CX3CR1 ( IC50 = 0.32 nM )

JMS-17-2（10 mg/kg；腹腔注射；每天两次，持续三天）显着减少 SCID 小鼠内脏和外周器官的肿瘤数量[1]。

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在上述靶点验证研究的基础上，我们利用非特异性趋化因子拮抗剂的重要药效学特征，并将其与G蛋白偶联受体配体的药物样元件结合，合成了CX3CR1的小分子拮抗剂JMS-17-2（图3A和方法）JMS-17-2以剂量依赖的方式强烈拮抗CX3CR1信号传导，如通过抑制ERK磷酸化所测量的（图3B和C）。有趣的是，在阻断FKN诱导的ERK磷酸化方面最有效的两种浓度的JMS-17-2也显著减少了乳腺癌症细胞在体外的迁移[1]。

JMS-17-2 (10 mg/kg；腹腔注射；每天两次，持续三)导致SCID小鼠周和内脏器官肿瘤显着减少[1]。 动物模型：SCID小鼠（~25g）MDA- 231 异种移植物[1] 剂量：10 mg/kg 给药方式：腹腔注射；每天两次，持续三周 结果：使骨骼和内脏器官的肿瘤显着减少。

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然后，研究人员试图在我们的相关临床前转移模型中确定JMS-17-2对乳腺CTC转化为骨骼DTC的影响。以10mg/Kg（i.p.）的剂量给小鼠服用JMS-17-2的药代动力学评估显示，给药后一小时血液中的药物水平为89ng/ml（210 nM），相当于该化合物在体外最低完全有效剂量（10nM，见图3B，C）的20倍。因此，第一组小鼠接受用10nMJMS-17-2预孵育的MDA-231癌症细胞，而第二组动物在IC接种癌症细胞前一小时和后三小时给予JMS-17-2（10mg/Kg；腹膜内）两次，以最大限度地靶向参与。值得注意的是，与用赋形剂治疗的对照组动物相比，两个实验组的DTC减少了约60%（图3D和E）

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减少肿瘤接种直接损害定植和生长[1]
在患者骨髓中检测到的乳腺DTC的预后价值已经确定，值越高，临床结果越差。由于JMS-17-2没有完全消除乳腺CTC向骨骼的接种，我们的目的是确定骨骼DTC的减少是否以及在多大程度上会转化为对肿瘤生长的长期抑制。为此，小鼠移植了MDA-231细胞，这些细胞经过工程改造，既能表达荧光标记，也能表达生物发光标记，并在IC注射后的两周内通过体内成像进行监测。此外，尸检后还通过多光谱荧光显微镜检查骨组织切片来评估骨骼肿瘤和DTC的存在。这些实验表明，与在骨骼和内脏部位出现多个肿瘤的对照动物相比，接受用JMS-17-2预孵育的癌症细胞的八只动物中有七只没有肿瘤（图4A和B）。值得注意的是，当使用基于多光谱显微镜的冷冻组织切片成像检查荧光信号时，这些动物也被发现没有微观肿瘤病灶和DTC（图4C）。

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为了确定CX3CR1与JMS-17-2的药物靶向是否会产生类似的效果，根据我们对该化合物的药代动力学研究结果，将MDA-231细胞移植小鼠，并在IC注射后的第一周随机给药，每天两次或10mg/KgJMS-17-2腹腔注射，持续三周。在实施安乐死之前，每周对动物进行一次成像，我们观察到用JMS-17-2治疗可以减少肿瘤病灶的数量和整体肿瘤负担，至少与使用CRISPRi观察到的效果一样有效（图5D和E）。总之，这些结果表明了CX3CR1在决定扩散性乳腺癌症细胞的接种、定植和进展中的关键作用。我们决定确定干扰CX3CR1功能是否会改变在肿瘤发生中起作用的基因的表达。因此，通过LCM收集的肿瘤组织（图5F）使用Nanostring技术询问730个基因的表达，包括606个调节13个典型信号通路的基因和124个癌症驱动基因（PanCancer Panel）。CRISPRi和JMS-17-2处理的比较分析表明，有9个基因发生了类似的改变，其中WNT5a是唯一上调的基因（图5G和表1）。值得注意的是，CX3CR1的药理学和基因组靶向都导致NOTCH3的强烈下调（表1）和Notch信号通路的显著失调（补充图S4）。

JMS-17-2的药效团设计与合成 先前发现一种有效的CCR1拮抗剂与巨细胞病毒受体US28结合。由于FKN也能与这种受体有效结合，我们推测这种化合物也会与CX3CR1结合。因此，我们合成了这种CCR1拮抗剂（命名为化合物-1），并发现它也是CX3CR1的功能性拮抗剂，IC50=268 nM，这是通过测量FKN刺激的ERK1/2磷酸化的抑制作用而建立的，该抑制作用是用平板法检测的。随后，我们通过修饰二苯基乙腈部分并将其与右侧芳基哌啶基序结合来优化化合物-1，从而发现了先导系列和化合物JMS-17-2（IC50=0.32 nM）。JMS-17-2显示的有利效力与CX3CR1对其他趋化因子受体（如CXCR2和CXCR1）的显著选择性相结合，对于这些受体，该化合物在高达1μM的浓度下缺乏活性，并且通过蛋白质印迹分析测试了CXCR4（最近发表了一项涵盖该化合物的专利[US no.8435993]，并在其他地方提交了一份手稿，详细报告了JMS-17-2的合成和药理学验证）。

CX3CR1的体外刺激和下游信号传导分析[1]
SKBR3人癌症细胞在暴露于50nM重组人FKN 5分钟之前血清饥饿4小时，之前与15μg/ml的CX3CR1中和抗体或JMS-17-2拮抗剂（10nM）在37°C下孵育30分钟。

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趋化性测定[1]
将MDA-231细胞（1×105）饥饿过夜，并放置在200μl无血清培养基的transwell插入物（孔径为8-μm的过滤器）的顶部腔室中。将插入物转移到24孔板中，每个孔含有700μl无血清培养基，含或不含重组人FKN（50nM）。使用10%FBS获得阳性对照。对于涉及JMS-17-2和CX3CR1中和抗体的实验，将细胞接种在含有JMS-17-2（1nM、10nM和100nM）或抗体（15μg/ml）的无血清培养基中，然后转移到含有JMS-17-2中或中和抗体加FKN的孔中。允许细胞在37°C下迁移6小时，在测定结束时，用尖头拭子擦洗两次，去除仍在过滤器顶部的细胞。迁移到过滤器底部的细胞用100%甲醇固定10分钟；然后用蒸馏水洗涤过滤器，从插入物中取出，使用含有DAPI的安装介质安装在盖玻片上进行核染色。每种情况进行两次重复实验，并使用五个随机显微镜场进行细胞计数，使用连接到Nuance多光谱成像系统的奥林巴斯BX51显微镜，使用2.4版分析软件（CRI）进行。进行了三个独立的实验，结果以每种条件下迁移的细胞与对照条件（无血清培养基）中迁移的细胞的比率表示。

SCID mice (~25g) with MDA-231 xenograft
10 mg/kg
Aministered i.p.; twice a day for three weeks

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Model of tumor seeding [1]
For the pre-incubation experiments, MDA-231 cells in suspension were exposed to either a CX3CR1 neutralizing antibody (15μg/ml) or the JMS-17-2 compound (10nM in 0.1% DMSO) for 30 minutes (10 minutes at room temperature plus 20 minutes on ice), before being delivered to mice in the same pre-incubation suspension to maximize target engagement. Species- and class-matched irrelevant immunoglobulins (Rabbit IgG, 15μg/ml) or DMSO were used for the control groups. For the experiments requiring administration of JMS-17-2, animals were then treated i.p. with the CX3CR1 antagonist dissolved in 4% DMSO, 4% Cremophor EL in sterile ddH20 or just vehicle twice, one-hour prior and three hours after being injected with cancer cells. The dosing regimen was selected based on results from pharmacokinetic analyses. Mice were killed 24 hours post-injection, except for the experiments described in Fig. 3 A-C, for which mice were killed at two weeks post-injection. Blue-fluorescent beads, 10μm-polystyrene in diameter were included in the injection medium and visualized by fluorescence microscopy to validate injection efficiency. Mice showing non-homogenous distribution of or lacking fluorescent beads in tissue sections of lungs and kidneys were removed from the study.

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Model of established metastases [1]
One week after IC cell injection, animals were randomly assigned to control and treated group and then imaged for tumors in the skeleton and soft-tissue organs. Vehicle or JMS-17-2 (10mg/Kg) was administered i.p. twice/day, respectively, for the entire duration of the study while animals were imaged weekly.

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Pharmacokinetic analyses [1]
Mice were administered with 10mg/Kg of JMS-17-2 in 10% dimethylacetamide (DMAC), 10% tetraethylene glycol and 10% Solutol HS15 in sterile ddH2O. Animals were then anesthetized as described above and 300μl of blood samples were collected by cardiac puncture at the designated time points and transferred in K2EDTA tubes. Blood samples were placed on ice and tested after dilution. The measurement of JMS-17-2 concentrations in blood and brain tissue was outsourced to Alliance Pharma (www.alliancepharmaco.com).

Pharmacokinetic evaluation of JMS-17-2 administered to mice at a dose of 10mg/Kg (i.p.) produced drug levels of 89ng/ml (210 nM) in blood measured one hour after dosing, which corresponds to a 20-fold increase over the lowest fully effective dose of this compound in vitro [1].

[1]. Novel Small-Molecule CX3CR1 Antagonist Impairs Metastatic Seeding and Colonization of Breast Cancer Cells. Mol Cancer Res. 2016 Jun;14(6):518-27.

Recent evidence indicates that cancer cells, even in the absence of a primary tumor, recirculate from established secondary lesions to further seed and colonize skeleton and soft tissues, thus expanding metastatic dissemination and precipitating the clinical progression to terminal disease. Recently, we reported that breast cancer cells utilize the chemokine receptor CX3CR1 to exit the blood circulation and lodge to the skeleton of experimental animals. Now, we show that CX3CR1 is overexpressed in human breast tumors and skeletal metastases. To assess the clinical potential of targeting CX3CR1 in breast cancer, a functional role of CX3CR1 in metastatic seeding and progression was first validated using a neutralizing antibody for this receptor and transcriptional suppression by CRISPR interference (CRISPRi). Successively, we synthesized and characterized JMS-17-2, a potent and selective small-molecule antagonist of CX3CR1, which was used in preclinical animal models of seeding and established metastasis. Importantly, counteracting CX3CR1 activation impairs the lodging of circulating tumor cells to the skeleton and soft-tissue organs and also negatively affects further growth of established metastases. Furthermore, nine genes were identified that were similarly altered by JMS-17-2 and CRISPRi and could sustain CX3CR1 prometastatic activity. In conclusion, these data support the drug development of CX3CR1 antagonists, and promoting their clinical use will provide novel and effective tools to prevent or contain the progression of metastatic disease in breast cancer patients.[1]

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A major clinical need is to identify effective treatment to delay disease progression in patients presenting with few metastatic lesions. It has been recently demonstrated that existing metastases function as active reservoirs of tumor cells, cross-seeding other metastases and generate additional lesions, which makes therapeutic treatments directed to effectively counteract cancer seeding urgently needed. The target validation achieved in vivo with CRISPRi silencing of CX3CR1 provided impetus to test the JMS-17-2 compound in animals reproducing early metastatic onset in patients. The results from these experiments are strongly indicative that impairing CX3CR1 can successfully limit metastatic cross-seeding. On the other hand, the unexpected observation that both JMS-17-2 treatment and CRISPRi drastically restricted the growth of single lesions and contained the overall tumor burden could not be justified by interfering with tumor seeding.
Therefore, to understand the mechanistic basis underpinning this role of CX3CR1 in regulating secondary tumor growth and identify the signaling pathway altered by targeting this receptor, we harvested tumor tissues from animals in the control, JMS-17-2 treated and CRISPRi experimental groups and conducted comparative transcriptome analyses using Nanostring technology. Using this uniquely informative approach, we found that nine genes were altered in a corresponding fashion by JMS-17-2 and CRISPRi-mediated gene silencing, thus revealing molecular mediators for the role of CX3CR1 in supporting survival and proliferation of disseminated breast cancer cells. Particularly relevant is the up-regulation of WNT5A, involved in non-canonical Wnt signaling and endowed with a suppressive activity on metastatic breast cancer. The down-regulation of SOST, highly implicated in bone-related disease nd PRLR, which promotes colonization of breast cancer cells in soft tissues, are equally compelling. Finally, the down-regulation of NOTCH3 and deregulation of Notch signaling pathway (Supplementary Fig. S4) are strongly indicative of a possible role of CX3CR1 antagonism mitigating the tumor-initiating properties regulated by this gene in breast cancer. Indeed, CX3CR1 transactivates the Epidermal Growth Factor signaling pathway in breast cancer cells, promoting cell proliferation in vitro and delaying mammary tumor onset in mouse models.
In conclusion, the work presented here introduces a conceptual shift in the treatment strategies for breast cancer patients. Furthermore, we have synthesized and functionally characterized the first lead compound in a novel class of potentially new drugs with novel mechanisms of action to be added to the arsenal of therapies to treat advanced breast adenocarcinoma.[1]

C25H26CLN3O

419.95

419.176

C, 71.50; H, 6.24; Cl, 8.44; N, 10.01; O, 3.81

1380392-05-1

JMS-17-2 hydrochloride; 2341841-07-2

57382073

White to off-white solid powder

1.3±0.1 g/cm3

603.8±55.0 °C at 760 mmHg

319.0±31.5 °C

0.0±1.7 mmHg at 25°C

1.661

4.8

28.5

0

2

5

30

585

0

ClC1C=CC(=CC=1)C1CCN(CCCN2C(C3=CC=CN3C3C=CC=CC2=3)=O)CC1

WOSMCMULWWHMIV-UHFFFAOYSA-N

InChI=1S/C25H26ClN3O/c26-21-10-8-19(9-11-21)20-12-17-27(18-13-20)14-4-16-29-23-6-2-1-5-22(23)28-15-3-7-24(28)25(29)30/h1-3,5-11,15,20H,4,12-14,16-18H2

5-[3-[4-(4-chlorophenyl)piperidin-1-yl]propyl]pyrrolo[1,2-a]quinoxalin-4-one

JMS-172; JMS-17-2; 1380392-05-1; 5-(3-(4-(4-chlorophenyl)piperidin-1-yl)propyl)pyrrolo[1,2-a]quinoxalin-4(5H)-one; 5-[3-[4-(4-chlorophenyl)piperidin-1-yl]propyl]pyrrolo[1,2-a]quinoxalin-4-one; JMS-17-21380392-05-1; 5-{3-[4-(4-chlorophenyl)piperidin-1-yl]propyl}-4h,5h-pyrrolo[1,2-a]quinoxalin-4-one; 5-{3-[4-(4-chlorophenyl)piperidin-1-yl]propyl}pyrrolo[1,2-a]quinoxalin-4-one; MFCD30489012; JMS172; JMS 17-2; JMS-17 2; JMS 17 2; JMS17-2

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: 25~40 mg/mL (59.5~95.2 mM)
Ethanol: 10 mg/mL

配方 1 中的溶解度: ≥ 2.08 mg/mL (4.95 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 (4.95 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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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；
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