CX3CR1 ( IC50 = 0.32 nM )

JMS-17-2 (10 mg/kg; intraperitoneal injection; twice daily for three days) significantly reduced the amount of tumors in the visceral and peripheral organs in SCID mice [1].

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Building on the target validation studies described above, we synthesized JMS-17-2, a small-molecule antagonist of CX3CR1, by exploiting important pharmacophoric features of non-specific chemokine antagonists and combining these with drug-like elements of G-protein coupled receptor ligands (Fig.3A and methods). JMS-17-2 potently antagonizes CX3CR1 signaling in a dose-dependent fashion, as measured by inhibition of ERK phosphorylation (Fig.3B and C). Interestingly, the two concentrations of JMS-17-2 most effective in blocking FKN-induced ERK phosphorylation also significantly reduced the migration of breast cancer cells in vitro [1].

JMS-17-2 (10 mg/kg; intraperitoneal injection; twice daily for three days) resulted in significant reductions in peripheral and visceral organ tumors in SCID mice [1]. Animal model: SCID mouse (~25g) MDA-231 xenograft [1] Dose: 10 mg/kg Administration: intraperitoneal injection; twice daily for three weeks Results: Significant tumor growth in bones and visceral organs reduce.

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Researchers then sought to ascertain the effects of JMS-17-2 on the conversion of breast CTCs into skeletal DTCs, in our relevant pre-clinical model of metastasis. 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 (10nM, see fig. 3B,C). Thus, a first group of mice received MDA-231 cancer cells pre-incubated with 10nM JMS-17-2, whereas a second group of animals was dosed with JMS-17-2 (10mg/Kg; i.p.) twice, one hour prior and three hours after the IC inoculation of cancer cells, to maximize target engagement. Remarkably, both experimental groups showed a reduction in DTCs of approximately 60% as compared to control animals treated with vehicle (Fig. 3D and E)

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Reducing tumor seeding directly impairs colonization and growth [1]
The prognostic value of breast DTCs detected in the bone marrow of patients has been established, with higher values corresponding to a poorer clinical outcome. Since JMS-17-2 did not completely abolish the seeding of breast CTCs to the skeleton, we aimed to determine if and to which extent the reduction in skeletal DTCs would translate into a long-term inhibition of tumor growth. To this end, mice were grafted with MDA-231 cells engineered to express both fluorescent and bioluminescent markers and monitored by in vivo imaging during the two weeks following IC injection. Furthermore, the presence of skeletal tumors and DTCs was also assessed post-necropsy by examining bone tissue sections by multispectral fluorescence microscopy. These experiments showed that, in contrast to control animals presenting multiple tumors both in the skeleton and visceral sites, seven of the eight animals that received cancer cells pre-incubated with JMS-17-2 were found free of tumors (Fig. 4A and B). Notably, these animals were also found devoid of microscopic tumor foci and DTCs when inspected for fluorescent signals with multispectral microscopy-based imaging of frozen tissue sections (Fig. 4C).

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To determine whether the pharmacologic targeting of CX3CR1 with JMS-17-2 would produce similar effects, mice were grafted with MDA-231 cells and at the first week post-IC injection were randomized and administered with either vehicle or 10mg/Kg JMS-17-2 i.p. twice daily for three weeks, based on the results of our pharmacokinetics studies with this compound. Animals were imaged weekly before being euthanized and we observed that treatment with JMS-17-2 led to a reduction in the number of tumor foci and overall tumor burden, at least as effectively as observed using CRISPRi (Fig. 5D and E). Taken together, these results point towards a crucial role of CX3CR1 in dictating seeding, colonization and progression of disseminated breast cancer cells. We decided to ascertain whether interfering with CX3CR1 functioning could alter the expression of genes with an established role in tumorigenesis. Thus, tumor tissues collected by LCM (Fig. 5F) were interrogated using Nanostring technology for the expression of 730 genes including 606 genes regulating 13 canonical signaling pathways and 124 cancer driver genes (PanCancer Panel). A comparative analysis of CRISPRi and JMS-17-2 treatment showed that nine genes were similarly altered, with WNT5a being the only up-regulated gene (Fig. 5G and table 1). Notably, both pharmacologic and genomic targeting of CX3CR1 resulted in a strong down-regulation of NOTCH3 (table 1) and significant deregulation of the Notch signaling pathway (Supplementary Fig. S4).

Pharmacophore design and synthesis of JMS-17-2 [1]
A potent CCR1 antagonist, was previously found to bind the cytomegalovirus receptor US28. Since FKN also binds potently to this receptor, we speculated that this compound would also engage CX3CR1. Thus, we synthesized this CCR1 antagonist (named Compound-1) and found it to be also a functional antagonist of CX3CR1 with an IC50 = 268 nM, established by measuring the inhibition of FKN-stimulated ERK1/2 phosphorylation detected with a plate-based assay. Subsequently, we optimized Compound-1 by modifying the diphenyl acetonitrile moiety and combining it with the right-hand aryl piperidine motif, which led to the discovery of a lead series and the compound JMS-17-2 (IC50 = 0.32 nM). The favorable potency shown by JMS-17-2 was combined with significant selectivity for CX3CR1 over other chemokine receptors such CXCR2 and CXCR1, for which this compound showed lack of activity at concentrations as high as 1μM as well as CXCR4 that was tested by Western blot analysis (a patent covering this compound was recently published [US no. 8,435,993] and a manuscript reporting synthesis and pharmacologic validation of JMS-17-2 in major detail has been submitted elsewhere).

In vitro stimulation of CX3CR1 and analysis of downstream signaling [1]
SKBR3 human breast cancer cells were serum starved for four hours before being exposed to 50nM recombinant human FKN for 5 minutes, with or without previous incubation with either a CX3CR1 neutralizing antibody used at 15μg/ml or the JMS-17-2 antagonist (10nM) for 30 minutes at 37 °C.

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Chemotaxis assay [1]
MDA-231 cells (1×105) were starved overnight and plated in the top chamber of transwell inserts (filters with 8-μm pore diameter) 200μl of serum-free culture medium. The inserts were transferred into a 24-well plate where each well contained 700μl of serum-free medium with or without recombinant human FKN (50nM). Positive controls were obtained using 10% FBS. For experiments involving JMS-17-2 and the CX3CR1 neutralizing antibody, cells were plated on the upper side of the filters in serum-free medium containing JMS-17-2 (1nM, 10nM and 100nM) or the antibody (15μg/ml) and then transferred to wells containing JMS-17-2 or the neutralizing antibody plus FKN. Cells were allowed to migrate at 37°C for 6 hours and at the end of the assay the cells still on the top of the filter were removed by scrubbing twice with a tipped swab. Cells migrated to the bottom of the filter were fixed with 100% methanol for 10 minutes; filters were then washed with distilled water, removed from the insert and mounted on cover glasses using mounting medium containing DAPI for nuclear staining. Two replicates were conducted for each condition, and five random microscope fields were used for cell enumeration, conducted with an Olympus BX51 microscope connected to the Nuance multispectral imaging system using version 2.4 of the analysis software (CRI). Three independent experiments were performed and results were presented as a ratio of cells that migrated under each condition relative to cells that migrated in control conditions (serum-free culture medium).

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).

Pharmacokinetics of JMS-17-2 were evaluated in mice by administering it at a dose of 10 mg/Kg (ip). One hour after administration, the drug concentration in the blood was 89 ng/ml (210 nM), which is equivalent to 20 times the lowest fully effective dose of the 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 suggests that cancer cells can circulate from established metastatic lesions even without a primary tumor, further disseminating and colonizing bone and soft tissue, thereby expanding the scope of metastasis and accelerating disease progression to terminal stages. We recently reported that breast cancer cells utilize the chemokine receptor CX3CR1 to leave the bloodstream and colonize the bones of experimental animals. Now, we have found that CX3CR1 is overexpressed in both human breast tumors and bone metastases. To evaluate the clinical potential of CX3CR1-targeted therapy for breast cancer, we first validated the functional role of CX3CR1 in metastasis and progression using a neutralizing antibody against this receptor and CRISPR interference (CRISPRi) technology. Subsequently, we synthesized and characterized JMS-17-2, a potent and selective small molecule antagonist of CX3CR1, and applied it to preclinical animal models of dissemination and established metastases. Importantly, inhibition of CX3CR1 activation hindered the colonization of circulating tumor cells in bone and soft tissue organs and negatively impacted the further growth of established metastases. In addition, the study found that nine genes under the action of JMS-17-2 and CRISPRi underwent similar changes, which can maintain the metastatic activity of CX3CR1. In summary, these data support the drug development of CX3CR1 antagonists, and promoting their clinical application will provide new and effective means to prevent or control the progression of metastatic disease in breast cancer patients. [1] An important clinical need is to find effective treatments to delay the progression of disease in patients with fewer metastatic lesions. Recent studies have shown that existing metastatic lesions can serve as active reservoirs of tumor cells, cross-disseminating to other metastatic lesions and generating new lesions. Therefore, there is an urgent need for treatments that effectively inhibit cancer cell dissemination. The in vivo targeting validation of CX3CR1 silencing using CRISPRi prompted us to test the compound JMS-17-2 in an animal model simulating early metastasis in patients. These experimental results strongly suggest that inhibiting CX3CR1 can effectively limit metastatic cross-dissemination. On the other hand, the unexpected finding that both JMS-17-2 treatment and CRISPRi can significantly inhibit the growth of individual lesions and control the overall tumor burden cannot be explained by interfering with tumor dissemination. Therefore, to understand the mechanism of CX3CR1 in regulating secondary tumor growth and to identify the signaling pathways altered by targeting this receptor, we collected tumor tissues from animals in the control, JMS-17-2 treatment, and CRISPRi experimental groups and performed comparative transcriptome analysis using Nanostring technology. Using this uniquely informative approach, we found that JMS-17-2 and CRISPRi-mediated gene silencing led to corresponding alterations in nine genes, revealing the molecular mechanism by which CX3CR1 plays a role in supporting the survival and proliferation of metastatic breast cancer cells. Of particular note was the upregulation of WNT5A, which participates in the non-canonical Wnt signaling pathway and has inhibitory activity against metastatic breast cancer. Also noteworthy were the downregulations of SOST (closely associated with bone-related diseases) and PRLR (promoting breast cancer cell colonization in soft tissues). Finally, the downregulation of NOTCH3 and the dysregulation of the Notch signaling pathway (Supplementary Figure S4) strongly suggest that CX3CR1 antagonism may function by attenuating the tumor initiation characteristics regulated by this gene in breast cancer. In fact, CX3CR1 can transactivate the epidermal growth factor signaling pathway in breast cancer cells, promote cell proliferation in vitro, and delay the occurrence of breast tumors in mouse models. In summary, the research results presented in this paper bring about a conceptual shift in the treatment strategy for breast cancer patients. In addition, we synthesized and functionally characterized the first potential new drug lead compound with a novel mechanism of action, which is expected to be added to the treatment regimen for 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

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

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.95 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 20.8 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: ≥ 2.08 mg/mL (4.95 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 20.8 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.)