area under the concentration-time curve (AUC) was calculated using trapezoidal method. AUC0−∞ = AUC0-t + Ct/ke, ke is elimination rate constant. Elimination half-life (T1/2) = 0.693/ke, mean residence time (MRT) = AUMC/AUC.

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Quantitation of FB23-2 in plasma [1]
Calibration curve concentrations ranged from 1.00 to 500 ng/ml for FB23-2 and FB23. 50 μl of rat plasma was precipitated by adding 150 μl acetonitrile immediately and vortexed to stabilize FB23-2 at each sample collection. 50 μl of study sample supernatant, 25 μl internal standard solution (probenecid and estrone-3-sulfate: 400/100 nmol/l), and 50.0 μl of 5 mM ammonium acetate solution (containing 0.1% formic acid) were added to a 1.5 ml polypropylene tube, then vortexed and centrifuged at 11,000 × g for 10 min, the supernatant was injected to LC-MS/MS. A LC-30AD liquid chromatographic system coupled to a Triple Quad 5500 mass spectrometer was used for acquiring LC-MS/MS data. Analytes were separated on an Eclipse Plus C18 column (100 mm × 4.6 mm I.D., 3.5 μm;). The mobile phases used for isocratic elution were 25% (A) 5 mM ammonium acetate-formic acid (100/0.1, v/v) and 75% (B) acetonitrile. The flow rate was 0.6 ml/min. The mass spectrometer was operated in the negative MRM mode. The parent-to-product transitions were m/z 390.2→318.0 for FB23-2, m/z 283.9→239.9 for probenecid (internal standard of FB23-2), m/z 375.2→298.2 for FB23, m/z 349.2→269.2 for estrone-3-sulfate (internal standard of FB23). The collision energy was set at −16, −30, −28, and −43 eV. The dwell time for each transition was set at 100 ms.

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Microsomal stability assay [1]
The assay was conducted as the previously reported (Di et al., 2006). Briefly, 3 μM FB23-2 was incubated with 0.5 mg/ml rat liver microsomal protein at 37 °C in the presence of 1 mM NADPH cofactor. After incubation for 0, 5, 15, 30 and 60 min, respectively, cold acetonitrile was added to terminate the reactions. The solution was centrifuged, and the supernatants were analyzed using LC–MS/MS method similar with the quantitation of FB23 and FB23-2 in AML cells. The parent-to-product transition of probenecid, internal standard of FB23-2, was m/z 283.9→239.9. The formula for calculation was T1/2 = −0.693/k, and the inherent clearance rate CLint = (0.693/in vitro T1/2) × (incubation volume/mg of microsomal protein) × (mg of microsomal protein/gram of liver) × (gram of liver/kg body weight). Each time-point group includes two repeats.

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Plasma protein binding [1]
The plasma protein binding assay was conducted with the Rapid Equilibrium Dialysis (RED) (Wang and Williams, 2013). 3 μM FB23-2 was added to rat plasma, which were vortexed well before placing 100 μl in the red chamber of the RED device. 300 μl of PBS (pH 7.4) was added to the corresponding white chamber, the base plate was covered with a gas-permeable membrane, and then incubated in a CO2 incubator set at 250 rpm and 37 °C for 4 hr. Samples collected from both chambers were analyzed with LC-MS/MS to determine the compound concentrations (C0 - initial concentration, Cf - ultrafiltrate and Cp - plasma). Percentage of protein binding (PPB) using the RED device was calculated with the formula, PPB (%) = (1- Cf /Cp) × 100%. The experiments were performed in triplicate.

FB23-2 is safe in mice [1]
To determine if FB23-2 is safe for in vivo treatment, we examined the toxic effects of multi-doses of FB23-2 in BALB/c mice over a two-week time frame. The BALB/c mice (n = 5) were treated by way of intraperitoneal injection (i.p.) daily with 10, 20, 40, and 80 mg/kg FB23-2 respectively, for 14 days. Under a dosing scheme of 20 mg/kg FB23-2, we observed no evidence of body weight loss (Figures 7A and S5A); nor was any physical damage observed on different organs (Figures 7A, S5A, and S5B). Blood was collected, and further hematology and plasma biochemistry analysis showed that no significant difference was observed in hematopoiesis among the vehicle control and the 20 mg/kg inhibitor-treated mice (Tables S5 and S6). These data indicate that FB23-2 in a dosage of 20 mg/kg is safe for exploring in vivo efficacy.

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Toxicity study [1]
6 to 8-week old BALB/c mice weighed 20 ± 2 g were used. A veterinary health check was performed to select healthy BALB/c mice. Mice were randomly grouped and treated daily with either vehicle control or FB23-2 intraperitoneally for 14 days. The mice were housed five per ventilated polysulfone cage and maintained under constant temperature (18 – 26 °C), humidity (30 – 70%) and lighting conditions (12 hr light and 12 hr dark). After 14 days, animals were euthanized. Then PB samples were collected for complete blood content analysis and plasma biochemical analysis. The vital organs (heart, kidney, lung, liver, and spleen) were collected and weighed.

[1]. Small-Molecule Targeting of Oncogenic FTO Demethylase in Acute Myeloid Leukemia. Cancer Cell. 2019 Apr 15;35(4):677-691.e10.

FTO, an mRNA N6-methyladenosine (m6A) demethylase, was reported to promote leukemogenesis. Using structure-based rational design, we have developed two promising FTO inhibitors, namely FB23 and FB23-2, which directly bind to FTO and selectively inhibit FTO's m6A demethylase activity. Mimicking FTO depletion, FB23-2 dramatically suppresses proliferation and promotes the differentiation/apoptosis of human acute myeloid leukemia (AML) cell line cells and primary blast AML cells in vitro. Moreover, FB23-2 significantly inhibits the progression of human AML cell lines and primary cells in xeno-transplanted mice. Collectively, our data suggest that FTO is a druggable target and that targeting FTO by small-molecule inhibitors holds potential to treat AML.[1]

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Few inhibitors for regulation of RNA methylation have been characterized, which exists in sharp contrast to factors of DNA and histone epigenetics. Here we report that through structure-based rational designs, we have successfully developed more effective small-molecule inhibitors of FTO. The MA-derived inhibitor FB23 displays significantly improved inhibitory activity on FTO demethylation of m6A-RNA in vitro. Next, we optimized the physicochemical property of FB23, thus leading to the identification of FB23-2 with a significantly improved ability to hinder the proliferation of a panel of AML cell lines, and also inhibits primary AML LSCs in PDX mice, thus suggesting that FTO might serve as a potential molecular target in LSCs in order to inhibit leukemogenesis. The discovery of FB23-2 and its anti-proliferative effects on AML would increase the current intense interest in RNA methylation, especially with regard to the pharmacology.[1]

Importantly, we tend to show our inhibitors target FTO and impair its demethylation, and by targeting FTO our inhibitor causes a significant biological impact. We validated that the effects of FTO inhibitors on AML are linked to certain downstream targets, e.g., MYC, CEBPA, RARA, and ASB2 RNA transcripts. It remains unknown whether FB23-2 impairs FTO’s binding to target transcripts in cells, however. The target engagement of current inhibitors needs further explorations with a more depth, which could show the potential for these inhibitors to help propel the field of epitranscriptomics forward.

In summary, we provide here a proof-of-concept that small-molecule targeting of oncogenic FTO demethylase may be an effective therapeutic strategy for the treatment of AML. Our study demonstrates the feasibility of attenuated FTO demethylation for the induction of differentiation of AML cells. This effect is likely achieved through specifically regulating expression of critical genes and signalling pathways as a result of elevated m6A levels in mRNA transcripts of these genes that are induced by FTO inhibitors. As FTO-mediated demethylation has also been linked to a variety of cancer types, our findings may have a broad impact on cancer therapy by targeting epitranscriptomic RNA methylation.

C18H15CL2N3O3

392.2360

391.05

C, 55.12; H, 3.85; Cl, 18.08; N, 10.71; O, 12.24

2243736-45-8

2243736-45-8

138454779

White to light yellow solid powder

4.9

87.4Ų

3

5

4

26

486

0

ILHNIWOZZKIBNW-UHFFFAOYSA-N

InChI=1S/C18H15Cl2N3O3/c1-9-16(10(2)26-23-9)11-7-13(19)17(14(20)8-11)21-15-6-4-3-5-12(15)18(24)22-25/h3-8,21,25H,1-2H3,(H,22,24)

2-((2,6-dichloro-4-(3,5-dimethylisoxazol-4-yl)phenyl)amino)-N-hydroxybenzamide

FB 23-2; FB232; FB23-2; FB-23-2; FB 232; FB-232

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 本产品在运输和储存过程中需避光。

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

DMSO: ~25 mg/mL (~63.7 mM)

配方 1 中的溶解度: ≥ 2.08 mg/mL (5.30 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: ≥ 2.08 mg/mL (5.30 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 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 3 中的溶解度: 10 mg/mL (25.49 mM) in 50% PEG300 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 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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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;
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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。